Synthesis and profiling of benzylmorpholine 1,2,4,5-tetraoxane analogue N205: Towards tetraoxane scaffolds with potential for single dose cure of malaria

Article abstract of DOI:10.1016/j.bmc.2018.05.006

A series of aryl carboxamide and benzylamino dispiro 1,2,4,5-tetraoxane analogues have been designed and synthesized in a short synthetic sequence from readily available starting materials. From this series of endoperoxides, molecules with in vitro IC50s versus Plasmodium falciparum (3D7) as low as 0.84 nM were identified. Based on an assessment of blood stability and in vitro microsomal stability, N205 (10a) was selected for rodent pharmacokinetic and in vivo antimalarial efficacy studies in the mouse Plasmodium berghei and Plasmodium falciparum Pf3D70087/N9 severe combined immunodeficiency (SCID) mouse models. The results indicate that the 4-benzylamino derivatives have excellent profiles with a representative of this series, N205, an excellent starting point for further lead optimization studies.

Full text of DOI:10.1016/j.bmc.2018.05.006

Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and profiling of benzylmorpholine 1,2,4,5-tetraoxane analogue
N205: Towards tetraoxane scaffolds with potential for single dose cure
of malaria
a
a
a
a,f
Paul M. O’ Neill a, , Paul A. Stocks , Sunil Sabbani , Natalie L. Roberts , Richard K. Amewu
,
⇑
Emma R. Shore a, Ghaith Aljayyoussi b, Iñigo Angulo-Barturén c, María Belén c, Jiménez-Díaz c,
Santiago Ferrer Bazaga c, María Santos Martínez c, Brice Campo d, Raman Sharma b, Susan A. Charman e,
Eileen Ryan e, Gong Chen e, David M. Shackleford e, Jill Davies b, Gemma L. Nixon a, Giancarlo A. Biagini b,
Stephen A. Ward b
a Department of Chemistry, University of Liverpool, Liverpool L69 7ZD, United Kingdom
b Research Centre for Drugs and Diagnostics, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom
c Tres Cantos Medicines Development Campus, DDW, GlaxoSmithKline, Severo Ochoa 2, 28760 Tres Cantos, Spain
d Medicines for Malaria Venture, ICC, Route de Pré-Bois 20, P.O. Box 1826, 1215 Geneva, Switzerland
e Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of aryl carboxamide and benzylamino dispiro 1,2,4,5-tetraoxane analogues have been designed
and synthesized in a short synthetic sequence from readily available starting materials. From this series
of endoperoxides, molecules with in vitro IC50s versus Plasmodium falciparum (3D7) as low as 0.84 nM
were identified. Based on an assessment of blood stability and in vitro microsomal stability, N205
(10a) was selected for rodent pharmacokinetic and in vivo antimalarial efficacy studies in the mouse
Plasmodium berghei and Plasmodium falciparum Pf3D70087/N9 severe combined immunodeficiency
(SCID) mouse models. The results indicate that the 4-benzylamino derivatives have excellent profiles
with a representative of this series, N205, an excellent starting point for further lead optimization studies.
Ó 2018 Published by Elsevier Ltd.
Received 30 January 2018
Revised 3 May 2018
Accepted 4 May 2018
Available online xxxx
1. Introduction
tive with improved pharmacokinetics and enhanced in vivo anti-
7–9
malarial activity.
1,2,4,5-Tetraoxanes are another class of
The emergence of malaria parasite resistance to most available
peroxide with excellent antimalarial profiles against both chloro-
quine-resistant and chloroquine-sensitive strains of Plasmodium
falciparum and oral activity in murine models of the disease.10–15
Previously in our group, RKA182 (3) (Fig. 1) was selected as a can-
didate for full preclinical development from a series of synthetic
tetraoxane derivatives; this compound shows superior in vitro
and in vivo activity compared to artemether and artesunate, has
good oral bioavailability in rodent models and is more stable than
arterolane in malaria infected human red blood cells.16,17 Although
the PK profile for RKA182 is compatible with that of a 3-day dosing
regimen, there is now a drive for the development of endoperox-
ides with PK/PD properties predicted to allow single dose cure in
humans. Three distinct tetraoxane templates were simultaneously
investigated; a representative described here, alongside the series
which led to the discovery of E209 (4) 17,18, both of which have
PK/PD characteristics that are compatible with a single-dose cure
(See Scheme 1).
1
drugs, including the semi-synthetic artemisinin derivatives arte-
2–4
mether and artesunate,
has led to efforts to create new syn-
thetic peroxides as potential antimalarial agents. Leading
examples of synthetic endoperoxides include OZ277 (arterolane)
5
(1),
a molecule deployed in combination with piperaquine
6
(known as Synriam), and OZ439 (2) a second generation deriva-
Abbreviations: AUC, area under the curve; BA, bioavailability; CLplasma, plasma
clearance; CLint, intrinsic clearance; CYP, cytochrome P450; DCM, dichloromethane;
DMF, dimethyl formamide; ED, effective dose; FaSSIF, fasted state simulated
intestinal fluid; FeSSIF, fed state simulated intestinal fluid; IC, inhibitory concen-
tration; IV, intravenous; LAH, lithium aluminium hydride; PD, pharmacodynamic;
PK, pharmacokinetic; PO, oral; SCID, severe combined immunodeficiency; THF,
tetrahydrofuran; Vdss, volume of distribution at steady state.
⇑
Corresponding author.
E-mail address: p.m.oneill01@liv.ac.uk (P.M. O’ Neill).
Present address: Department of Chemistry, University of Ghana, P. O. Box LG56,
f
Legon, Ghana, Accra.
https://doi.org/10.1016/j.bmc.2018.05.006
0968-0896/Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: O’ Neill P.M., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.05.006

2
P.M. O’ Neill et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Fig. 1. Structures of synthetic peroxides OZ277, OZ439, RKA182 and E209.
Scheme 1. (a) Tf2O, NEt3, ꢀ25 °C- rt, 12 h, (b) Pd(OAc)2, CO, atm, DIPEA, dppp, DMF/MeOH, 70 °C, 5hr, rt, o/n, (c) oxalyl chloride, AlCl3, DCM, 0 °C- rt o/n, 0 °C, MeOH, pyridine,
rt, 3 h, (d) Formic acid/HCl, 50% H2O2, acetonitrile, 0 °C, 30–60 min (e) 2-adamantanone, Re2O7, DCM, rt, 1 h (f) MeOH, KOH, 70 °C (g) NEt3, 0 °C, CH3COCl, 1 h, NHR1R2, 0 °C, 30
min then rt, 1.5 h (h) THF, LAH, 0 °C, 30 min (i) NEt3, THF, 0 °C, methane sulfonyl chloride, 1.5 h (j) NEt3, DCM, 0 °C, 10 min, then amine, rt 3.5 h.
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P.M. O’ Neill et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
3
2. Results and discussion
Herein we describe the design and synthesis of a new series of
tetraoxanes (Templates 1 and 2, Fig. 2) and present data on their
in vitro and in vivo antimalarial activity profiles. The new series
were designed to increase the lipophilicity (CLogP/LogD) compared
with RKA182 (by inclusion of an aromatic ring in the side-chain)
and enhance blood stability (rodent and human) in addition to
enhancing PK/PD properties in appropriate animal models. For
the benzyl series, we focused on the use of morpholine and fluo-
ropiperidine to enable direct comparisons with OZ439 and E209.
