Bridged bicyclic 2,3-dioxabicyclo[3.3.1]nonanes as antiplasmodial agents: Synthesis, structure-activity relationships and studies on their biomimetic reaction with Fe(II)

Article abstract of DOI:10.1016/j.bioorg.2019.103020

Despite recent advancements in its control, malaria is still a deadly parasitic disease killing millions of people each year. Progresses in combating the infection have been made by using the so-called artemisinin combination therapies (ACTs). Natural and synthetic peroxides are an important class of antimalarials. Here we describe a new series of peroxides synthesized through a new elaboration of the scaffold of bicyclic-fused/bridged synthetic endoperoxides previously developed by us. These peroxides are produced by a straightforward synthetic protocol and are characterized by submicromolar potency when tested against both chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum strains. To investigate their mode of action, the biomimetic reaction of the representative compound 6w with Fe(II) was studied by EPR and the reaction products were characterized by NMR. Rationalization of the observed structure-activity relationship studies was performed by molecular docking. Taken together, our data robustly support the hypothesized mode of activation of peroxides 6a-cc and led to the definition of the key structural requirements responsible for the antiplasmodial potency. These data will pave the way in future to the rational design of novel optimized antimalarials suitable for in vivo investigation.

Full text of DOI:10.1016/j.bioorg.2019.103020

Accepted Manuscript
Bridged bicyclic 2,3-dioxabicyclo[3.3.1]nonanes as antiplasmodial agents: syn-
thesis, structure-activity relationships and studies on their biomimetic reaction
with Fe(II)
Sarah D'Alessandro, Gloria Alfano, Luisa Di Cerbo, Simone Brogi, Giulia
Chemi, Nicola Relitti, Margherita Brindisi, Stefania Lamponi, Ettore Novellino,
Giuseppe Campiani, Sandra Gemma, Nicoletta Basilico, Donatella Taramelli,
Maria Camilla Baratto, Rebecca Pogni, Stefania Butini
PII:
S0045-2068(19)30397-9
https://doi.org/10.1016/j.bioorg.2019.103020
103020
DOI:
Article Number:
Reference:
YBIOO 103020
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
13 March 2019
15 May 2019
28 May 2019
Please cite this article as: S. D'Alessandro, G. Alfano, L. Di Cerbo, S. Brogi, G. Chemi, N. Relitti, M. Brindisi, S.
Lamponi, E. Novellino, G. Campiani, S. Gemma, N. Basilico, D. Taramelli, M. Camilla Baratto, R. Pogni, S. Butini,
Bridged bicyclic 2,3-dioxabicyclo[3.3.1]nonanes as antiplasmodial agents: synthesis, structure-activity
relationships and studies on their biomimetic reaction with Fe(II), Bioorganic Chemistry (2019), doi: https://doi.org/
1
0.1016/j.bioorg.2019.103020
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Bridged bicyclic 2,3-dioxabicyclo[3.3.1]nonanes as antiplasmodial agents: synthesis,
structure-activity relationships and studies on their biomimetic reaction with Fe(II)
a,
b,
b
b,
b
Sarah D’Alessandro, † Gloria Alfano, † Luisa Di Cerbo, Simone Brogi, Giulia Chemi, Nicola
b
b
b
c
,b
Relitti, Margherita Brindisi, Stefania Lamponi, Ettore Novellino, Giuseppe Campiani,* Sandra
Gemma,* Nicoletta Basilico, Donatella Taramelli, Maria Camilla Baratto, Rebecca Pogni,
,
b
a
d
a
a
a
Stefania Butini
a. Department of Biomedical, Surgical and Dental Sciences, University of Milan, Via Pascal 36,
0133 Milan, Italy
2
b. Department of Biotechnology, Chemistry and Pharmacy (DoE 2018-2022), University of Siena,
via Aldo Moro 2, 53100 Siena, Italy
c. Dipartimento di Farmacia (DoE 2018-2022), University of Naples Federico II, via D. Montesano
4
9, 80131 Naples, Italy
d. Department of Pharmacological and Biomolecular Sciences, University of Milan, Via Pascal 36,
2
0133 Milan, Italy
†
These authors equally contributed to the present work

Current address: Department of Pharmacy, University of Pisa, via Bonanno 6, 56126 Pisa, Italy
Corresponding Author: Department of Biotechnology, Chemistry and Pharmacy, Siena, Italy; E-
*
mail: gemma@unisi.it
Corresponding Author: Department of Biotechnology, Chemistry and Pharmacy, Siena, Italy; E-
*
mail: campiani@unisi.it
Abstract. Despite recent advancements in its control, malaria is still a deadly parasitic disease
killing millions of people each year. Progresses in combating the infection have been made by
using the so-called artemisinin combination therapies (ACTs). Natural and synthetic peroxides are
an important class of antimalarials. Here we describe a new series of peroxides synthesized
through a new elaboration of the scaffold of bicyclic-fused/bridged synthetic endoperoxides
previously developed by us. These peroxides are produced by a straightforward synthetic protocol
and are characterized by submicromolar potency when tested against both chloroquine-sensitive
and chloroquine-resistant Plasmodium falciparum strains. To investigate their mode of action, the
biomimetic reaction of the representative compound 6w with Fe(II) was studied by EPR and the
reaction products were characterized by NMR. Rationalization of the observed structure-activity
relationship studies was performed by molecular docking. Taken together, our data robustly
support the hypothesized mode of activation of peroxides 6a-cc and led to the definition of the
key structural requirements responsible for the antiplasmodial potency. These data will pave the
way in future to the rational design of novel optimized antimalarials suitable for in vivo
investigation.
Keywords: Plasmodium falciparum; synthetic endoperoxides; antimalarials; bioactivation reaction;
C-centered radicals; docking studies
1
Introduction
The decline of malaria burden observed in the last years in malaria-endemic countries is due to a
combination of several factors, including better diagnosis, extended use of artemisinin-based
combination therapy (ACT), improved drug accessibility, wide distribution of insecticide-treated
1
bed nets, and indoor residual spray measures. Despite these recent progresses, malaria is still a
deadly disease killing millions of people each year and resistance or decreased susceptibility to

artemisinins has been observed in some regions.2,3 Due to the importance of dihydroartemisin
DHA, 1, Figure 1) and semisynthetic derivatives, intense research efforts have been dedicated to
(
the development of synthetic peroxides. Artefenomel (2) and arterolane (3) are two
representative synthetic endoperoxides, undergoing clinical investigation and introduced in
4
,5
therapy, respectively. Synthetic peroxides are not only a more economical alternative to
artemisinins, but are also suitable for pharmacokinetic optimization. In this regard, improvement
of pharmacokinetic properties of endoperoxides, and in particular an increase in their half-life
compared to artemisinin has recently emerged as a critical factor that could improve efficacy
against mutant K13 parasites showing decreased susceptibility or resistance to artemisinins.6-8 The
mode of action of artemisinins and related synthetic peroxides is still debated and not completely
clarified. One of the hypothesis regarding their mode of action suggest that organic peroxides,
endowed with specific structural features, react with Fe(II)-heme, present as a by-product of the
hemoglobin metabolism inside the parasite food vacuole. Based on this hypothesis, the activity of
peroxides is related to two critical events, first the interaction of the peroxide bond with Fe(II)-
heme and, second, the reductive cleavage of the peroxide system with the formation of an O-
centered radical. The subsequent evolution of the latter to a C-centered radical results in damage
9
-12
of proteins and membranes of the parasite.
Despite several studies points to Fe(II)-catalyzed
activation of artemisinin and peroxide-containing compounds to form transient toxic species,
there is no consensus about the actual Fe(II)-source required for bioactivation or the actual toxic
1
3-17
species responsible for parasite killing.
Moreover, it has also been suggested that peroxide-
containing compounds are able to overwhelm redox homeostasis in the parasite by inducing rapid
oxidation of flavin cofactors used by flavoenzymes for maintaining reduced glutathione levels.18,19
Since several years, we have been engaged in the total synthesis of dihydroplakortin, a naturally
2
0
occurring endoperoxide with antimalarial activity. Moreover the design and synthesis of novel
synthetic endoperoxides characterized by diverse bicyclic scaffolds has also been carried out.20-24
A
first series of compounds characterized by a fused-bicyclic endoperoxide system typified by 4 was
designed.

Me
H
H N
2
H
O
O
N
O
O
O
O
Me
O
O
O
3
, arterolane
O
H
O
Me
O
N
1
, DHA OH
2, artefenomel
O
Me
O
O
O
H
O
O
FeII
O
4
, Fused bicyclic
system
hydrophobic
interactions with
Fe(II)PPIX
Scaffold
morphing
H
Me
H
Me
Me
RO
Me
4
1
O
O
O
O
OR1
O
O
Me
bridged
O
5
, Bridged
trioxabicyclononane
dioxabicyclononane
Fe(II)
6a-cc, Table 1
Me
H
Me
O
H
Me Me
H
O
Me
Me
Me
Me
Me
Me
Me
Me
Me
O
O
O
9
O
O
Me O
O
0a-c
O
7
Me
O
O
Me
1
R
8
Figure 1. Structure of reference (1-5) and newly designed (6-10) compounds. Bold and dashed
lines indicate relative configuration of each carbon forming the bridge. Bridged bicyclic derivatives
are obtained as racemic mixtures.
Based on the hypothesis that our compounds, analogously to what previously observed for
2
5
plakortin and dihydroplakortin, could react with Fe(II)-heme and being activated to form a C-
centered radical, we performed docking studies on the fused bicyclic structure of 4 and analogues,
which suggested that the peroxide moiety is able to correctly interact with Fe(II)-heme and that
the binding mode is stabilized by the formation of appropriate hydrophobic interactions between
the C-skeleton of the peroxide and the protoporphyrin ring system.24 To improve the activity of
the compounds, we converted the fused-bicyclic core into a bridged-bicyclic scaffold
(
representative compound 5, Figure 1) which is also present in other natural endoperoxides such
2
3
as Yangsozhou A. Through a scaffold morphing approach, we present herein a further
elaboration of the bridged bicyclic scaffold in which the oxygen adjacent to peroxide functionality
was shifted from an endocyclic to an exocyclic position (compounds 6a-cc, Figure 1). This
modification would result in a simplification of the overall synthetic strategy, while maintaining
appropriate hydrophobic contacts of the carbon-skeleton with the protoporphyrin ring and
making possible straightforward exploration of a wide variety of R1 substituents on the exocyclic
oxygen for scaffold decoration and investigation of the structure-activity relationships. We also
performed modification of the bicyclic scaffold through introduction of extra-methyl groups such

as in compounds 7 and 8. The introduction of dimethyl substituents on the scaffold was
accomplished in order to validate the hypothesized binding mode of the compounds to free heme.
Finally, we hybridized scaffolds 4 and 7 leading to the design and synthesis of compounds 9 and
1
0a-c.
2
Results and discussion
2
.1 Chemistry
The title compounds 6a-bb and 7 were synthesized by a straightforward 3-step procedure
highlighted in Scheme 1.
Scheme 1. a. CuI, isopropenylmagnesium bromide, dry THF, -78 °C, 3 h; b. Co(acac) , Et SiH, t-
2
3
BuOOH, O , 1,2-DCE, 25 °C, 5 h; c. from 13a: ROH 14a-bb, p-TsOH, dry DCM, 25 °C, 1 h; d. from
2
1
3b: 14w, p-TsOH, dry DCM, 25 °C, 1 h; d. LiOH, H O/MeCN, 25 °C. Bold and dashed lines indicate
2
relative configuration of each carbon forming the bridge. Bridged bicyclic derivatives are obtained
as racemic mixtures.
Cyclohexenones 11a,b were treated with propenylmagnesium bromide in the presence of CuI to
afford the addition products 12a,b. The Mukaiyama protocol was used to install the
triethylsilylperoxide moiety. Due to the low steric hindrance of the propylene system, the Co-
based catalyst Co(acac) could be used instead of the more expensive Co(thd) catalyst used in
2
2
2
0,26
previous protocols.
Silylperoxides 13a,b were then reacted under acidic conditions with the
suitable alcohol to obtain peroxides 6a-bb (Table 1) and 7 (Figure 1). Due to ring constraints, only
the regioisomer bearing each carbon forming the bridge cis to each other is formed. Accordingly,
cyclized compounds are obtained as racemic mixtures. Compound 6cc was obtained from
compound 6bb by a saponification reaction with LiOH. Non-commercially available alcohols
necessary for the synthesis of final compounds listed in Table 1 have been prepared as described
in the Supporting Information file (Table S1 and Schemes S1-5). Reaction yields of the last
synthetic steps ranged from 10 to 55% yields.
Table 1. Antiplasmodial Activity of Compounds 6a-cc, and of Reference Compounds DHA (1) and
CQ.