2.1. Chemistry
Two routes were explored for the synthesis of key ester 4c. Ini-
tially, we examined the conversion of commercially available 4-(4-
hydroxyphenyl)cyclohexanone 4a into the corresponding triflate
4b. This was then subjected to a palladium mediated carbonylation
reaction in the presence of methanol to provide methyl ester 4c.
This key intemediate could be converted into the tetraoxane ester
6 according to the procedure developed by Dussault et al.19 An
alternative approach to 4c involves the use of a modified Friedel-
Crafts procedure on 4-phenyl cyclohexanone; this latter route
has advantages in terms of scale up of chemistry for production
of multi-gram quantities. For the synthesis of template 1 analogues
the ester 6 was first hydrolysed to the carboxylic acid 6b and con-
verted to target amides via a mixed anhydride intermediate. For
template 2 analogues, the ester 6 was reduced with LAH to the
alcohol 8 and treated with methane sulfonyl chloride to form the
mesylate 9 which was then allowed to react with morpholine (or
4-fluoro cyclohexanone for N214, 10b).
Fig. 2. Plasma concentrations in male Sprague Dawley rats following IV and PO
administration of 1.6 and 8.1 mg/kg, respectively. Data represent the mean of n = 2
rats except for the point marked with * where only one measurement was available.
the plasma at the 24 h timepoint (>100 ng/ml). These results are
superior data recorded for the tetraoxane version of OZ439 tested
previously in the same animal model where the mean survival was
15 days with no cures 21. Based on these results, N205 was selected
for more extensive profiling to determine if comparative studies
should be performed in the humanized mouse model of malaria.
OZ439 was used as the benchmark compound throughout these
studies.
Table 3 shows data for the measured solubility of the mesylate
salt of N205. Compound 10a is more soluble in water than OZ439
mesylate but is less soluble in 0.01 and 0.1 M HCl, FaSSIF and FeS-
SIF media. Overall, the profile points towards lower overall solubil-
ity than OZ439 (it should be noted that the final salt form and
levels of crystallinity will influence solubility data in these assays).
N205 (10a) exhibited degradation in human, rat and mouse
liver microsomes with rates generally being fastest in rat and slow-
est in mouse (Table 4). The rates of degradation were similar in
human and rat liver microsomes at substrate concentrations of 1
and 5 mM, however the rate of degradation in mouse liver micro-
somes appeared to be somewhat lower at the higher substrate con-
centration suggesting a possible concentration dependency.
Metabolite identification (see Supplementary Material) for
N205 revealed that the major route of metabolism for N205 is
hydroxylation of the adamantane ring accounting for almost 50%
of the turnover observed in human liver microsomes (based on
peak area only). Minor metabolites observed included products
stemming from tetraoxane ring cleavage (M-182) and morpholine
ring cleavage (M-26). Thus the metabolic weak spot in these struc-
tures is the lipophilic admantane ring system and improvement in
the DMPK profile may be possible through chemical substitution
within the adamantylidene portion of the molecule.
2.2. Biological assessment
All compounds synthesized were tested in vitro against the 3D7
strain of Plasmodium falciparum.20 With the exception of the amino
cyclobutane 7d and thiomorpholine analogue 7e, all of the tetraox-
ane analogues displayed potent single digit nanomolar activity
with several compounds more potent than OZ439 positive control.
No correlation was seen with calculated physicochemical parame-
ters such as CLogP, LogD or calculated solubility (Table 1) consis-
tent with previous reports for trioxolane derivatives. 5,8. Due to
the importance of blood stability for enhancing overall drug expo-
sure in this class, compounds depicted in Table 1 were screened for
stability in human and rat blood and it was shown that the amide
series was unstable particularly in rat blood (half-life < 4 h, data
not shown). Amides 7 h, and 7i (all of which have higher ClogP val-
ues than RKA182) were selected for in vivo analysis along with the
benzylamino analogues 10a and 10b (the latter compounds
demonstrated comparatively better rodent and human blood
stability).
Data presented in Table 2 summarise the results in terms of
cure and mean survival time following a single 30 mg/kg oral dose
treatment of Plasmodium berghei (P. berghei) infected mice. The
performance of the amides, although superior to artesunate and
equivalent to RKA182 and OZ277, was comparatively poor relative
to OZ439. In contrast, the benzylamino analogues performed better
than the amides with a 26 day mean survival for N205 (10a) and
13 day average survival for fluoropiperdine analogue 10b. For the
benzyl morpholine analogue N205, 2/3 mice were cured; use of
the mesylate salt of N205 in a standard suspension vehicle (SSV)
was next examined to see if a better performance could be
obtained with the salt form. A similar result was obtained with a
66% single dose cure rate and average survival of 25 days. Snapshot
PK data for this latter study revealed significant levels of N205 in
As noted previously, an important feature of OZ439 is its
7
enhanced blood stability thought to be due at least in part to
reduced degradation in the presence of Fe(II). Table 5 shows data
on the stability in human and rat blood, predicted plasma clear-
ance and rat pharmacokinetic data for N205 and OZ439. N205
has similar human blood stability compared to OZ439 but is less
stable in rat blood with a measured half-life of approximately 8
h. The high plasma protein binding for both N205 and OZ439 in
human plasma may have some impact on the observed blood sta-
bility and further studies are in progress to explore this. Predicted
human clearance values for OZ439 and N205 are similar with
higher clearance predicted in rats for the tetraoxane analogue.22
In head to head comparisons of tetraoxanes with 1,2,4-trioxolanes
in our laboratory, it is generally observed that rodent microsomal
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P.M. O’ Neill et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Table 1
In vitro 3D7 IC50, CLogD, calculated solubility, CLog P.
Compound
Structure
IC50 3D7 (nM)
7.1 0.4
CLogD*
3.96
Calculated* Solubility (mg/mL)
0.02
ClogP*
7.4
7a
3.22 0.81
7b
7c
7d
5.3 0.6
1.31
1.36
1.11
0.029
0.083
0.013
4.66 0.80
3.85 0.80
2.59 0.80
0.84 0.03
33.0 1.0
7e
15.0 1.0
4.58
0.01
4.10 0.90
7f
6.4 0.6
3.93
1.70
0.10
3.68 0.84
3.61 0.84
7g
0.82 0.11
0.034
7h
7i
3.0 0.4
2.76
1.11
0.13
3.18 0.86
3.37 0.79
4.2 0.45
0.027
7j
1.10 0.1
1.07
0.69
3.39 0.90
10a (N205)
10b (N214)
OZ439
1.3 0.1
1.8 0.7
8.0 0.3
4.42
5.02
4.83
0.076
0.025
0.55
4.50 0.82
5.36 0.86
4.63 0.70
*Log D, log P and Solubility values were calculated using the Virtual Computational Chemistry Laboratory (VCCLAB); http://www.vcclab.org.
Table 2
Table 3
Percentage activity and mean mouse survival time following 30 mg/kg single oral
Overall solubility results for N205 (10a) mesylate at 37 °C after 4 h. All solubility
values refer to the free base equivalent.
dose in the P. berghei model.
Compound Number
% Activity
Mean survival time (days) following
30 mg/kg oral dose
Medium
Solubility (mg/mL) after 4 h at 37 °C
OZ439 mesylate
N205 mesylate
7h
7i
99.0
99.0
10.0 (9, 10, 11)
8.0 (8, 8, 8)
Water
6800
>8000
34
240
Not meas.
120
>9000
>9000
17
172
<0.1
18
10a (N205)
10a (N205 mesylate)
10b (N214 mesylate)
OZ277
RKA182
OZ439
99.42
99.30
99.98
99.98
99.98
99.40
99.09
–
26.3 (16, 30, 30)
25.0 (15, 30, 30)
13 (12, 13, 14)
10.2 (8, 10, 8, 10, 15)
11.4 (14, 15, 7, 7, 14)
30 (30, 30, 30)
6.8 (6, 7, 7, 7, 7)
4.0 (4, 4, 4)
pH 2.0 buffer
0.1 N HCl pH 1.0
0.01 N HCl pH 2.0
pH 7.4 PBS
FaSSIF pH 6.5
FeSSIF pH 5.0
>1500
760
Artesunate
Untreated Control
Both plasma and microsomal protein binding of N205 and OZ439 were found to be
very high (>99.9% bound for both compounds in each matrix).
clearance is faster for the adamantylidene tetraoxane scaffold. The
higher predicted rat hepatic clearance and lower rat blood stability
translates into a comparatively worse performance for N205 in
terms of rat pharmacokinetics (Table 5 and Fig. 2). N205 exhibited
higher clearance and lower oral bioavailability of 52% compared
with approximately 100% for OZ439. Both IV and PO half-lives of
OZ439 were superior to N205, although the 17 h oral half-life of
N205 demonstrates a large improvement over all other endoperox-
ides examined in this class (e.g. PO half-life of RKA182 at same
dose was 3.5 h).16
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P.M. O’ Neill et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
5
Table 4
In vitro Metabolism in Liver Microsomes.
Compound Details
Species Substrate
Concentration (
Degradation half-
life (min)
In vitro CLint (mL/min/
mg protein)
Microsome-
Predicted EH
l
M)
Human
Rat
1
34 (33, 35)
43 (44, 41)
15 (13, 16)
20 (17, 23)
48 (42, 54)
87 (93, 82)
51 (53, 50)
41 (39, 42)
118 (130, 105)
89 (101, 76)
37 (41, 32)
20 (19, 21)
0.67 (0.68, 0.66)
0.62 (0.61, 0.63)
0.75 (0.77, 0.73)
0.69 (0.72, 0.66)
0.44 (0.47, 0.41)
0.30 (0.29, 0.31)
5
1
5
1
5
Mouse
N205 (CDCO_01)
Human
Rat
1
5
1
25 (26, 23)
66 (73, 59)
95 (86, 103)
141 (75, 207)
126
71 (66, 75)
27 (24, 29)
18 (20, 17)
16 (23, 8)
14
0.74 (0.72, 0.75)
0.51 (0.49, 0.54)
0.32 (0.34, 0.30)
0.27 (0.37, 0.18)
0.23
5
Mouse
1a
5
197
9
0.16
OZ439 (OZ-439/PC-276/02)
Data represent the mean values of two technical replicates (individual values in parenthesis), except for OZ439 in mouse liver microsomes where only one value is available.
aData reported previously.21
Table 5
AUCED90 < 0.75
l
gꢁhꢁml-1ꢁfollowing single oral dose administra-
Human/rat blood stability, predicted plasma clearance and rat pharmacokinetic
parameters of N205 versus OZ439. PK parameters for OZ439 are from7.
tion. In contrast to studies in P. berghei, the dose levels adminis-
tered were not able to cure mice even at the top dose of 100 mg/
Property
N205
Mesylate
OZ439 mesylate
kg. The in vivo data confirms that N205 has outstanding antimalar-
7,18
ial activity within the same region as OZ439 and E209.
In this
In vitro blood stability (37 °C, 4 h)
T1/2 (h) in rat blood
T1/2 (h) in human blood
Pred CLplasma (mL/min/mg)*
Human
model, an ED90 of 10 mg/kg was obtained for artesunate after four
daily doses indicating that a single dose of N205 has similar oral
potency to multiple doses of artesunate.
ꢂ8
>15
ꢂ10% loss
No degradation detected
14
50
53
15
21
28
Rat
Mouse
3. Experimental methods
Rat PK
CLplasma (mL/min/kg)
Vdss (L/kg)
3.1. Biological assessment
77
11
40
18
Estimated IV T1/2
(h)BA (%)
6.352
32
ꢂ100
Please see Supplementary Information for solubility, plasma
protein binding and blood stability experimental methods.
3.1.1. In vitro sensitivity assays
Drug susceptibilities were assessed at the Liverpool School of
Tropical Medicine by the measurement of fluorescence after the
Additional profiling in human liver microsomes to determine
CYP450 inhibition (Table 6) revealed no concerns (IC50 > 20 mM
for each isoform).
addition of SYBR Green I as previously described by Smilkstein
20
et al.
Drug IC50s were calculated from the log of the dose/
In order to provide an assessment of the therapeutic efficacy of
N205 against P. falciparum Pf3D70087/N9, potency was assessed by
administering a single oral dose (2.5, 5, 15, 30, 50 and 100
mgꢁkgꢀ1) at day 3 after infection and measuring the effect on blood
parasitemia by flow cytometry (Fig. 3A–C and Table 7).23,24 The
parameters of efficacy estimated in the study were a) the dose of
N205 that reduces parasitemia at day 7 after infection by 90% with
respect to vehicle-treated mice (parameter denoted as ED90) and b)
the estimated average daily exposure in whole blood necessary to
reduce P. falciparum parasitemia in peripheral blood at day 7 after
infection by 90% with respect to vehicle-treated mice (parameter
used to measure the potency of the compound and denoted as
AUCED90). In the experimental conditions used in the assay N205
is efficacious against P. falciparum, with ED90 = 8.6 mgꢁkg-1 and
response relationship as fitted with Grafit software (Erithacus Soft-
ware, Kent, United Kingdom). Results are given as the mean of at
least three separate experiments.
For the fluorescence assay, after 48 h of growth, 100
ll of SYBR
Green I in lysis buffer (0.2 l of SYBR Green I/ml of lysis buffer) was
l
added to each well, and the contents were mixed until no visible
erythrocyte sediment remained. After 1 h of incubation in the dark
at room temperature, fluorescence was measured with a Varioskan
fluorescence multiwell plate reader from Thermo Electron Corpo-
ration with excitation and emission wavelengths of 485 and 530
nm, respectively.
3.1.2. In vitro physicochemical and ADME studies and in vivo animal
experiments
3.1.2.1. In vitro ADME and in vivo PK. The in vitro ADME studies and
in vivo PK studies were conducted at the Centre for Drug Candidate
Optimisation, Monash University (Australia). Methods for solubil-
ity, metabolite identification, plasma and microsomal protein
binding, and plasma analysis are described in the Supplementary
Information. Microsomal stability and CYP inhibition were con-
ducted as described previously 25 using liver microsomes from Sek-
isui XenoTech (Kansas City, KS). Rat PK and rat blood stability
Table 6
IC50 values against five drug-metabolising CYP isoforms using a substrate specific
approach in human liver microsomes.