Cpd
R
D10
W2
6.0
2.6
R
D10
W2
1.4
IC50 (μM)a
IC50 (μM)a
6
a
b
8.5
5.8
6p
6q
2.4
1.2
6
0.70
6
c
3.4
1.7
1.5
1.1
6r
6s
0.42
12.5
0.34
8.1
6
d
e
6
0.80
0.51
0.49
0.40
6t
0.80
1.31
0.43
6
f
6u
0.622

6
g
0.90
0.52
6v
0.67
0.25
6
h
0.33
0.24
0.27
0.16
6w
6x
0.74
0.89
0.63
0.44
6
i

6
j
0.58
0.44
6y
3.0
1.5
6
k
1.5
2.5
1.6
0.83
1.5
6z
0.98
2.4
0.70
1.6
6
l
6aa
6bb
6
m
0.92
0.78
0.53
6
n
1.32
0.75
0.83
0.46
6cc
2.4
4.5
6
o
DHA, 1
CQ
0.0049
0.0046
0.409
0
.022
aIC50 values are the mean of at least three experiments in duplicate; SD were within 30% of the
mean.
To optimize the reaction yields for the deprotection/cyclization reaction, different conditions were
explored as reported in Table 2. Different acidic catalysts were used: sulfonic acids or Lewis acids
such as trimethylsilyl triflate, boron trifluoride, indium chloride or titanium chlorides. We noted
that Lewis acids generally provided the worst yields, allowing the formation of final compound in
trace amount or with yields lower than 20%, while better or comparable results were obtained by
using sulfonic acids such as pTsOH or CSA.
Table 2. Yield optimization attempts for the reaction of 13 with 14w to afford 6w.

Entry
Acid
Solvent Temperature
Time
Yield
1
pTsOH
DCM
25 °C
1 h
30%
2
3
pTsOH + MS 4 Å
DCM
DCM
25 °C
25 °C
3 h
5 h
45%
31%
CSA
4
5
CSA + MS 4 Å
DCM
DCM
25 °C
24 h
41%
17%
TMSOTf
-78 °C
15 min
6
7
8
9
BF
PPTS
InCl
TiCl
3
(OEt)
2
DCM
DCM
MeCN
DCM
25 °C
25 °C
25 °C
25 °C
40 min
20 h
24 h
2 h
6%
Traces
Traces
5%
3
4
In both cases addition of 4 Å molecular sieves to remove the water formed during the reaction
improved of the reaction yields. The main byproduct was the silylated alcohol. However, even
when increasing the amount of alcohol, no better yields than those reported in Table 2, entries 2
and 4 were obtained.
The synthesis of peroxide 8 is reported in Scheme 2. Starting from cyclohexanone 11a,
methylation of the olefin was achieved through a two-step protocol. Firstly, -iodination of
cyclohexanone 11a to 15 was accomplished using I in the presence of K CO and N,N-
2
2
3
dimethylaminopyridine (DMAP). In the next step, alkenylation of the methylmagnesium bromide
was performed in the presence of Fe(III) catalyst. Normant alkylcuprate addition on 16 was
followed by regioselective -alkylation of the resulting cyclohexanone to afford 17. This latter
intermediate was finally submitted to the hydroperoxysilylation-cyclization protocol to afford the
final compound 8.
Scheme 2. a. I , K CO , DMAP, THF, H O, 0-25 °C, 3 h; b. Fe(acac) , MeMgBr, NMP, THF, 0-25 °C, 1
2
2
3
2
3
h; c. CuI, isopropenylmagnesium bromide, THF, -78 °C, 2.5 h; d. NaH, MeI, THF, reflux, 4 h; e.
Co(acac) , Et SiH, t-BuOOH, O , 1,2-DCE, 25 °C, 5 h; f. 14w, p-TsOH, dry DCM, 25 °C, 1 h.
2
3
2
Scheme 3 describes the synthetic procedure used for the preparation of peroxides 9 and 10a-c.
The key intermediate for the synthesis of 9 is the peroxide 21, that was obtained by alkylation of

-methylketon 20 with 3-bromo-2-methylpropene in the presence of lithium diisopropylamide
(LDA) and subsequent peroxysilylation of 20. Intermediate 24, necessary for the synthesis of 10a-c

was prepared starting from the -ketoester 22 that was alkylated to 23, and then decarboxylated
and hydroperoxyslylated. Compounds 21 and 24 were cyclized to the corresponding final
compounds following the usual protocol.
Scheme 3. a. 3-bromo-2-methylpropene, LDA, THF, -78 °C, 7.5 h; b. Co(acac) , Et SiH, t-BuOOH, O ,
2
3
2
1
1
,2-DCE, 25 °C, 5 h; c. 3-bromo-2-methylbut-2-ene; K CO , THF, 90 °C, 24 h; d. KOH, MeOH, reflux,
h; e. 14a for 10a, 14h for 10c and 14w (for 9 and 10b), p-TsOH, dry DCM, 25 °C, 1 h.
2
3
2
.2 Antiplasmodial evaluation and structure-activity relationships
The in vitro antiplasmodial activity of the compounds was evaluated against a chloroquine-
sensitive (CQ-S) D10 and a chloroquine-resistant (CQ-R) W2 strain of P. falciparum, as previously
2
4,27
reported.
DHA was used as the control drug. The results are reported in Tables 1,3.
Table 3. Antiplasmodial activity of compounds 7-9 and 10a-c
Cpd
R
D10
W2
IC50 (μM)a
7
-
>16
8.9
8
9
-
-
8.2
3.4
>30
>30
1
0a
0b
>30
>30
>30
>30
1
1
0c
>30
>30
DHA, 1
CQ
0.0049 0.0046
0.022 0.409
aIC50 values are the mean of at least three experiments in duplicate; SD were within 30% of the
mean.
The simplest compounds of the series bear small alkyl chains as in 6a-c. More branched R side
1
chains were introduced using alicyclic rings, spaced from the peroxyketal moiety by 1 to 3 carbon
atoms (6d-j). Next, we introduced unsaturated groups with various spacers exploring terminal (6l-
m), trisubstituted (6n-p) or disubstituted (6q-r) olefins. In compound 6s, an activated Michael
acceptor group was also introduced. Moreover, we sampled appended aromatic or heterocyclic
substituents (6t-aa). Finally, an extended side chain bearing an ester or carboxylic acid group was

also explored (6bb,cc). Overall, all peroxides here described presented an increased activity
against the CQ-R over the CQ-S P. falciparum strain in line with the behavior of artemisinins and
related synthetic endoperoxides. The most potent compounds were characterized by the presence
of the adamantane moiety spaced by 1 to 3 carbon atoms (6h-j,r), with IC50s lower than 0.24 μM.
Also the compounds with an aromatic substituent showed interesting activities (6t,v-x,z,bb), with
IC50 higher than those of compounds with the adamantane moiety but still lower than 1 μM. On
the other hand, as outlined in Table 3, peroxides 7-9 and 10a-c showed lower potency with
respect to the original series.
To understand whether the antiplasmodial potency of our synthetic peroxides could be related to
the lipophilicity of the substituent, the lipophilicity for compounds 6a-cc was predicted in silico
using ChemDraw with the “Chemical Properties” function, ACD/ChemSketch through the
“
Calculate logP” function, and QikProp calculation considering the QPlogPo/w parameter (Table S2
of the Supporting Information). Subsequently a correlation analysis between activity and
lipophilicity was performed obtaining R values clearly highlighting that potency of the peroxides
2
was not merely dependent on lipophilicity for both CQ-S D10 and CQ-R W2 P. falciparum strains
(Table S2 and S3 of the Supporting Information).
2
.3 Cytotoxicity evaluation
Cell viability on a selected set of analogues was evaluated in vitro against NIH3T3 after 24 h of
contact, by Neutral Red Uptake (NRU) test. The cell line has been chosen because is a standard
fibroblast cell line. Results of cytotoxicity tests are reported in Table 4. All compounds tested
showed TC values in the high micromolar range well above the antiplasmodial IC value.
5
0
50
Table 4. TC values (M) for a selected set of endoperoxides
5
0
a
Cpd
TC50 (μM)
6
c
70
37.5
>150
140
>140
95
6
6
d
e
6
f
6
h
6
k
6
l
47.5
28
6
m
6
6
6
n
x
y
160
140
140
>140
(
DHA, 1)
aData are expressed as mean of three experiments repeated in six replicates. SD are within 5% of
the mean.
2
.4 Study of the mode of activation of the peroxide moiety by Fe(II)
Several studies demonstrated that artemisinins and endoperoxides such as dioxanes, trioxanes
and tetraoxanes could exert their mode of action by C-radical generation.1
1,12,28-30
To examine if
the generation of a C-radical is also possible for our bridged bicyclic dioxanes, we studied their
reaction with Fe(II) salts using biomimetic reaction conditions.1
1,12,28-30
2
.4.1 Biomimetic reaction: analysis of the reaction products
The Fe(II) reaction of the representative compound 6w was investigated in a biomimetic system
consisting of a mixture of MeCN/H O in the presence of Fe(II) salts. We chose 6w since it is
2
endowed with submicromolar antimalarial activity and presents a tolyl moiety that would make
easier to detect and structurally characterize the Fe(II)-mediated decomposition products. When
peroxide 6w was treated with 5 equivalents of FeCl or FeSO , the formation of the same reaction
2
4