CYP isoform
IC50 (
lM)
N205
Reference Inhibitor
CYP1A2
CYP2C9
CYP2C19
CYP2D6
>20
>20
>20
>20
>20
>20
3.6 (Furafylline)
0.72(Sulfaphenazole)
0.48 (Ticlopidine)
0.025 (Quinidine)
0.022 (Ketoconazole)
0.013 (Ketoconazole
7
studies were performed as described previously in accordance
with the Australian Code of Practice for the Care and Use of Ani-
mals for Scientific Purposes, and the study protocols were
approved by the Monash Institute of Pharmaceutical Sciences Ani-
CYP3A4(Midazolam 10-hydroxylation)
CYP3A4(Testosterone 6b-hydroxylation)
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P.M. O’ Neill et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Fig. 3. (A) Parasitemia in peripheral blood of mice infected with P. falciparum Pf3D70087/N9. Data shown correspond to individual parasitemia values for mice treated with
N205 or vehicle (n = 2). (B) Dose-response relationship for N205; data are presented as log10 [percentage of parasitemia at day 7 after infection] of individual mice versus the
dose in mgꢁkgꢀ1. Parasitemias lower than the limit of detection of flow cytometry (0.01%) are computed and plotted as 0.01% for the dose-response curve fitting. (C) Upper
panels show peripheral blood smears stained with Giemsa and lower panels show flow cytometry dot plots from samples of peripheral blood stained with TER-119-
Phycoerythrine and SYTO-16. Dots inside the polygonal region represent P. falciparum-infected human erythrocytes.
mal Ethics Committee. The intravenous formulation for N205 was
prepared in 5% w/v glucose solution, whilst the oral formulation
Table 7
PK Parameters for N205 in the humanized Pf SCID mouse model at five different
was prepared as a solution in aqueous vehicle containing 0.5% w/
v hydroxypropylmethyl cellulose, 0.5% v/v benzyl alcohol and
0.4% v/v Tween 80.
doses.
Blood PK Parameters in humanized mouse
Dose (mg/kg)
Cmax (mg/mL)
5
15
0.1480
0.0099
30
0.1480
0.0049
50
0.6070
0.0121
100
0.2500
0.0025
0.1090
0.0218
C
max/Dose (mg/mL per
3.1.2.2. In vivo antimalarial screening (Plasmodium berghei). In vivo
efficacy studies in P. berghei-infected mice were conducted at the
Swiss Tropical and Public Health Institute (Basel, Switzerland),
adhering to local and national regulations of laboratory animal
welfare in Switzerland (permission No. 1731). The tetraoxanes
and OZ439 were dissolved or suspended in a vehicle consisting
of 0.5% w/v hydroxypropylmethyl cellulose, 0.5% v/v benzyl alco-
hol, 0.4% v/v Tween 80, and 0.9% w/v sodium chloride in water,
and administered orally on day 1 post infection. Antimalarial activ-
ity was measured as a percent reduction in parasitemia on day 3
mg/kg)
tmax (h)
AUC(0-t) (mg.h/mL)
DNAUCd(0-t) (mgꢁh/mL per
mg/kg)
1
1
1
2
0.5
1.2801a 0.7489b 0.9399b 2.9973b 2.3211c
0.2560
0.0499
0.0313
0.0599
0.0232
Efficacy Parameters in humanized mouse
ED90 mgꢁkgꢀ1
8.6/6.2*
<0.75/0.68*
-/-*
AUCED90 mgꢁhꢁmlꢀ1
AUCPCC mgꢁhꢁmlꢀ1
t = 4 h; bt = 8 h; ct = 23 h; dDNAUC, dose normalized AUC0-t *data for OZ439.
a
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P.M. O’ Neill et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
7
post infection. Animals were considered cured if there were no
detectable parasites on day 30 post infection. The onset of action
was determined after a single oral dose of compounds to mice
(n = 3) on day 3 post infection, resulting in a high initial para-
sitemia to allow the onset of action to be assessed. The reduction
in parasitemia was initially monitored at 12 h post treatment,
and the time of recrudescence was assessed by daily blood smears
for 14 d followed by intermittent assessment up to 30 days. All
groups, including an untreated control group, were infected simul-
taneously with P. berghei. Parasitemia was determined on day 3
post infection, and compared with values in control animals.
using Merck 938S Kieselgel 60 Silica gel. IR spectra were run using
a Perkin-Elmer 298 infrared spectrophotometer. Solid samples
were dissolved in CHCl3 and liquids/oils applied neat on to sodium
chloride discs.
1H NMR spectra were recorded using a Bruker 400 MHz NMR
spectrophotometer. Spectra were referenced to the residual sol-
vent peak and chemical shifts expressed in ppm from TMS as the
internal reference peak. All NMR experiments were performed at
room temperature. The following annotations are used to describe
multiplicity; s, singlet, bs, broad-singlet, d, doublet, t, triplet, q,
quartet, m, multiplet and coupling constants are expressed in
Hertz.
3.1.2.3. In vivo efficacy (Pf SCID mice). Humanised mouse efficacy
and pharmacokinetic studies in Pf SCID mice were conducted at
GSK Tres Cantos, Madrid. Studies of murine P. falciparum infection
were ethically reviewed and carried out in accordance with Euro-
pean Directive 2010/63/EU and the GSK Policy on the Care, Welfare
and Treatment of Laboratory Animals. In vivo efficacy against P. fal-
ciparum was conducted 23 in age-matched female immunodeficient
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (8–10 weeks of age; 22–24
gm) supplied by Charles River, UK, under license of The Jackson
Laboratory, Bar Harbor. Mice were engrafted with human erythro-
cytes (Red Cross Transfusion Blood Bank in Madrid, Spain) by daily
intraperitoneal injection with 1 mL of a 50% hematocrit erythro-
cyte suspension (RPMI 1640 (Invitrogen), 25 mM HEPES (Sigma),
25% decomplemented AB+ human serum (Sigma) and 3.1 mM
hypoxanthine (Sigma)). Mice with ꢂ40% circulating human ery-
throcytes were intravenously infected with 2 ꢃ 107P. falciparum
Pf3D70087/N9-infected erythrocytes (day 0). Efficacy was assessed
by administering one oral dose of N205 (2.5, 5, 15, 30, 50 and
100 mg.kg-1) at day 3 after infection. Treatment group assignments
were allocated randomly. Parasitemia was measured by flow
cytometry in samples of peripheral blood stained with the fluores-
cent nucleic acid dye SYTO-16 (Molecular Probes) and anti-murine
erythrocyte TER119 monoclonal antibody (Becton Dickinson) in
Mass spectra were recorded between 20 and 70 eV using a
VG7070E and/or Micromass LCT mass spectrometers.