1
product 25 was observed in almost quantitative yields after 2 h (Scheme 4). The H- and COSY
bidimensional- NMR experiments are reported in Figure S1 of the Supporting Information.
Scheme 4. Hypothesized reaction mechanism
2
.4.2 EPR experiments
To detect the reaction species generated during Fe(II)-induced decomposition of 6w, EPR
experiments were performed. Low temperature X-band EPR spectra of the reaction mixture
recorded at different reaction times are reported in Figure 2. The first acquisition time is 2 h after
the reaction starts (Figure 2b) and, in agreement with the above-described reaction time, Fe(III)
species with the signal at g = 4.3 due to the orthorhombic contribution, are formed and survived
after 4 h and 36 h of reaction time (Figure 2c,d). In Figure 2a, the EPR spectrum of the Fe(II) salts in
the MeCN/H O solvent is reported as a control.
2
a)
4
.3
b)
c)
d)
5
40
75
110
145
180
215
250
285
320
355
390
Magnetic Field (mT)
Figure 2. CW X-band EPR spectra of: a) Fe(II) salts in MeCN/H O mixture, b) the reaction after 2 h
2
of incubation, c) the reaction after 4 h of incubation and d) the reaction after 36 h of incubation.
Experimental conditions:  = 9.388GHz, 5mW microwave power, 1 mT for a) and 0.2 mT for b-d)
modulation amplitude, T = 8K

Subsequently, peroxide 6w was combined with FeSO in the presence of the 2-methyl-2-
4
nitrosopropane (MNP) C-radical spin trap in order to detect the radical species formed in concert
with the Fe(III) formation. In Figure 3, the EPR spectra at room temperature of the biomimetic
reaction mixture (6w + Fe(II) + MNP in MeCN/H O) have been reported at different acquisition
2
times. The spectrum shows that a C-radical species is trapped by the MNP with g = 2.0064 
0
.0001 and a nitrogen coupling constant A = 1.5  0.1 mT; the adduct lasts for 2 h. Thus the
N
3
1
radical is a C tertiary radical as shown by the reaction mechanism proposed in Scheme 4.
a)
b)
c)
d)
3
46
348
350
352
354
356
Magnetic Field (mT)
Figure 3. X-band EPR spectra of the reaction in presence of the spin trap MNP at different reaction
times: a) t = 2 h, b) t = 3 h, c) t = 4 h and d) t = 18 h. Experimental conditions:  = 9.86GHz, 5mW
microwave power, 0.1 mT modulation amplitude, T = 298 K.
To exclude the presence of artifacts, MNP was reacted with Fe(II) and peroxide separately in the
absence of 6w and no EPR signal was detected (Figure S2 of the Supporting Information).
Given together, these data suggest that the formation of olefin 25 can be explained assuming the
reductive cleavage of the peroxide bond with the formation of an intermediate radical at O1.
3
2,33
Following a -scission mechanism,
is formed along with a radical at C4.
and a rearrangement of electrons, a terminal double bond
2
.5 Molecular docking
The above results prompted us to hypothesize that the difference in antiplasmodial potency of our
peroxides could be related to a different ability of the compounds to interact with Fe(II)-heme. To
verify this hypothesis, we investigated in depth the interaction of peroxides with Fe(II)-heme by
performing computational studies based on molecular docking and relative ligand binding energies
estimation. In particular, we analyzed the interaction of the peroxide moiety of compounds 6a–cc
with Fe(II)-heme by molecular docking studies employing Glide software, estimated the relative
ligand binding energies by employing Prime software and performed ab initio calculations
employing Jaguar software. The protocol was used as previously reported.24 Full details of our
calculation are shown in Table S4. We observed a nice correlation among the distance of the
peroxide oxygen atoms from the Fe(II) center of heme and the ligand binding energies with the
antiplasmodial potency. The hydrophobic interaction established by the R substituents and the
1
protoporphyrine ring system can be visualized in Figure S3.
Docking of one of the most active compounds (3i, IC50 0.24 µM against D10 and 0.16 µM against
W2) is reported in Figure 4A. For this compound a strong metal coordination bond was found
between oxygen atoms and Fe(II), since the O1-Fe and O2-Fe distances were 2.24 Å and 2.34 Å,
respectively, and a ΔGbind of -42.38 kcal/mol. The adamantyl group is able to stabilize the peroxide-
Fe(II) coordination bond by forming hydrophobic interactions with the protoporphyrin ring (Figure
S3). Compound 6w (Figure 4B) is slightly less potent than 6i (IC 0.74 µM against D10 and 0.63 µM
5
0
against W2), in agreement with the less negative ΔGbind of -40.05 kcal/mol, although its peroxide

bond was still able to coordinate Fe(II) (distances O1-Fe and O2-Fe of 2.39 Å and 2.34 Å,
respectively). The significant drop of antiplasmodial potency found for 6s (Figure 4C, IC 12.5 µM
5
0
against D10 and 8.1 µM against W2) can be explained by considering the increased distances
between O1-Fe and O2-Fe (3.10 Å and 3.00 Å, respectively) paralleled by a ΔGbind value of -24.26
kcal/mol. As can be observed in the potential bioactive conformation of 6s (Figure 4C) the R1
substituent does not correctly interact with the protoporphyrin ring establishing less hydrophobic
contacts with it (Figure S3).
Figure 4. Docked poses of compounds: 6i (orange sticks in panel A), 6w (yellow sticks in panel B)
and 6s (grey sticks in panel C) in complex with heme (green balls and sticks), charged iron was
coloured cyan and represented by the CPK model. The potential ligand–metal coordination bonds
are reported as red dotted lines while the distances are reported in magenta dotted lines. The
picture was generated by Maestro.
3
Conclusions
Here we presented a novel class of endoperoxide antimalarials characterized by a bridged-bicyclic
scaffold. A straightforward 3-steps protocol for the synthesis of these compounds was used that
allowed the development of a significant number of analogues bearing functionalization at R1.
Several compounds showed submicromolar antimalarial potency. Chemical and EPR studies under
biomimetic conditions are consistent with a mode of activation of the peroxide system by Fe(II)
leading to the formation of a C-centered radical intermediate. The observed differences in
antiplasmodial potency were rationalized by in silico docking studies. Accordingly, we were able to
relate the potency of the compounds with the distances of endoperoxide oxygen atoms from the
Fe(II)-heme center and the relative ligand binding energies. The role of the side chain R is the
1
stabilization of the O-O----Fe(II) coordination bond by forming specific interactions with the
protoporphyrin ring. The study of the mode of action of the bridged-bicyclic peroxides and the
comprehension of the key determinant for their appropriate interaction with Fe(II)-heme suggest
that their physicochemical and ADME properties, could be modulated in the future by rational
modifications at the R substituent.
1
4
Experimental section
4
.1 Chemistry
Starting materials and solvents were purchased from commercial suppliers and used without
further purification. Reaction progress was monitored by TLC using silica gel 60 F254 (0.040-0.063
mm) with detection by UV. Silica gel 60 (0.040-0.063 mm) was used for column chromatography.
1
13
Yields refer to purified materials and are not optimized. H NMR and C NMR spectra were
recorded on a Varian 300 MHz or a Bruker 400 MHz spectrometer using the residual signal of the
deuterated solvent as internal standard. Splitting patterns are described as singlet (s), doublet (d),
triplet (t), quartet (q), and broad (br); chemical shifts (δ) are given in ppm and coupling constants
(J) in hertz (Hz). Mass spectra were recorded utilizing electrospray ionization (ESI). All moisture-

sensitive reactions were performed under argon atmosphere using oven-dried glassware and
anhydrous solvents. R* and S* indicate relative configuration at each carbon forming the bridge.
4
.1.1 3-(1-Propen-2-yl)cyclohexanone (12a).
To a -78 °C solution of isopropenylmagnesium bromide (0.5 M in THF, 6.66 mL, 3.33 mmol) in dry
THF (10.0 mL) CuI (634.17 mg, 3.33 mmol) was added and the mixture was stirred for 1.5 h. 2-
Cyclohexenone 11a (200 mg, 2.08 mmol) was added and the resulting solution was stirred at -78
°
C for another 1.5 h. A saturated solution of NH Cl (10.0 mL) was added dropwise and the mixture
4
was allowed to cool to room temperature in 15 min. The blue residue was filtered through
Celite and the solvent was removed in vacuo. Thereafter, the aqueous phase was extracted with
TM
DCM (3x10 mL) and the organic layer was dried over anhydrous Na SO , filtered and concentrated.
2
4
The crude material was purified by flash chromatography (1:100 ethyl acetate/petroleum ether).
1
Compound 12a was obtained as pale volatile yellow oil (50% yield); H NMR (300 MHz, CDCl ) δ
3
4
1
2
.77 (s, 1H), 4.72 (s, 1H), 2.48 – 2.15 (m, 5H), 2.15 – 1.99 (m, 1H), 1.99 – 1.84 (m, 1H), 1.73 (s, 3H),
1
3
.71 – 1.49 (m, 2H); C NMR (75MHz, CDCl ) δ 211.8, 147.7, 110.3, 46.9, 45.9, 41.5, 30.2, 25.4,
3
0.8.
4
.1.2 4,4-Dimethyl-3-(1-propen-2-yl)cyclohexanone (12b).
Starting from 11b, the title compound was prepared following the procedure described for 12a
1
(11% yield); H NMR (300 MHz, CDCl ) 4.93 – 4.86 (m, 1H), 4.67 – 4.61 (m, 1H), 2.59 – 2.34 (m, 2H),
3
1
3
2
2
.32 – 2.13 (m, 3H), 1.82 – 1.50 (m, 5H), 1.05 (s, 3H), 0.99 (s, 3H); C NMR (75 MHz, CDCl ) δ
12.0, 145.3, 113.7, 53.4, 43.4, 40.8, 38.1, 33.4, 29.5, 23.3, 20.6.
3
4
.1.3 3-(2-((Triethylsilyl)peroxy)propan-2-yl)cyclohexanone (13a).
To a stirred solution of 12a (174 mg, 1.26 mmol) and Co(acac) (0.324 g, 1.26 mmol) in dry 1,2-DCE
2
(
5.0 mL), under nitrogen atmosphere, triethylsilane (0.293 gg, 2.52 mmol) was added. Afterwards
the atmosphere was saturated with oxygen and a drop of tert-butylhydroperoxide (5.0 M in
nonane) was added. The resulting green mixture was stirred at 25 °C for 4 h, until consumption of
the starting material. Thereafter the solvent was evaporated, and the resulting residue was
purified by flash chromatography on silica gel (1:15 ethyl acetate/n-hexane) to afford 13a as a
1
colorless oil (90% yield); H NMR (300 MHz, CDCl ) δ 2.49 – 1.80 (m, 6H), 1.66 – 1.22 (m, 3H), 1.14
3
+
(
d, J = 10.9 Hz, 6H), 1.00 – 0.84 (m, 9H), 0.69 – 0.48 (m, 6H); ESI-MS m/z 287 [M + H] , 309 [M +
+
Na] .
4
.1.4 4,4-Dimethyl-3-(2-((triethylsilyl)peroxy)propan-2-yl)cyclohexanone (13b).
Starting from 12b, the title compound was prepared following the procedure described for 13a.
The title compound was not isolated but was immediately used in the next reaction.
4
.1.5 General procedure for the synthesis of 1-alkyl-4,4-dimethyl-2,3-
dioxabicyclo[3.3.1]nonanes (6a-bb).
To a solution of compound 13a (20 mg, 0.07 mmol) and the appropriate alcohol 14a-bb (0.07
mmol) in dry DCM (3.0 mL), p-toluensulfonic acid (13 mg, 0.07 mmol) was added. The mixture was
stirred for 1 to 6 h at 25 °C, then quenched with a saturated solution of NaHCO and the aqueous
3
phase was extracted with DCM (3 x 3.0 mL). The organic extracts were dried over anhydrous
Na SO , filtered and concentrated. The title compounds were isolated as pure compounds by flash
2
4
chromatography on silica gel.
4
1
1
.1.6 (±)-(1R*,5S*)-1-Methoxy-4,4-dimethyl-2,3-dioxabicyclo[3.3.1]nonane (6a).
1
:10 ethyl acetate/ n-hexane; 55% yield; H NMR (300 MHz, CDCl ) 3.38 (s, 3H), 2.29 – 2.18 (m,
3
1
3
H), 2.13 – 1.72 (m, 3H), 1.69 – 1.52 (m, 2H), 1.52 – 1.38 (m, 3H), 1.34 (s, 3H), 1.20 (s, 3H);
C
NMR (75 MHz, CDCl ) δ 104.0, 82.1, 49.3, 39.7, 32.2, 32.0, 25.9 (2C), 22.3, 21.3; ESI-MS m/z 209
3
+
[
M+Na] .