3.3.1. Preparation of 4-(4-oxocyclohexyl)phenyl
trifluoromethanesulfonate 4b26
To a stirred solution of 4a (10 g, 52 mmol)) in dry DCM (75 mL)
at ꢀ78 °C was added triethylamine (10 mL). To this mixture was
added triflic anhydride (10.6 mL (density = 1.67 g/ml, 63 mmol))
drop-wise over 30 min. After this time the solution was allowed
to warm to room temperature and stirred overnight. The reaction
mixture was washed with water (30 mL), dried over MgSO4 and
concentrated. Purification by flash column chromatography using
ethyl acetate/hexane (20/80) afforded the pure triflate 4b in 91%
yield as off-white foam. 1H NMR (400 MHz, CDCl3) dH, 1.81–1.99
(m, 2H, CH2), 2.18–2.28 (m, 2H, CH2), 2.48–2.58 (m, 4H, CH2),
3.02–3.15 (m, 1H, CH), 7.22 (d, 2H, J = 7.2 Hz, Ar), 7.32 (d, 2H, J =
7.2 Hz, Ar). 13CNMR (100 MHz, CDCl3), dC 34.2, 41.5, 42.6, 117.6,
121.9, 128.9, 145.7, 148.5, 210.6 MS (ES+), [M + Na] (1 0 0), 345.0
HRMS calculated 345.0384 for C13H13O4Na found 345.0392.
27
3.3.2. Preparation of methyl 4-(4-oxocyclohexyl)benzoate 4c
To a solution of the triflate 4b (2.41 g, 0.007 mol)) in DMF (25
mL) and MeOH (12 mL) was added di-isopropylethylamine (2.7
mL) followed by Pd(OAc)2 (84 mg, 0.376 mmol) and dppp (155
mg, 0.376 mmol). A stream of carbon monoxide (CO) gas was bub-
bled into the solution for 5 min then a balloon filled with CO was
added to the top of the reflux condenser. After allowing to stir
for 16 h at 80–90 °C, the reaction mixture was allowed to cool to
room temperature, diluted with ethyl acetate (250 mL), washed
with saturated bicarbonate solution (50 mL), water (50 mL), brine
(50 mL) and dried over sodium sulphate. Purification by flash chro-
serial 2 ll blood samples taken every 24 h until assay completion.
The ED90 was estimated by fitting a four parameter logistic equa-
tion using GraphPad 6.0 Software
3.2. Systemic exposure in infected Pf SCID mice
The levels of N205 were evaluated in whole blood in order to
determine standard pharmacokinetic parameters in the individual
animals used in the efficacy study. Peripheral blood samples (25
mL) were taken at different times (0.25, 0.5, 1, 2, 4, 6, 8 and 23
h) after drug administration, mixed with 25 ml of Milli-Q water
and immediately frozen on dry ice. The frozen samples were stored
at -80 °C until analysis. Vehicle-treated mice experienced the same
blood-sampling regimen. Blood samples were processed by liquid–
liquid extraction. Quantitative analysis by Liquid chromatography-
tandem mass spectrometry (LC-MS/MS) was performed using a
Waters UPLC system and Sciex API4000 mass spectrometer. The
lower limit of quantification in this assay was 5 ng/ml. Blood con-
centration vs time was analyzed by non-compartmental analysis
(NCA) using Phoenix ver.6.3 (from Pharsight), from which expo-
sure-related values (Cmax and AUC0-23, AUC0-t) and tmax were
estimated.
27
matography (EtOAc/Hex, 20/80) gave 4c in 72% mp = 94 °C lit
mp = 93–94 °C 1HNMR (400 MHz, CDCl3) dH, 1.81–2.04 (m, 2H,
CH2), 2.18–2.28(m, 2H, CH2), 2.48–2.58 (m, 4H, CH2), 3.02–3.15
(m, 1H, CH), 3.90 (s, 3H, CH3), 7.34 (d, 2H, J = 7.2 Hz, Ar), 8.04 (d,
2H, J = 7.2 z, Ar). 13CNMR (100 MHz, CDCl3), dC 34.06, 41.62, 43.2,
52.51, 127.37, 128.97, 130.38, 150.48, 167.24, 211.11, MS (ES+),
[M + Na] (1 0 0), 255.1 HRMS calculated 255.0997 for C14H16O3Na
found 255.0396.
3.3.3. Alternative Preparation of methyl 4-(4-oxocyclohexyl)benzoate
4c
A solution of oxalyl chloride 4.51 mL (53.3 mmol) in DCM (50
mL) was added to a suspension of 4-phenylcyclohexanone (7g,
40 mmol) and AlCl3 (16.07 g, 120 mmol) in DCM (150 mL) at 0 °C.
The reaction mixture was stirred at 0 °C for 1 h then at room tem-
perature for 2 h. A mixture of methanol (10 mL) and pyridine (8.1
mL) was added drop wise to the reaction mixture and left to stand
overnight. The reaction mixture was then washed with water, 3 N
HCl, NaHCO3, dried over NaSO4, filtered and concentrated. Purifica-
tion by flash column chromatography gave 4c in 65% yields with
identical spectroscopic and physical properties to that described
above.
3.3. Chemistry
With exception of those stated all reagents were obtained from
commercial suppliers. Dichloromethane, triethylamine and THF
were freshly distilled before use. Analytical thin layer chromatog-
raphy was performed on pre-coated silica gel (0.25 mm layer of sil-
ica gel F254) aluminium sheets. UV light (254 nm) was used for all
visualizations and flash column chromatography was performed
Please cite this article in press as: O’ Neill P.M., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.05.006

8
P.M. O’ Neill et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
3.3.4. Preparation of methyl 4-[(10’r,30’r,50’R,70’R)-dispiro[cyclohexane-
1,30-[1,2,4,5]tetroxane-60,20’-tricyclo[3.3.1.13,7]decan]-4-yl]benzoate
6a
492.2372.% C, H, N calculated; C = 69.06, H = 7.51, N = 2.98; found
C = 69.40, H = 7.85, N = 3.21
To a solution of the ketone 4c (4g, 17 mmol) in acetonitrile (75
mL) at 0 °C was added formic acid (8 mL) and 50% H2O2 (16 mL).
The resulting reaction mixture was allowed to stir for 30 min at
0 °C, then allowed to warm to room temperature and diluted with
water (30 mL). The resulting mixture was extracted in DCM (3 x 50
mL), dried over MgSO4 and concentrated to give the crude gem-
bishydroperoxide 5 which was used without further purification.