4
1
2
3
2
.1.7 (±)-(1R*,5S*)-1-Isopropoxy-4,4-dimethyl-2,3-dioxabicyclo[3.3.1]nonane (6b).
1
:20 ethyl acetate/petroleum ether; 30% yield; H NMR (300 MHz, CDCl ) δ 4.26 - 4.18 (m, 1H),
3
.24 - 2.18 (m, 1H), 2.07 - 1.90 (m, 3H), 1.80 - 1.76 (m, 1H), 1.63 - 1.36 (m, 4H), 1.32 (s, 3H), 1.21 (s,
1
3
H), 1.20 - 1.18 (m, 6H); C NMR (75 MHz, CDCl ) δ 104.1, 81.9, 64.6, 39.7, 33.8, 33.3, 26.0, 25.9,
3
+
+
4.9, 24.7, 22.4, 21.5; ESI-MS m/z 236 [M+Na] , 253 [M+K] .
4
1
2
.1.8 (±)-(1R*,5S*)-1-Isobutoxy-4,4-dimethyl-2,3-dioxabicyclo[3.3.1]nonane (6c).
1
:20 ethyl acetate/petroleum ether; 30% yield; H NMR (300 MHz, CDCl ) δ 3.46-3.35 (m, 2H),
3
.28-2.22 (m, 1H), 2.07-1.84 (m, 3H), 1.81-1.75 (m, 2H), 1.63-1.57 (m, 2H), 1.52-1.39 (m, 2H), 1.33
1
3
(s, 3H), 1.20 (s, 3H), 0.90 (d, J = 6.9 Hz, 6H); C NMR (75 MHz, CDCl ) δ 103.8, 82.0, 70.0, 68.3,
3
+
+
3
9.7, 33.1, 32.4, 29.0, 26.0, 22.3, 21.4, 19.6, 19.1; ESI-MS m/z 229 [M+H] , 251 [M+Na] , 267
+
[
M+K] .
4
1
2
.1.9 (±)-(1R*,5S*)-1-(Cyclopentylmethoxy)-4,4-dimethyl-2,3-dioxabicyclo[3.3.1]nonane (6d).
1
:100 ethyl acetate/petroleum ether, 11% yield; H NMR (300 MHz, CDCl ) δ 3.47 - 3.37 (m, 2H),
3
.29 – 2.20 (m, 1H), 2.17 - 1.87 (m, 5H), 1.80 - 1.65 (m, 4H), 1.56 - 1.29 (m, 8H), 1.26 (s, 3H), 1.13
1
3
(s, 3H); C NMR (75 MHz, CDCl ) δ 103.5, 81.7, 65.8, 53.4, 39.7, 39.4, 32.9, 32.1, 29.6, 29.4, 25.8,
3
2
5.4, 25.3, 22.1, 21.1.
4
1
2
.1.10 (±)-(1R*,5S*)-1-(Cyclohexylmethoxy)-4,4-dimethyl-2,3-dioxabicyclo[3.3.1]nonane (6e).
1
:50 ethyl acetate/petroleum ether; 11% yield); H NMR (400 MHz, CDCl ) δ 3.39 – 3.30 (m, 2H),
3
.17 (d, 1H, J = 12.4 Hz), 1.99 - 1.85 (m, 3H), 1.73 – 1.30 (m, 12H), 1.26 (s, 3H), 1.23 – 1.04 (m, 7H);
1
3
C NMR (75 MHz, CDCl ) 103.5, 81.7, 67.1, 53.4, 39.5, 38.3, 32.9, 32.1, 30.1, 30.0, 26.7, 25.9, 25.8,
3
+
2
5.7, 22.1, 21.14; ESI-MS m/z 269 [M+H] .
4
1
2
1
.1.11 (±)-(1R*,5S*)-1-(2-Cyclohexylethoxy)-4,4-dimethyl-2,3-dioxabicyclo[3.3.1]nonane (6f).
1
:50 ethyl acetate/petroleum ether; 18% yield); H NMR (400 MHz, CDCl ) δ 3.62 - 3.52 (m, 2H),
3
.19 - 2.16 (m, 2H), 1.99 - 1.86 (m, 3H), 1.64 - 1.52 (m, 7H), 1.39 - 1.35 (m, 8H), 1.25 (s, 3H), 1.20 -
+
+
08 (m, 5H); ESI-MS m/z 283 [M+H] , 305 [M+Na] .
4
.1.12 (±)-(1R*,5S*)-1-(Adamantan-2-ylmethoxy)-4,4-dimethyl-2,3- dioxabicyclo[3.3.1]nonane
(
6g).
1
1
2
1
:90 diethyl ether/n- hexane; 21% yield; H NMR (400 MHz, CDCl ) δ 3.67 - 3.59 (m, 2H), 2.22 -
3
.13 (m, 1H), 2.06 - 1.96 (m, 1H), 1.94 - 1.64 (m, 12H), 1.59 - 1.31 (m, 10H), 1.30 – 1.25 (m, 3H),
+
.16 - 1.12 (m, 3H); ESI-MS m/z 343 [M+Na] .
4
.1.13 (±)-(1R*,5S*)-1-(Adamantan-1-ylmethoxy)-4,4-dimethyl-2,3- dioxabicyclo[3.3.1]nonane
(
6h).
1
1
:100 diethyl ether/n-hexane; 45% yield; H NMR (400 MHz, CDCl ) δ 3.20 - 3.08 (m, 2H), 2.18 (d,
3
2
1
H, J = 20,0 Hz), 2.06 - 1.80 (m, 6H), 1.73 - 1.52 (m, 10H), 1.44 - 1.32 (m, 6H), 1.30 – 1.25 (s, 3H),
.16 - 1.12 (s, 3H); ESI-MS m/z 343 [M+Na] .
+
4
.1.14 (±)-(1R*,5S*)-1-(3-((1R,3S,5R,7R)-Adamantan-2-yl)propoxy)-4,4-dimethyl-2,3-
dioxabicyclo[3.3.1]nonane (6i)
:50 ethyl acetate/petroleum ether; 19% yield; H NMR (300 MHz, CDCl ) δ 3.71 - 3.52 (m, 2H),
1
1
2
3
1
3
.30-2.19 (m, 1H), 2.10 - 1.74 (m, 16H), 1.74 - 1.38 (m, 11H), 1.32 (m, 3H), 1.21 (s, 3H); C NMR
(
2
75 MHz, CDCl ) δ 103.6, 81.8, 77.2, 62.0, 39.4, 39.3, 38.4, 32.8, 31.8, 31.6, 29.7, 28.9, 28.4, 28.3,
8.1, 25.7, 22.1, 21.1; ESI-MS m/z 349 [M+H] 371 [M+Na] .
3
+
+
4
1
.1.15 (±)-(1R*,5S*)-4,4-Dimethyl-1-(2-propyn-1-yloxy)-2,3-dioxabicyclo[3.3.1]nonane (6k).
1
:20 ethyl acetate/petroleum ether; 54% yield; H NMR (300 MHz, CDCl ) δ 4.33 (s, 1H), 2.41
3
(
2
3
s,1H), 2.29-2.25 (m, 1H), 2.16-1.87 (m, 3H), 1.79-1.74 (m, 1H), 1.67-1.48 (m, 3H), 1.43-1.34 (m,
1
3
H), 1.25 (s, 3H), 1.17 (s, 3H); C NMR (75 MHz, CDCl ) δ 104.7, 82.2, 80.9, 73.5, 49.9, 39.5, 32.7,
3
1.9, 25.7, 25.4, 21.9, 21.2.

4
1
5
1
.1.16 (±)-(1R*,5S*)-1-(Allyloxy)-4,4-dimethyl-2,3-dioxabicyclo[3.3.1]nonane (6l).
1
:20 ethyl acetate/petroleum ether; 50% yield; H NMR (300 MHz, CDCl ) δ 6.00 - 5.87 (m, 1H),
3
.27 (d, J = 17.4 Hz, 1H), 5.13 (d, J = 10.2 Hz, 1H), 4.19 - 4.12 (m, 2H), 2.28 - 2.23 (m, 1H), 2.09 -
1
3
.89 (m, 3H), 1.81 - 1.75 (m, 1H), 1.66 - 1.36 (m, 4H), 1.34 (s, 3H), 1.19 (s, 3H); C NMR (75 MHz,
CDCl ) δ 135.6, 116.6, 104.2, 82.2, 63.1, 39.7, 33.1, 32.4, 26.0, 25.9, 22.3, 21.4; ESI-MS m/z: 235
3
+
[
M+Na] .
4
1
5
1
.1.17 (±)-(1R*,5S*)-1-(3-Buten-1-yloxy)-4,4-dimethyl-2,3-dioxabicyclo[3.3.1]nonane (6m).
1
:20 ethyl acetate/petroleum ether; 52% yield; H NMR (300 MHz, CDCl ) δ 5.80-5.69 (m, 1H),
3
.04-4.92 (m, 2H), 3.68-3.53 (m, 2H), 2.29-2.21 (m, 2H), 2.19-2.17 (m, 1H), 2.00-1.81 (m, 3H), 1.72-
+
.57 (m, 1H), 1.54- 1.30 (m, 4H), 1.28 (s, 3H), 1.10 (s, 3H); ESI-MS m/z 249 [M+Na] .
4
.1.18 (±)-(1R*,5S*)-4,4-Dimethyl-1-((3-methylbut-2-en-1-yl)oxy)-2,3-dioxabicyclo[3.3.1]nonane
(
6n).
1
1
4
5
:90 ethyl acetate/petroleum ether; 25% yield; H NMR (300 MHz, CDCl ) δ 5.36 - 5.31 (m, 1H),
3
.17 - 4.15 (m, 2H), 2.33 - 2.25 (m, 1H), 2.07 - 1.88 (m, 3H), 1.72 (s, 3H), 1.67 (s, 3H), 1.64 - 1.37 (m,
H), 1.33 (s, 3H), 1.20 (s, 3H).
4
.1.19 (±)-(1R*,5S*)-1-((Z)-2-(Adamantan-2-ylidene)ethoxy)-4,4-dimethyl-2,3-
dioxabicyclo[3.3.1]nonane (6o).
:20 diethyl ether/n-hexane; 10% yield; H NMR (300 MHz, CDCl ) δ 5.23 (t, 1H, J = 6.6 Hz) 4.25-
1
1
4
1
3
3
.15 (m, 2H), 2.83 (s, 1H), 2.37 (s, 1H), 2.28 (d, 1H, J = 12.6 Hz), 2.17-1.40 (m, 20H), 1.33 (s, 3H),
1
3
.20 (s, 3H); C NMR (75 MHz, CDCl ) δ 151.6, 113.4, 103.8, 81.8, 57.4, 40.3, 39.6, 39.5, 38.8, 37.2,
3
+
3.0, 32.4, 32.2, 31.6, 30.9, 28.4, 25.7, 22.6, 22.1, 21.2; 14.1; ESI-MS m/z: 355 [M+Na] , 370.1
+
[
M+K] .
4
.1.20 (±)-(1R*,5S*)-1-(1-Cyclohexen-1-ylmethoxy)-4,4-dimethyl-2,3- dioxabicyclo[3.3.1]nonane
6p).
(
1
1
1
:20 diethyl ether/n-hexane; 11% yield); H NMR (300 MHz, CDCl ): δ 5.70 (s, 2H), 4.02 (t, 2H, J =
3
2.3 Hz), 2.27 (dq, 1H, J = 2.4 Hz, J = 9.9 Hz), 2.10-1.86 (m, 6H), 1.77 (d, 1H, J = 12.4 Hz), 1.67-1.36
1
2
1
3
(m, 8H), 1.34 (s, 3H), 1.19 (s, 3H); C NMR (75 MHz, CDCl ): 135.2, 124.3, 103.8, 81.8, 66.3, 53.4,
3
+
3
9.4, 32.9, 32.2, 25.9, 25.8, 25.0, 22.5, 22.4, 22.1, 21.2; ESI-MS m/z 289 [M+Na] .
4
1
6
3
.1.21 (±)-(1R*,5S*)-1-(Cinnamyloxy)-4,4-dimethyl-2,3-dioxabicyclo[3.3.1]nonane (6q).
1
:25 ethyl acetate/petroleum ether; 10% yield; H NMR (400 MHz, CDCl ) δ 7.31 - 7.14 (m, 5H),
3
.53 (d, J = 16.0 Hz, 1H), 6.27 - 6.20 (m, 1H), 4.33 - 4.24 (m, 2H), 2.25 - 2.22 (m, 2H), 2.04 - 1.85 (m,
+
H), 1.74 - 1.71 (m, 2H), 1.59 - 1.33 (m, 2H), 1.29 (s, 3H), 1.14 (s, 3H); ESI-MS m/z 311 [M+Na] , 327
+
[
M+K] .
4
.1.22 (±)-(1R*,5S*)-1-(((E)-3-((Adamantan-2-yl)allyl)oxy)-4,4-dimethyl-2,3-
dioxabicyclo[3.3.1]nonane (6r).
:50 diethyl ether/n-hexane; 25% yield; H NMR (300 MHz, CDCl ) δ 5.91 (dd, 1H, J = 6.3 Hz, J =
1
1
1
2
1
2
3
1
2
5.6 Hz), 5.62-5.53 (m, 1H), 4.25-4.12 (m, 2H), 2.40 (d, 1H, J = 5.4 Hz), 2.27 (d, 1H, J = 12.3 Hz),
1
3
.10-1.69 (m, 15H), 1.66-1.36 (m, 7H), 1.33 (s, 3H), 1.20 (s, 3H); C NMR (75 MHz, CDCl ) δ 137.81,
3
26.04, 103.86, 81.83, 63.11, 46.68, 39.46, 38.68(3), 38.05, 32.96, 32.67, 32.29, 32.00 (3), 28.03,
+
+
7.81, 25.72, 22.09, 21.18; ESI-MS m/z 369 [M+Na] , 385 [M+K] .
4
.1.23 (±)-(1R*,5S*)-Ethyl-2-(((4,4-dimethyl-2,3-dioxabicyclo[3.3.1]nonan-1-
yl)oxy)methyl)acrylate (6s).
:30 diethyl ether/n-hexane; 13% yield; H NMR (300 MHz, CDCl ) δ .28 (s, 1H), 5.92 (s, 1H), 4.39
1
1
3
(
t, 2H, J = 15.0 Hz), 4.21 (q, 2H, J = 7.2 Hz), 2.29 (d, 1H, J = 10.2 Hz), 2.10 - 1.92 (m, 3H), 1.80 - 1.64
1
3
(m, 2H), 1.62 - 1.37 (m, 4H),1.34 (s, 3H), 1.29 (t, 3H, J = 7.2 Hz), 1.19 (s, 3H); C NMR (75 MHz,
CDCl ) δ 165.8, 137.9, 125.3, 104.2, 82.0, 60.6, 60.0, 39.5, 32.8, 32.1, 25.7, 25.6, 22.0, 21.2, 14.2;
3
+
+
ESI-MS m/z: 307 [M+Na] , 323 [M+K] .

4
1
4
1
8
.1.24 (±)-(1R*,5S*)-4,4-Dimethyl-1-(benzyloxy)-2,3-dioxabicyclo[3.3.1]nonane (6t).
1
:90 ethyl acetate/petroleum ether; 20% yield; H NMR (300 MHz, CDCl ) δ 7.41 – 7.27 (m, 5H),
3
.72 (d, J = 3.5 Hz, 2H), 2.33 (m, 1H), 2.18 – 1.87 (m, 3H), 1.88 – 1.73 (m, 1H), 1.70 – 1.41 (m, 4H),
1
3
.37 (s, 3H), 1.22 (s, 3H); C NMR (75 MHz, CDCl ) δ 139.1, 128.5 (2C), 127.9 (2C), 127.5, 104.4,
3
+
+
2.3, 64.0, 39.8, 33.3, 32.5, 26.0, 25.9, 22.3, 21.5; ESI-MS m/z: 285 [M+Na] , 301 [M+K] .
4
1
3
1
1
.1.25 (±)-(1R*,5S*)-4,4-Dimethyl-1-(phenethoxy)-2,3-dioxabicyclo[3.3.1]nonane (6u).
1
:90 ethyl acetate/petroleum ether; 26% yield; H NMR (300 MHz, CDCl ) δ 7.35 – 7.09 (m, 5H),
3
.92 – 3.72 (m, 2H), 2.89 (t, J = 7.6 Hz, 2H), 2.32 – 2.16 (m, 1H), 2.14 – 1.84 (m, 3H), 1.77 (m, 1H),
1
3
.68 – 1.53 (m, 1H), 1.52 – 1.36 (m, 3H), 1.34 (s, 3H), 1.19 (s, 3H); C NMR (75 MHz, CDCl ) δ
3
39.1, 129.2 (2C), 128.5 (2C), 126.4, 104.0, 82.1, 62.8, 39.7 (2C), 37.1, 33.0, 32.3, 25.9, 22.3, 21.4;
+
+
ESI-MS m/z: 299 [M+Na] , 315 [M+K] .
4
1
3
1
3
.1.26 (±)-(1R*,5S*)-4,4-Dimethyl-1-(3-(o-tolyl)propoxy)-2,3-dioxabicyclo[3.3.1]nonane (6v).
1
:50 ethyl acetate/petroleum ether; 28% yield; H NMR (300 MHz, CDCl ) δ 7.17 – 7.04 (m, 4H),
3
.76 – 3.58 (m, 2H), 2.66 (m, 2H) 2.28 (s, 3H), 2.12 – 1.72 (m, 5H), 1.69 – 1.38 (m, 6H), 1.33 (s, 3H),
1
3
.20 (s, 3H); C NMR (75 MHz, CDCl ) δ 140.3, 136.0, 130.0, 128.8, 125.8, 103.6, 81.8, 65.8, 39.4,
3
2.8, 32.2, 30.6, 29.6, 25.7, 25.7, 22.1, 19.2.
4
1
7
1
1
.1.27 (±)-(1R*,5S*)-4,4-Dimethyl-1-((2-methylbenzyl)oxy)-2,3-dioxabicyclo[3.3.1]nonane (6w).
1
:25 ethyl acetate/petroleum ether; 22% yield; H NMR (300 MHz, CDCl ) δ 7.37 – 7.33 (m, 1H),
3
.18 – 7.12 (m, 3H), 4.64 (q, 2H, J = 11.7 Hz), 2.35 (s, 3H), 2.17 – 1.90 (m, 3H), 1.85 – 1.74 (m, 1H),
1
3
.69 – 1.39 (m, 5H), 1.37 (s, 3H), 1.21 (s, 3H); C NMR (75 MHz, CDCl ) δ 136.7, 136.5, 130.1,
3
28.6, 127.6, 125.8, 104.2, 82.0, 62.0, 39.5, 32.9, 32.1, 25.8, 25.7, 22.1, 21.2, 18.8.
4
1
1
1
.1.28 (±)-(1R*,5S*)-1-((2-Ethylbenzyl)oxy)-4,4-dimethyl-2,3-dioxabicyclo[3.3.1]nonane (6x).
1
:50 ethyl acetate/petroleum ether; 30% yield); H NMR (300 MHz, CDCl ) δ 7.36 (d, J = 7.2 Hz,
3
H), 7.17 (m, 3H), 4.73 (q, 2H, J = 11.1 Hz), 2.71 (q, 2H, J = 7.5 Hz), 2.34 (d, 1H, J = 12.3 Hz), 2.16-
1
3
.90 (m, 3H), 1.80 (d, 1H, J = 14.4 Hz), 1.69-1.40 (m, 4H), 1.37 (s, 3H), 1.25 – 1.19 (m, 6H); C NMR
(75 MHz, CDCl ) δ 142.4, 135.9, 129.1, 128.4, 127.8, 125.8, 104.1, 81.9, 61.6, 53.4, 39.5, 32.9, 32.1,
3
2
5.7, 25.2, 22.1, 21.2, 15.3.
4
.1.29 (±)-(1R*,5S*)-1-((2-Isopropylbenzyl)oxy)-4,4-dimethyl-2,3- dioxabicyclo[3.3.1]nonane
(
6y).
1
1
7
:20 ethyl acetate/petroleum ether; 24% yield; H NMR (300 MHz, CDCl ) δ 7.35 - 7.28 (m, 3H),
3
.17 - 7.11 (m, 1H), 4.75 (q, 2H, J = 11.1 Hz), 3.25 (q, 1H, J = 7.2 Hz), 2.36 - 2.32 (m, 1H), 2.10 - 1.40
(m, 8H), 1.37 (s, 3H), 1.24 (s, 3H), 1.22-1.16 (m, 6H).
4
.1.30 (±)-(1R*,5S*)-4,4-Dimethyl-1-(thiophen-2-ylmethoxy)-2,3-dioxabicyclo[3.3.1]nonane (6z).
1
:100 ethyl acetate/petroleum ether), to afford the final compound 55 as a colorless oil (19%
1
yield); H NMR (400 MHz, CDCl ) δ 7.18 (d, 1H, J = 5.2 Hz), 6.93 (s, 1H), 6.88 (t, 1H, J = 3.2 Hz) , 4.86
3
-
4
5
4.78 (m, 2H), 2.26 (d, 1H, J = 11.4 Hz), 2.08 - 1.83 (m, 3H), 1.72 (d, 1H, J = 16 Hz), 1.61 - 1.33 (m,
1
3
H), 1.31 (s, 3H), 1.14 (s, 3H); C NMR (75 MHz, CDCl ): δ 141.6, 126.3, 125.8, 125.3, 104.4, 81.7,
3
+
8.7, 39.5, 32.9, 31.6, 25.8, 25.6, 22.0, 21.2; ESI-MS m/z: 291 [M+Na] .
4
1
4
1
1
.1.31 (±)-(1R*,5S*)-1-(Furan-2-ylmethoxy)-4,4-dimethyl-2,3-dioxabicyclo[3.3.1]nonane (6aa).
1
:20 diethyl ether/n-hexane; 15% yield; H NMR (300 MHz, CDCl ) δ 7.39 (s, 1H), 6.32 (s, 2H), 4.71 -
3
.62 (m, 2H), 2.36 – 2.25 (m, 1H), 2.11-1.90 (m, 3H), 1.78 (dd, 1H, J = 2.4 Hz, J = 14.7 Hz), 1.68-
1
2
1
3
.40 (m, 4H), 1.36 (s, 3H), 1.20 (s, 3H); C NMR (75 MHz, CDCl ) δ 152.1, 142.6, 110.3, 108.7,
04.3, 82.1, 56.2, 39.5, 32.8, 32.0, 25.8, 25.5, 22.0, 21.2; ESI-MS m/z 275 [M+Na] , 291 [M+K] .
3
+
+