The gem-bishydroperoxide 5 was dissolved in CH2Cl2 (50 mL) and
added to a stirring solution of the required adamantanone (1.5
equiv.) and rhenium (VII) oxide (0.02 eqv) in CH2Cl2 (50 mL) at
room temperature. The reaction mixture was stirred for 1 h, fil-
tered through a plug of silica and concentrated. Purification by
flash column chromatography gave 6a in 48% as a white foam. 1H
NMR (400 MHz, CDCl3-d6) dH 7.89 (d, 2H, J = 8.3 Hz, Ar), 7.22 (d,
2H, J = 8.3 Hz, Ar), 3.83 (s, 3H, CH3), 2.60 (tt, 1H, J = 11.5 Hz, 3.9
Hz, CH), 2.06–1.44 (m, 23H, CH2/CH) 13C NMR (100 MHz, CDCl3-
d6) dc 167.4, 151.6, 130.2, 128.7, 127.3, 111.0, 107.7, 52.5, 44.1,
3.3.8. Preparation of 4-[(10’r,30’r,50’R,70’R)-dispiro[cyclohexane-1,30-
[1,2,4,5]tetroxane-60,20’-tricyclo[3.3.1.13,7]decan]-4-yl]-N-(piperidin-
4-ylmethyl)benzamide 7b
Off white powder (Yield 77%) Mpt = 132–134 °C 1H NMR (400
MHz, CDCl3-d6) dH 8.41 (t, 1H, J = 5.9 Hz, NH), 7.77 (d, 2H, J = 8.3
Hz, Ar), 7.32 (d, 2H, J = 8.3 Hz, Ar), 3.11 (t, 2H, J – 5.9 Hz, NCH2),
2.98 (t, 4H, J = 10.6 Hz, CH2N), 2.83 (t, 2H, J = 6.3 Hz, CH2), 2.78–
2.70 (m, 1H, CH), 2.55–2.45 (m, 1H, CH), 1.95–0.94 (m, 26H, CH/
CH2) 13C NMR (100 MHz, CDCl3-d6) dc 177.5, 149.2, 133.0, 127.7,
126.9, 110.1, 107.6, 51.5, 46.5, 45.3, 42.5, 40.5, 38.8, 36.6, 32.9,
+
29.9, 27.2, 26.8 MS (ES+), [M + H] (1 0 0) 497.3 HRMS calculated
for 497.3015 C29H41N2O5, found 497.3017;% C, H, N calculated; C
= 70.13, H = 8.12, N = 5.64; found C = 70.05, H = 7.95, N = 5.42
3.3.9. Preparation of 4-[(10’r,30’r,50’R,70’R)-dispiro[cyclohexane-1,30-
[1,2,4,5]tetroxane-60,20’-tricyclo[3.3.1.13,7]decan]-4-yl]-N-[2-
(methylamino)ethyl]benzamide 7c
+
37.4, 34.7, 33.6, 29.9, 27.5. MS (ES+), [M + Na] (1 0 0) 437.2
White foam (Yield, 64%) Mpt 60–62 °C 1H NMR (400 MHz,
CDCl3-d6) dH 8.81 (t, 1H, J = 5.5 Hz, NH), 7.88 (d, 2H, J = 8.2 Hz,
Ar), 7.40 bs, 1H, NH), 7.34 (d, 2H, J = 8.2 Hz, Ar), 3.60 (q, 2H, J =
5.9 Hz, NCH2), 3.07 (t, 2H, J = 5.9 Hz, CH2N), 2.79–2.72 (m, 1H,
3.3.5. Preparation of 4-[(10’r,30’r,50’R,70’R)-dispiro[cyclohexane-1,30-
[1,2,4,5]tetroxane-60,20’-tricyclo[3.3.1.13,7]decan]-4-yl]benzoic acid 6b
A solution of 6a (3.86 mmol) in 10% w/v potassium hydroxide/
methanol (12.6 mL) was stirred at reflux for 90 min. The solution
was allowed to cool to room temperature and concentrated under
reduced pressure. The resulting residue was taken up in water (15
mL) and washed with diethyl ether (3 ꢃ 12 mL). The aqueous layer
was acidified with concentrated hydrochloric acid and a white pre-
cipitate formed. Diethyl ether (18 mL) was added to dissolve the
precipitate and the aqueous phase extracted with diethyl ether
(2 ꢃ 12 mL). The combined organic phases were washed with brine
(10 mL), dried over Na2SO4, filtered and concentrated under
reduced pressure to give a white solid. Recrystallization from etha-
nol gave the carboxylid acid 6b as a white solid in 91% yield. 1H
NMR (400 MHz, CDCl3-d6) dH 8.04 (d, 2H, J = 8.4 Hz, Ar), 7.34 (d,
2H, J = 8.4 Hz, Ar), 2.75–2.66 (m, 1H, CH), 2.12–1.45 (m, 22H, CH/
CH2) 171.3, 155.3, 132.8, 131.0, 129.1, 127.3, 126.8, 114.4, 44.5,
CH), 2.56 (s, 3H, NCH3), 1.99–1.57 (m, 22H, CH/CH2) MS (ES+),
+
[M + H]
(1 0 0) 457.3 HRMS calculated for 457.2702
C26H37N2O5, found 457.2701.% C, H, N Calculated; C = 68.40, H =
7.95, N = 6.14; found C = 68.10, H = 7.62, N = 5.92
3.3.10. Preparation of N-(3-aminocyclobutyl)-4-[(10’r,30’r,50’R,70’R)-
dispiro[cyclohexane-1,30-[1,2,4,5]tetroxane-60,20’-tricyclo[3.3.1.13,7
decan]-4-yl]benzamide 7d
]
White foam (Yield 82%) Mpt = 116–118 °C 1H NMR (400 MHz,
CDCl3-d6) dH 8.74 (s, 1H, NH), 7.88 (bs, 2H, NH2), 7.56 (d 2H, J =
8.3 Hz, Ar), 7.34 (d, 2H, J = 8.3 Hz, Ar), 3.55–3.50 (m,1H, CH),
3.45–3.24 (m, 5H, CH/CH2), 3.20–3.18 (m, 1H, CH), 1.99–1.52 (m,
CH/CH2) 13C NMR (100 MHz, CDCl3-d6) dc 169.5, 149.4, 128.2,
127.0,l 110.1, 107.5, 42.5, 40.7, 36.5, 32.9, 31.5, 26.8 MS (ES+),
+
[M + H]
(1 0 0) 455.3 HRMS calculated for 455.2546
-
C26H35N2O5, found 455.2532.% C, H, N Calculated; C = 69.21, H =
7.74, N = 5.98; found C = 69.01, H = 7.38, N = 5.63
36.7, 33.8, 32.9, 27.9, 25.8 MS (ES+), [M - H] (1 0 0) 399.2 HRMS
calculated for 399.1808 C23H27O6, found 399.1808.
3.3.11. Preparation of {4-[(10’r,30’r,50’R,70’R)-dispiro[cyclohexane-1,30-
[1,2,4,5]tetroxane-60,20’-tricyclo[3.3.1.13,7]decan]-4-yl]phenyl}
(thiomorpholin-4-yl)methanone 7e
3.3.6. General procedure for the amide formation (7a-j)
To a solution of the acid 6b (2.33 mmol) in dry DCM (30 mL)
was added triethylamine (0.7 mmol, 1.5 eq) and ethylchlorofor-
mate (2.33 mmol, 1.0 eq). The reaction was allowed to stir for 60
min at 0 °C. (2.33 mmol, 1.0 eq) of the required amine was added,
and after stirring for 30 min, the reaction mixture was allowed to
warm to room temperature and and then allowed to stir for a fur-
ther 90 min. The reaction mixture was then diluted with water and
extracted with DCM (3 x 30 mL). The combined organic extracts
were washed with brine, dried over anhydrous Na2SO4 and con-
centrated. Purification by flash column chromatography afforded
the required amide.