4
.1.32 (±)-(1R*,5S*)-Methyl-4-(4-((((4,4-dimethyl-2,3-dioxabicyclo[3.3.1]nonan-1-yl)oxy)methyl)
phenoxy)-2-butenoate (6bb).
1
:10 ethyl acetate/n-hexane; 13% yield; 1H NMR (300 MHz, CDCl ) δ 7.29(d,2H,
3
J=8.4Hz),7.07(dt,1H, J = 3.9 Hz, J = 15.9 Hz), 6.85 (d, 2H, J = 8.4 Hz), 6.18 (d, 1H, J = 15.6 Hz), 4.68-
1
2
4
1
1
.64 (m, 4H), 3.75 (s, 3H), 2.31 (d, 1H, J = 12.3 Hz), 2.16 - 1.89 (m, 3H), 1.80 (d, 1H, J = 15.0 Hz),
1
3
.68 - 1.41 (m, 4H), 1.36 (s, 3H), 1.21 (s, 3H); C NMR (75 MHz, CDCl ) δ 166.5, 157.4, 142.8, 131.7,
3
29.3(2), 121.5, 114.5(2), 104.1, 82.0, 66.4, 63.3, 51.6, 39.5, 33.1, 32.2, 25.7, 25.6, 22.1, 21.2.
4
.1.33 (±)-(1R*,5S*)-4-(4-(((4,4-Dimethyl-2,3-dioxabicyclo[3.3.1]nonan-1- yl)oxy)methyl)
phenoxy)-2-butenoic acid (6cc).
Compound 6bb (10 mg, 0.03 mmol) was dissolved in MeCN (1.0 mL) and subsequently a solution
of LiOH (1 mg, 0.03 mmol) in H O (500 μL) was added. The reaction was stirred at 25 °C for 12 h,
2
then MeCN was evaporated and the aqueous residue (pH= 8) was extracted with EtOAc to
eliminate impurities. Consequently, the aqueous phase was acidified by adding HCl 1 N at 0 °C
dropwise and extracted with ethyl acetate (3 x 3 mL). Combined organics were dried over
anhydrous Na SO , filtered and concentrated to give the desired compound 6cc as an amorphous
2
4
1
white solid (50% yield). H NMR (300 MHz, CD OD) δ 7.26 (d, 2H, J = 8.4Hz),7.06 (dt,1H, J = 4.2 Hz,
3
1
J2 = 15.6 Hz), 6.91 (d, 2H, J = 8.4 Hz), 6.11 (d, 1H, J = 15.9 Hz), 4.72 (s, 2H), 4.61 (d, 2H, J = 3.6 Hz),
2
3
1
.33 (d, 1H, J = 12.3 Hz), 2.11 - 1.85 (m, 3H), 1.82 (d, 1H, J = 14.4 Hz), 1.67-1.48 (m, 4H), 1.34 (s,
1
3
H), 1.19 (s, 3H); C NMR (75 MHz, CD OD) δ 168.1, 157.6, 143.2, 131.5, 129.1, 128.9(2), 121.4,
3
14.1(2), 104.1, 81.7, 66.1, 63.0, 39.4, 32.9, 31.5, 25.2, 24.6, 20.9.
4
1
7
1
.1.34 4,4,6,6-Tetramethyl-1-((2-methylbenzyl)oxy)-2,3-dioxabicyclo[3.3.1]nonane (7)
1
:30 ethyl acetate/petroleum ether; 31% yield; H NMR (300 MHz, CDCl ) δ 7.39 – 7.30 (m, 1H),
3
.20 – 7.07 (m, 3H), 4.67 (q, 2H, J = 11.2 Hz), 2.35 (s, 3H), 2.31 – 2.19 (m, 1H), 1.98 - 1.82 (m, 3H),
.80 – 1.64 (m, 1H), 1.56 (s, 3H), 1.42 – 1.21 (m, 2H), 1.15 (s, 3H), 1.13 (s, 3 H), 1.08 (s, 3H);
1
3
C
NMR (75 MHz, CDCl ) δ 136.7, 136.6, 130.1, 128.7, 127.6, 125.8, 105.0, 84.5, 62.0, 51.6, 33.7, 33.5,
3
3
+
0.9, 30.4, 29.3, 28.3, 27.7, 25.9,18.8; ESI-MS m/z 327 [M+Na] .
4
.1.35 2-iodocyclohex-2-enone (15)
To a solution of 11a (1000 mg, 10.40 mmol) in THF/H2O (25 mL:25 mL) cooled at 0 °C, iodine (5.3
g, 20.80 mmol), K CO (1.72 g, 12.5 mmol) and DMAP (254 mg, 2.08 mmol) were added. The
2
3
reaction was stirred at 25 °C for 3 h, then it diluted with EtOAc. The organic layer was extracted,
washed with a saturated solution of Na S O and a 1 N solution HCl. The resulting solution was
2
2 3
dried over anhydrous Na SO , filtered and concentrated. The residue was purified by flash
2
4
chromatography on silica gel (1:5 ethyl acetate/petroleum ether) to afford the desired compound
1
1
5 as a pale-yellow solid (87% yield). H NMR (300 MHz, CDCl ) δ 7.76 (t, J = 4.4 Hz, 1H), 2.72 –
3
2
.59 (m, 2H), 2.51 – 2.35 (m, 2H), 2.16 – 1.95 (m, 2H).
4
.1.36 2-Methylcyclohex-2-enone (16)
To a solution of 15 (2.46 g, 11.08 mmol) in dry THF (25 mL), Fe(acac) (195 mg, 0.55 mmol) was
3
added. Then the mixture was cooled to 0 °C and NMP (9.6 mL, 99.72 mmol) and MeMgBr (3 M in
diethyl ether, 6.3 mL 18.84 mmol) were added dropwise. The color of the solution switched from
orange to yellowish. The reaction was stirred at 25 °C for 1 h, then a 1 M solution of HCl was
added and the mixture was extracted with diethyl ether (3 x 20 mL). The combined organics were
washed with a saturated solution of NaHCO , water and brine, dried over anhydrous Na SO ,
3
2
4
filtered and concentrated. The resulting residue was purified by flash chromatography on silica gel
(
1:10 ethyl acetate/pentane) to afford to give the desired compound 16 as volatile dark yellow oil
1
(78% yield). H NMR (300 MHz, CDCl ) δ 6.74 (s, 1 H), 2.46 – 2.37 (m, 2H), 2.37 – 2.26 (m, 2H), 2.07
3
1
3
–
1.90 (m, 2 H), 1.77 (s, 3H); C NMR (75 MHz, CDCl ) δ 200.0, 145.6, 135.7, 38.3, 26.0, 23.3, 16.0.
3