White solid (Yield 76%) 1HNMR (400 MHz, CDCl3) dH, 7.40 (d,
2H, J = 8 Hz, Ar), 7.27 (d, 2H, J = 8 Hz, Ar), 3.4–3.9 (m, 4H, CH2),
3.14–3.40 (m, 2H, CH), 2.58–2.7 (m, 1H, CH), 1.60–2.10 (m, 20H,
CH2/CH), 13CNMR (100 MHz, CDCl3), dC 170.8, 169.5, 148.3, 133.6,
127.8, 127.4, 110.9, 107.7, 43.9, 42.9, 39.6, 37.3, 34.6, 33.6, 32.2,
30.5, 27.8, 27.5 MS (ES+), [M + Na] 508.2 (1 0 0), HRMS calculated
for 508.2134 C27H35NO5NaS found, 508.2138.% C, H, N Calculated;
C = 66.78, H = 7.26, N = 2.88; found C = 66.39, H = 6.98, N = 2.47
3.3.12. Preparation of {4-[(10’r,30’r,50’R,70’R)-dispiro[cyclohexane-1,30-
[1,2,4,5]tetroxane-60,20’-tricyclo[3.3.1.13,7]decan]-4-yl]phenyl}(4-
methylpiperazin-1-yl)methanone 7f
3.3.7. Preparation of {4-[(10’r,30’r,50’R,70’R)-dispiro[cyclohexane-1,30-
[1,2,4,5]tetroxane-60,20’-tricyclo[3.3.1.13,7]decan]-4-yl]phenyl]
(morpholin-4-yl)methanone 7a
White solid (Yield 81%) mpt 108–110 °C 1HNMR (400 MHz,
CDCl3) dH, 7.4 (d, 2H, J = 9 Hz, Ar), 7.27 (d, 2H, J = 9 Hz, Ar), 3.4–
3.9 (m, 4H, CH2), 3.14–3.34 (m, 2H, CH), 2.6–2.8 (m, 1H, CH),
2.42 (s, 3H, CH3), 1.50–2.10 (m, 20H, CH2/CH) 13CNMR (100 MHz,
CDCl3), dC, 170.4,150.6, 147.9, 133.2, 129.9, 127.3, 127.0, 126.7,
110.5, 107.3, 45.0, 43.6, 43.4, 41.2, 34.2, 33.1, 31.7, 29.5, 27.0 MS
(ES+), [M + H] 483.3 (1 0 0), HRMS calculated for 483.2859
White solid (Yield 87%) Mpt = 123–124 °C 1H NMR (400 MHz,
CDCl3) dH, 7.38 (d, 2H, J = 8 Hz, Ar), 7.29 (d, 2H, J = 8 Hz, Ar), 3.4–
3.9 (m, 4H, CH2), 3.14–3.40 (m, 2H, CH), 2.6–2.7 (m, 1H, CH),
1.60–2.10 (m, 20H, CH2/CH), 13CNMR (100 MHz, CDCl3), dC 170.9,
169.5, 148.3, 127.8, 127.4, 117.7, 110.9, 107.8, 67.3, 43.9, 39.6,
37.6, 34.7, 33.6, 32.3, 30.6, 29.9, 27.8MS (ES+), [M + Na] 492.2 (1
C28H39N2O5 found, 483.2858.% C, H, N Calculated; C = 69.68, H =
0 0), HRMS calculated for 292.2362
C27H35NO6Na found,
7.94, N = 5.80; found C = 69.41, H = 7.42, N = 5.49
Please cite this article in press as: O’ Neill P.M., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.05.006

P.M. O’ Neill et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
9
+
3.3.13. Preparation of 1,4-diazepan-1-yl{4-[(10’r,30’r,50’R,70’R)-dispiro
[cyclohexane-1,30-[1,2,4,5]tetroxane-60,20’-tricyclo[3.3.1.13,7]decan]-
4-yl]phenyl}methanone 7g
37.4, 36.2, 34.7, 33.6, 30.1, 27.5 MS (ES+), [M + Na] (1 0 0) 409.2
HRMS 409.1991 calculated for C23H30NO5Na found 409.1990.
White powder (Yield 58%) 1HNMR (400 MHz, CDCl3) dH, 7.24–
7.4 (m, 4H, Ar), 3.5–3.8 (m, 6H, CH/CH2), 3.1–3.4 (m, 4H, CH),
2.65–2.57 (m, 1H, CH), 1.48–2.11 (m, 22H, CH2/CH), 13CNMR
(100 MHz, CDCl3), dC 172.2, 147.8, 134.9, 127.4, 127.1, 126.8,
110.9, 107.8, 80.2, 45.2, 43.8, 29.9, 28.8, 27.5, 27.0 MS (ES+), [M
+ H] 483.3 (1 0 0), HRMS calculated for 483.2859 C28H39N2O5
found, 483.2856.% C, H, N calculated; C = 69.68, H = 7.94, N =
5.80; found C = 69.41, H = 7.65, N = 5.51
3.3.18. Preparation of 4-[(10’r,30’r,50’R,70’R)-dispiro[cyclohexane-1,30-
[1,2,4,5]tetroxane-60,20’-tricyclo[3.3.1.13,7]decan]-4-yl]benzyl
methanesulfonate 9
Methanesulfonyl chloride (3.36 mmol) and triethylamine (3.62
mmol) were added at 0 °C under nitrogen atmosphere to a solution
of 8 (1.81 mmol) in dry DCM (50 mL). The mixture was allowed to
stir for 60 min at the same temperature, washed with aqueous 5%
NaHCO3 and water, and dried over Na2SO4. Evaporation of the sol-
vent gave the mesylate 9 as colourless oil (0.8, 95%). 1H NMR (400
MHz, CDCl3-d6) dH 7.36(d, 2H, 8.1 Hz, Ar), 7.27 (d, 2H, J = 8.1 Hz,
Ar), 5.21 (s, 2H, CH2O), 2.92 (s, 3H, SO2CH3), 2.64 (tt, 1H, J = 11.4
3.3.14. Preparation of {4-[(10’r,30’r,50’R,70’R)-dispiro[cyclohexane-1,30-
[1,2,4,5]tetroxane-60,20’-tricyclo[3.3.1.13,7]decan]-4-yl]phenyl}
(piperazin-1-yl)methanone 7h
+
Hz, 3.8 Hz, CH), 2.09–1.56 (m, 22H, CH2/CH) MS (ES+), [M + Na]
White solid (Yield 78%) 1HNMR (400 MHz, CDCl3) dH, 7.24–7.41
(m, 4H, Ar), 3.8–4.2 (m, 4H, CH2), 3.1–3.4 (m, 2H, CH), 2.6–2.8 (m,
1H, CH), 1.50–2.2 (m, 20H, CH2/CH), 13CNMR (100 MHz, CDCl3), dC,
171.4, 156.1, 148.9, 132.9, 128.0, 127.7, 110.9, 107.7, 66.2, 53.3,
43.9, 42.5, 37.3, 33.6, 30.2, 27.4 MS (ES+), [M + H] 469.3 (1 0 0),
HRMS calculated for 468.2624 C27H37N2O5 found, 468.2631.% C,
H, N calculated; C = 69.21, H = 7.74, N = 5.98; found C = 69.36, H
= 7.85, N = 5.61
(1 0 0) 487.3 HRMS calculated for 487.1766 C24H32NO7Na, found
487.1765.
3.3.19. General procedure for the amine formation (10a-b)
To a solution of mesylate 9 (1.08 mmol 1 eq) in dichloro-
methane (25 mL) were added triethylamine (2.15 mmol, 2 eq) fol-
lowed by the amine (2.15 mmol 2, eq) at 0 °C temperature. The
reaction mixture was stirred at room temperature over a period
of 12 h. The resulting reaction mixture was diluted with dichloro-
methane (50 mL), washed with water (3 ꢃ 20 mL), brine (10 mL)
and dried over sodium sulphate. The crude product obtained was
purified by flash column chromatography to the required amine.