4
.1.37 2,2-Dimethyl-3-(prop-1-en-2-yl)cyclohexanone (17)
Starting from 16, 2-methyl-3-(propen-2-yl)cyclohexanone was prepared following the procedure
described for 12a. The crude material was purified by flash chromatography on silica gel (1:20
1
diethy ether/petroleum ether). Yield: 39%; H NMR (300 MHz, CDCl ) δ 4.89 (s, 1H), 4.65 (s, 1H),
3
1
3
2
1
6
.70 – 1.89 (m, 5H), 1.88 – 1.60 (m, 6H), 0.97 (d, J = 7.1 Hz, 3H); C NMR (75 MHz, CDCl ) δ 215.1,
44.7, 111.7, 48.0, 47.0, 38.3, 24.7, 24.3, 22.5, 11.7; MS (IE) m/z 95 (100); 137; 123; 109; 95; 81;
7; 53. 2-A solution of Methyl-3-(propen-2-yl)cyclohexanone (90 mg, 0.60 mmol) in dry THF (1 mL)
3
was added to a suspension of NaH (60 % in mineral oil, 24 mg, 0.60 mmol) in dry THF (1 mL). The
mixture was stirred at reflux for 1 h, then cooled down to 25 °C and MeI (170 mg, 1.20 mmol) was
added. The reaction was stirred at 25 °C for 4 h then brine was added, and the mixture was
extracted with diethyl ether (3 x 5 mL). The combined organics were washed with a saturated
solution of NaHCO , water and brine, dried over anhydrous Na SO , filtered and concentrated. The
3
2
4
resulting residue was purified by flash chromatography on silica gel (1:30 ethyl acetate/petroleum
1
ether) to give the desired compound 17 as a colorless volatile oil (10% yield). H NMR (300 MHz,
CDCl ) δ 4.94 – 4.90 (m, 1 H), 4.68 – 4.65 (m, 1H), 2.61 – 2.46 (m, 1H), 2.37 – 2.23 (m, 1H), 2.16
3
(
3
dd, J1 = 11.1 Hz, J2 = 3.6 Hz, 1 H), 2.07 – 1.82 (m, 2H), 1.78 – 1.56 (m, 5H), 1.10 (s, 3H), 1.06 (s,
H); MS (IE) m/z 166; 151; 123; 109; 95; 81; 67 (100).
4
.1.38 2,2-Dimethyl-3-(2-((triethylsilyl)peroxy)propan-2-yl)cyclohexanone (18)
Starting from 17, the title compound was prepared following the procedure described for 13a. The
title compound was isolated as pure compounds by flash chromatography on silica gel (1:50 ethyl
1
acetate/petroleum ether) to give the desired compound 18 as a colorless oil (33% yield). H NMR
(
9
300 MHz, CDCl ) δ 2.47 – 2.34 (m, 2H), 2.08 – 1.85 (m, 3H), 1.77 – 1.62 (m, 2H), 1.33 – 1.26 (m,
H), 1.18 (s, 3H), 0.96 (t, J = 7.8 Hz, 9H), 0.71 – 0.58 (m, 6H); ESI-MS m/z 337 [M+Na] .
3
+
4
.1.39 (±)-(1S*, 5R*)-4,4,9,9-Tetramethyl-1-((2-methylbenzyl)oxy)-2,3-
dioxabicyclo[3.3.1]nonane (8)
Starting from 18, the title compound was prepared following the general procedure described for
compounds 6a-bb, 7. The title compound was isolated as pure compounds by flash
chromatography on silica gel (1:50 ethyl acetate/petroleum ether) to give the desired compound 5
1
as a colorless oil (20% yield). H NMR (300 MHz, CDCl ) δ 7.43 – 7.29 (m, 1H), 7.23 – 7.05 (m, 3H),
3
4
6
1
1
.70 (d, J = 11.8 Hz, 1H), 4.50 (d, J = 11.8 Hz, 1H), 2.30 (s, 3H), 2.19 – 2.06 (m, 1H), 2.04 – 1.58 (m,
1
3
H), 1.45 (s, 3H), 1.40 (s, 3H), 1.35 (s, 3H), 1.17 (s, 3H); C NMR (75 MHz, CDCl ) δ 137.2, 136.2,
3
29.8, 127.6, 127.1, 125.6, 107.0, 83.8, 61.3, 48.7, 39.0, 29.5, 27.2, 26.5, 26.4, 25.5, 21.7, 20.0,
+
8.8; ESI-MS m/z 327 [M+Na] .
4
.1.40 2-Methyl-2-(2-methylallyl)cyclopentanone (20)
A solution of DIPA (1.04 mL, 7.41 mmol) in dry THF (9 mL) was cooled at -78 °C and a 2.5 M
solution of n-BuLi (2.69 mL, 6.74 mmol) in THF was added. The resulting mixture was stirred at the
same temperature for 30 min, followed by the addition of 19 (825 mg, 8.41 mmol). The reaction
was stirred at -78 °C for 3 h, and 3-bromo-2-methylpropene (1.7 mL, 16.82 mmol) was added and
the mixture was allowed to reach 25 °C and stirred for 4 h. Thereafter, a saturated solution of
NH Cl (10 mL) was added and the mixture was extracted with diethyl ether (3 x 10 mL). The
4
combined organics were dried over anhydrous Na SO , filtered and concentrated. The resulting
2
4
residue was purified by flash chromatography on silica gel (1:80 diethyl ether/petroleum ether) to
1
give the desired compound 20 as a colorless oil (43% yield). H NMR (300 MHz, CDCl ) δ 4.83 (s,
3
1
3
H), 4.68 (s, 1H), 2.33 – 2.24 (m, 1H), 2.24 – 2.09 (m, 3H), 2.05 – 1.78 (m, 4H), 1.67 (s, 3H), 0.99 (s,
1
3
H). C NMR (75 MHz, CDCl ) δ 223.3, 142.2, 114.7, 48.3, 44.6, 37.4, 35.3, 24.2, 22.6, 18.6.
3
4
.1.41 2-Methyl-2-(2-methyl-2-((triethylsilyl)peroxy)propyl)cyclopentanone (21)
Starting from 20, the title compound was prepared following the procedure described for 13a. 21
was isolated by flash chromatography on silica gel (1:20 diethyl ether/petroleum ether) as a

1
colorless oil (73% yield). H NMR (300 MHz, CDCl ) δ 2.37 – 2.12 (m, 3H), 2.01 – 1.86 (m, 2H), 1.84
3
–
9
1.77 (m, 1H), 1.73 (d, 1H, J = 15.2 Hz), 1.59 (s, 1H), 1.23 (s, 3H), 1.14 (s, 3H), 1.00 (s, 3H), 0.96 (t,
H, J = 7.9 Hz), 0.65 (q, 6H, J = 7.9 Hz); ESI-MS m/z 323 [M+Na] .
+
4
.1.42 3,3,4a-Trimethyl-7a-((2-methylbenzyl)oxy)hexahydro-3H-cyclopenta[c][1,2]dioxine (9)
Starting from 21, the title compound was prepared following the procedure described for 6a-bb,7.
The title compound purified by flash chromatography on silica gel (1:30 ethyl acetate/petroleum
1
ether) to give the desired compound 9 as a colorless oil (7% yield). H NMR (300 MHz, CDCl ) δ
3
7
4
.41 (m, 2H), 7.15 (m, 2H), 4.78 (d, 1H, J = 12.3 Hz), 4.40 (d, 1H, J = 12.3 Hz), 2.32 (s, 3H), 2.04 (m,
H), 1.60 (m, 4H), 1.16 (s, 6H), 1.07 (s, 3H); ESI-MS m/z 313 [M+Na] .
+
4
.1.43 Ethyl 1-(3-methylbut-2-en-1-yl)-2-oxocyclopentanecarboxylate (23)
To a solution of 22 (500 mg, 3.20 mmol) in dry THF (10 mL), K CO (1.77 g, 12.8 mmol) was added.
2
3
The mixture was stirred at 25 °C for 30 min, and 4-bromo-2-methyl-2-butene (2.59 g, 17.3 mmol)
was added and the reaction was stirred at 90 °C for 24 h. The mixture was filtered through paper
and a saturated solution of NH Cl (20 mL) was added, the mixture was extracted with DCM (3 x 10
4
mL). The combined organics were dried over anhydrous Na SO , filtered and concentrated. The
2
4
resulting residue was purified by flash chromatography on silica gel (1:30 ethyl acetate/petroleum
1
ether) to give the desired compound 23 as a yellow oil (70% yield). H NMR (300 MHz, CDCl ) δ
3
5
.00 (t, 1H, J = 7.5 Hz), 4.15 (q, 2H, J = 7.1 Hz), 2.60 (dd, 1H, J = 14.4 Hz, J = 7.6 Hz), 2.34 (ddt, 4H,
1
2
J = 35.2 Hz, J =26.9 Hz, J = 9.2 Hz), 2.03 – 1.82 (m, 3H), 1.69 (s, 3H), 1.61 (s, 3H), 1.24 (t, 3H, J = 7.1
1
2
3
+
Hz); ESI-MS m/z 247 [M+Na] .
4
.1.44 2-(3-Methyl-3-((triethylsilyl)peroxy)butyl)cyclopentan-1-one (24)
To a solution of 23 (449 mg, 2.00 mmol) in methanol (4 mL) a 4 M solution of KOH (1.72 mL) was
added. The reaction was stirred at 90 °C for 1 h, then it was diluted with water (5 mL) and
extracted with DCM (3 x 10 mL). The combined organics were dried over anhydrous Na SO ,
2
4
filtered and concentrated. The resulting residue was purified by flash chromatography on silica gel
(
1:20 ethyl acetate/petroleum ether) to give 2-(3-methylbut-3-en-1-yl)cyclopentanone as a yellow
1
oil (47% yield). H NMR (300 MHz, CDCl ) δ 5.09 (d, 1H, J = 7.2 Hz), 2.46 – 2.24 (m, 2H), 2.05 (ddd,
3
6
3
H, J = 38.8 Hz, J = 23.3 Hz, J = 15.0 Hz), 1.76 (dd, 1H, J = 18.3 Hz, J = 8.2 Hz), 1.69 (s, 3H), 1.61 (s,
H).
1
2
3
1
2
From this latter compound, following the procedure described to get compound 13a the title
compound was synthetized. 24 was isolated by flash chromatography on silica gel (1:40 ethyl
1
acetate/petroleum ether) as a colorless oil (54% yield). H NMR (300 MHz, CDCl ) δ 2.25 (ddd, 2H,
3
J = 18.1 Hz, J = 16.5 Hz, J = 6.9 Hz ), 2.09 (dd, 1H, J = 17.9, J = 9.2 Hz), 2.05 – 1.92 (m, 2H), 1.87 –
1
2
3
1
2
1
.67 (m, 2H), 1.64 – 1.45 (m, 3H), 1.34 – 1.20 (m, 1H), 1.18 (s, 3H), 1.16 (s, 3H), 0.97 (t, 9H, J = 7.9
+
Hz), 0.65 (q, 6H, J = 7.9 Hz); ESI-MS m/z 324 [M+Na] .
4
.1.45 8a-Methoxy-3,3-dimethyloctahydrocyclopenta[c][1,2]dioxepine (10a)
Starting from 24 and 14a, the title compound was prepared following the procedure described for
6
a-bb,7. 10a was isolated by flash chromatography on silica gel (1:30 ethyl acetate/petroleum
1
ether) as a colorless oil (71% yield). H NMR (300 MHz, CDCl ) δ 3.31 (s, 3H), 2.21 – 2.10 (m, 1H),
3
2
.00 – 1.83 (m, 2H), 1.78 – 1.64 (m, 4H), 1.62 – 1.50 (m, 4H), 1.39 (s, 3H), 1.12 (s, 3H).
4
.1.46 3,3-Dimethyl-8a-((2-methylbenzyl)oxy)octahydrocyclopenta[c][1,2]dioxepine (10b)
Starting from 24 and 14w, the title compound was prepared following the procedure described for
6
a-bb,7. 10b was isolated by flash chromatography on silica gel (1:20 ethyl acetate/petroleum
1
ether) as a colorless oil (7% yield). H NMR (300 MHz, CDCl ) δ 7.44 (m, 1H), 7.16 (m, 3H), 4.72 (d,
3
1
H, J = 11.9 Hz), 4.45 (d, 1H, J = 11.9 Hz), 2.36 (s, 2H), 2.28 (m, 2H), 1.96 (m, 4H), 1.64 (m, 6H), 1.25
1
3
(s, 3H), 1.12 (s, 3H); C NMR (75 MHz, CDCl ) δ 137.0, 136.3, 129.9, 128.2, 125.7, 117.8, 82.4, 62.2,
3
+
4
9.1, 39.2, 31.9, 31.6, 29.7, 27.1, 26.5, 24.4, 22.7, 18.9; ESI-MS m/z 313 [M+Na] .

4
.1.47 8a-((Adamantan-1-yl)methoxy)-3,3-dimethyloctahydrocyclopenta[c][1,2]dioxepine (10c)
Starting from 24 and 14h, the title compound was prepared following the procedure described for
6
a-bb,7. 10c was isolated by flash chromatography on silica gel (1:100 ethyl acetate/petroleum
1
ether) as a colorless oil (18% yield). H NMR (300 MHz, CDCl ) δ 3.19 (d, J = 9.2 Hz, 1H), 2.91 (d, J =
3
9
–
.2 Hz, 1H), 2.15 (d, J = 12.0 Hz, 2H), 1.90 – 1.75 (m, 2H), 1.66 (dt, 10H, J = 11.4 Hz, J =6.6 Hz), 1.60
1.53 (m, 9H), 1.53 – 1.38 (m, 3H), 1.36 (s, 3H), 1.08 (s, 3H).
1
2
4
.1.48 Biomimetic reaction: 2-methylbenzyl hex-5-enoate (25)
To a solution of 6w (10 mg, 0.036 mmol) in a mixture of MeCN/water (4:1, 2 mL) a suspension of
FeCl ·4H O (36 mg, 0.18 mmol) in a mixture of MeCN/water (4:1, 2 mL) was added. The reaction
2
2
was stirred into dark for 2 h. The mixture was extracted with Ethyl acetate (3 x 5 mL) and the
combined organic were dried over anhydrous Na SO , filtered and concentrated. The resulting
2
4
residue was purified by flash chromatography on silica gel (1:50 ethyl acetate/petroleum ether) to
1
give the desired compound 25 as a white solid (76% yield). H NMR (300 MHz, CDCl ) δ 7.39 – 7.10
3
(
m, 4H), 5.87 – 5.63 (m, 1H), 5.11 (s, 2H), 5.07 – 4.90 (m, 2H), 2.45 – 2.25 (m, 5H), 2.18 – 1.96 (m,
1
3
2
1
1
H), 1.83 – 1.63 (m, 2H); C NMR (75 Hz, CDCl ): δ 173.4, 137.6, 136.9, 133.9, 130.3, 129.2, 128.5,
3
26.0,
+
15.4, 64.5, 33.5, 33.0, 24.0, 18.8; ESI-MS m/z 241 [M+Na] .
4
.2 EPR experiments
CW X-band ( = 9GHz) EPR measurements were carried out with an E580 Elexsys Series
spectrometer (Bruker Biospin GmbH, Rheinstetten, Germany), Bruker ER 049X microwave bridge
with a 4122SHQE/0208 cavity. For low temperature spectra an Oxford helium continuous flow
cryostat (ESR 900, Oxford Instruments) was used. 3x4 mm I.D.xO.D. suprasil tubes were used for
the EPR experiments.
Sample preparation: biomimetic reaction of 6w with FeSO were run as described in 4.1.40. For
4
spin trap experiments, to a solution of 6w (10 mg, 0.036 mmol) and MNP (0.36 mmol) in a mixture
of MeCN/water (4:1, 2 mL), a suspension of FeCl ·4H O (36 mg, 0.18 mmol) in a mixture of
2
2
MeCN/water (4:1, 2 mL) was added. The reaction was stirred into dark and aliquots were taken up
at regular interval times (2, 3, 4 and 18 h) for EPR measurements.
4
.3 In vitro antiplasmodial evaluation
3
4
P. falciparum parasited were cultured according to the method of Trager and Jensen, with minor
3
5
modifications. CQ sensitive (D10) or CQ resistant (W2) strains were maintained at 5% hematocrit
of human type A-positive red blood cells in RPMI 1640 medium (EuroClone; Celbio) containing 1%
AlbuMAX II (lipid-rich bovine serum albumin), 24 mM sodium bicarbonate, 0.01% hypoxanthine,
2
0 mM HEPES, and 2 mM glutamine. All the cultures were maintained at 37°C in a standard gas
mixture consisting of 1% O2, 5% CO2, and 94% N2.
Compounds were dissolved in dimethyl sulfoxide (DMSO) and then diluted with medium to
achieve the required concentrations (final DMSO concentration <1%, nontoxic to the parasites).
Serial dilutions were made in a final volume of 100 μL/well in 96-well plates. Asexual parasite
stages derived from asynchronous cultures with parasitemia of 1 to 1.5% were distributed into the
plates (100 μl/well; final hematocrit, 1%) and incubated for 72 h at 37°C. Chloroquine (CQ) and
dihydroartemisinin (DHA) were used as a reference control drugs. Parasite growth was
determined by measuring the activity of the parasite lactate dehydrogenase (pLDH), according to a
modified version of the spectrophotometric Makler's method.36 Briefly, the drug-treated culture
was resuspended, and 20 μl/well was transferred to a plate containing 100 μl of Malstat reagent
(
0.11% [vol/vol] Triton-100, 115.7 mM lithium L-lactate, 30.27 mM Tris, 0.62 mM 3-acetylpyridine
adenine dinucleotide [APAD] [Sigma-Aldrich], adjusted to pH 9 with 1 M HCl) and 25 μl of PES/NBT
1.96 mM nitroblue tetrazolium chloride-0.24 mM phenazine ethosulfate). After few minutes
incubation in the dark, absorbance was read (wavelength 650 nm) using a microplate reader,
(

Synergy4 (BioTek). The antiplasmodial activity is expressed as the 50% inhibitory concentration
IC ). Each IC value presented in Table 1-3 is the mean and standard deviation of three
(
5
0
50
independent experiments performed in duplicate.
4
.4 Cytotoxicity
The in vitro acute toxicity of test compounds was evaluated using direct contact tests following the
3
7
procedure proposed by ISO 10995-5. As the in vitro acute toxicity does not depend on the
product’s intended use, mouse fibroblasts NIH3T3 were used to test samples.
NIH3T3 were propagated in DMEM supplemented with 10% fetal calf serum, 1% L-glutamine-
penicillin-streptomycin solution and 1% non-essential amino acid solution and incubated at 37 °C
in a humidified atmosphere containing 5% CO . Once at confluence, cells were washed twice with
2
0
.1 mol/l phosphate buffer saline, detached with trypsin-ethylenediaminetetraacetic acid solution
and centrifuged at 1500 r.p.m. (rounds per minute) for 5 min. The pellet was then suspended in
complete fresh medium (dilution 1:15) in order to have 1.5 × 10 cells/mL.
4
1
mL of cell suspension was then seeded in each well of a 24-well plate and incubated at 37 °C in
an atmosphere of 5% CO . When cells reached 50% of confluence, the test compounds were
2
added to each well. The tested concentrations of each samples ranged from 7 to 160 µM. All
samples were set up in six replicates. Complete medium was used as a negative control. Cells were
incubated in the test compounds for 24 h and then cell viability was evaluated through Neutral
Red Uptake test (NRU).
4
.4.1 Evaluation of NIH3T3 viability: NRU test
The following solutions were prepared in order to determine the percentage of viable cells after
2
1
2
3
4 h of contact with the test samples:
. Neutral Red (NR) stock solution: 0.33 g NR dye powder in 100 ml sterile H2O
. NR medium: 1.0 ml NR stock solution+99.0 routine culture medium pre-warmed to 37°C
. NR desorb solution: 1% glacial acetic acid solution + 50% ethanol + 49% H O
2
At the end of incubation, the routine culture medium was removed from each plate and the cells
were carefully rinsed with 1 ml pre-warmed PBS 0.1 M. Plates were then gently blotted with paper
towels. 1.0 ml NR medium was added to each dish and further incubated at 37°C, 95% humidity,
5
.0% CO for 3 h. The cells were checked during incubation for NR crystal formation. After
2
incubation, the NR medium was removed and the cells were carefully rinsed with 1 ml pre-
warmed D-PBS 0.1M. PBS was decanted and blotted from the dishes and exactly 1 ml NR desorb
solution was added to each sample. Plates were placed on a shaker for 20-45 minutes to extract
NR from the cells and form a homogeneous solution. During this step the samples were covered to
protect them from light. Five minutes after removal from the shaker, absorbance was read at 540
nm with a UV/visible spectrophotometer (Lambda 25, Perkin Elmer).
4
.5 Computational details
4
.5.1 Fe(II)-heme and ligands preparation
The heme was firstly extracted from the Protein Data Bank (PDB) structure 1CTJ and prepared
removing all the parts of the pdb crystal structure except for the porphyrin ring, and the Fe ion
charged as +2 and prepared, as specified in the next paragraph, for acquiring appropriate starting
complex for the subsequent computational analyses.
Compounds 6a-cc were designed in Maestro Schrödinger environment and subsequently treated
3
8
by means of LigPrep application, generating the most plausible ionization state at cellular pH
value (7.4 ± 0.2). For retrieving the lower energy starting structures to use as input in molecular
docking experiments, the prepared structures were analyzed by means of MacroModel.39 The
calculation was performed using OPLS-2005 as force field. The Generalized-Born/Surface-Area
(
GB/SA) model for simulating the solvent effects was used. No cutoff for non-bonded interactions
was used. PRCG method was employed with 1000 maximum iterations and 0.001 gradient

convergence threshold for performing the molecular energy minimizations. MCMM (Monte Carlo
Multiple Minimum) was employed as torsional sampling method for the conformational searches,
performing automatic setup with 21 kJ/mol (5.02 Kcal/mol) in the energy window for saving
structure and 0.5 Å was used as cutoff distance for redundant conformers.22
4
.5.2 Ab initio QM Calculations
2
4
The endoperoxides and the heme structure were treated using a protocol already described. The
4
0
optimization was performed by Jaguar, using B3LYP hybrid density functional method in
combination with the LACVP* basis set, which is composed of 6-31G* description for all light
atoms and of LANL2DZ for metal center such as iron. LACVP basis set has been successfully used in
calculation on metalloporphyrins and derivatives as well as for transition metal complexes. The
calculations were performed taking into account the solvation effects. Solvation was included by
solving the Poisson-Boltzmann equations with a realistic molecular surface (van der Waals radius
plus solvent radius about each atom) using the Jaguar solvation model (PBF).41 We used a
dielectric constant of 80.37 and a probe radius of 1.4 Å for water. The properties established were
ESP charges. These results are considered for docking calculation performed by Glide (see next
paragraph).
4
.5.3 Molecular docking
4
2
Molecular Docking was carried out by Glide software using the ligands and the Fe(II)-heme
prepared as above-mentioned, applying Glide extra precision (SP) method. The charges calculated
and derived from the QM calculation were considered for docking calculations by applying the
OPLS_2005 force field. The grid was generated considering the iron atom as the center of it and
restricting it to the length of the ligands used in the present study in order to establish its size. The
compounds were docked with default parameters, with the constraint of a metal charged
acceptor. The number of poses entered to post-docking minimization was set to 10. Glide SP score
was then evaluated. A single best conformation of each ligand was considered for the further
evaluation of the binding energy.
4
.5.4 Prime/MM-GBSA simulation
4
3
The Prime/MM-GBSA method implemented in Prime software consists in computing the change
between the free and the complex state of both the ligand and the heme after energy
minimization. The technique was applied using the docking complexes obtained in the docking
protocol. The software was used to calculate the free-binding energy (ΔGbind) of each ligand, as
4
4-47
recently reported by us.
5
Acknowledgments
Financial contribution from MIUR is kindly acknowledged (Project PRIN 20154JRJPP)
6
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Graphical abstract

Highlights





Bridged bicyclic endoperoxides were designed and synthesized as antiplasmodial agents
The new endoperoxides were prepared using a straightforward synthetic protocol
Activation of peroxides was investigated through a biomimetic reaction with Fe(II)
C-centered radical species formed upon reaction with Fe(II) were detected by EPR
Docking studies to Fe(II)-heme were performed to rationalize observed SAR