3.3.15. Preparation of (4-aminopiperidin-1-yl){4-[(10’r,30’r,50’R,70’R)-
dispiro[cyclohexane-1,30-[1,2,4,5]tetroxane-60,20’-tricyclo[3.3.1.13,7
decan]-4-yl]phenyl}methanone 7i
]
White powder (Yield 86%) Mpt = 116–118 °C. 1HNMR (400
MHz, CDCl3) dH, 7.24–7.4 (m, 4H, Ar), 3.7–4.1 (m, 7H, CH/CH2),
3.3.20. Preparation of 4-{4-[(10’r,30’r,50’R,70’R)-dispiro[cyclohexane-
1,30-[1,2,4,5]tetroxane-60,20’-tricyclo[3.3.1.13,7]decan]-4-yl]
benzyl}morpholine 10a (N205)
3.1–3.4 (m, 4H, CH), 2.6 (m, 1H, CH), 1.60–2.1 (m, 20H, CH2/CH), 13
-
CNMR (100 MHz, CDCl3), dC 172.0, 155.6, 149.3, 132.2, 128.0, 127.9,
126.7, 110.9, 107.7, 53.7, 43.9, 43.8, 41.1, 37.3, 33.5, 27.4 MS (ES+),
[M + H] 483.3 (1 0 0), HRMS calculated for 483.2814 C28H39N2O5
found, 483.2805.% C, H, N Calculated; C = 69.68, H = 7.94, N =
5.80; found C = 69.41, H = 7.81, N = 5.71
White powder (Yield, 62%). Mtp 138–140 °C. 1H NMR (400 MHz,
CDCl3-d6) dH 7.24 (d, 2H, J = 8.1 Hz, Ar), 7.17 (d, 2H, J = 8.1 Hz, Ar),
3.73–3.68 (m, 4H, NCH2), 3.46 (s, 2H, ArCH2N), 2.67–2.54 (m, 1H,
CH), 2.43 (bs, 4H, CH2O), 2.09–1.58 (m, 22H, CH/CH2) 13C NMR
(100 MHz, CDCl3-d6) dc 145.2, 136.0, 129.7, 127.1, 110.9, 107.9,
3.3.16. Preparation of N-(2-aminoethyl)-4-[(10’r,30’r,50’R,70’R)-dispiro
[cyclohexane-1,30-[1,2,4,5]tetroxane-60,20’-tricyclo[3.3.1.13,7]decan]-
4-yl]benzamide 7j
67.4, 63.6, 54.0, 43.8, 37.4, 34.8, 33.6, 33.4, 32.3, 30.1, 27.5 MS
+
(ES+), [M + H]
(1 0 0) 456.3 HRMS calculated for 456.2750
C27H38NO5, found 456.2762; C28H39NO4F, found 472.2859.% C, H,
White powder (Yield, 76%). 1H NMR (400 MHz, CDCl3-d6) dH
8.74 (t, 1H, J = 5.5 Hz, NH), 8.12 (bs, 2H, NH2), 7.86 (d, 2H, J = 8.3
Hz, Ar), 7.34 (d, 2H, J = 8.3 Hz, Ar), 3.52 (q, 2H, J = 5.8 Hz, NCH2),
2.97 (t, 2H, J = 5.8 Hz, CH2N),2.80–2.71 (m, 1H, CH), 1.99–1.52
(m, 22H, CH/CH2) 13C NMR (100 MHz, CDCl3-d6) dc 167.3, 157.1,
149.8, 132.0, 128.4, 110.3, 107.8, 52.4, 42.5, 41.1, 38.9, 37.5, 35.6,
33.5, 32.8, 28.9, 27.1, 26.8 MS (ES+), [M + H ]+ (1 0 0) 443.3 HRMS
calculated for 443.2546 C25H35N2O5, found 443.2545.% C, H, N cal-
culated; C = 67.85, H = 7.74, N = 6.33; found C = 67.40, H = 7.34, N
= 6.21
N Calculated; C = 71.18, H = 8.18, N = 3.07; found C = 70.92, H =
7.99, N = 3.10
3.3.21. Preparation of 1-{4-[(10’r,30’r,50’R,70’R)-dispiro[cyclohexane-
1,30-[1,2,4,5]tetroxane-60,20’-tricyclo[3.3.1.13,7]decan]-4-yl]benzyl}-4-
fluoropiperidine 10b
White powder (Yield, 60%), Mtp 122–124 °C 1H NMR (400 MHz,
CDCl3-d6) dH 7.23 (d, 2H, J = 8.1 Hz, Ar),7.17 (d, 2H, J = 8.1 Hz, Ar),
4.79–4.53 (m, 1H, CHF), 3.46 (s, 2H, CH2N), 2.64–2.53 (m, 5H,
NCH2/CH), 2.40–2.31 (m, 4H, CH2), 2.06–1.54 (m, 22H, CH2/CH)
13C NMR (100 MHz, CDCl3-d6) dc 145.1, 136.6, 129.6, 127.1,
110.9, 107.9, 90.0, 88.3, 63.1, 50.0, 43.8, 37.4, 34.7, 33.6, 32.3,
3.3.17. Preparation of {4-[(10’r,30’r,50’R,70’R)-dispiro[cyclohexane-1,30-
[1,2,4,5]tetroxane-60,20’-tricyclo[3.3.1.13,7]decan]-4-yl]phenyl}
methanol 8
+
31.9, 30.6, 27.5 MS (ES+), [M + H] (1 0 0) 472.3 HRMS calculated
for 472.2863 C28H39NO4F, found 472.2859.% C, H, N Calculated; C
= 71.31, H = 8.12, N = 4.03; found C = 70.97, H = 7.91, N = 3.80
To a stirred solution at 0 °C of methyl benzoate 6 (1.5 g, 3.65
mmol) in THF (50 mL) was added LiAlH4 (0.28 g, 7.29 mmol)).
The suspension was allowed to stir at 0 °C and was monitored by
TLC to determine the consumption of the benzoate. The reaction
mixture was quenched with 1 N HCl and was then extracted with
ethyl acetate (3 x 50 mL). The combined organic layers were dried
over anhydrous sodium sulphate, filtered and concentrated under
reduced pressure. Purification by flash column chromatography
afforded the required alcohol 8 as a white powder (1.36 g, 97%).
1H NMR (400 MHz, CDCl3-d6) dH 7.27 (d, 2H, J = 8.2 Hz, Ar), 7.21
(d, 2H, J = 8.2 Hz, Ar), 4.62 (s, 2H, ArCH2OH), 2.62 (tt, 1H, J = 11.4
Hz, 4.0 Hz, Ar), 2.08–1.57 (m, 22H, CH2/CH) 13C NMR (100 MHz,
CDCl3-d6) dc 145.9, 139.3, 127.6, 110.9, 107.9, 65.6, 60.8, 43.8,
3.3.22. Preparation of 4-{4-[(10’r,30’r,50’R,70’R)-dispiro[cyclohexane-
1,30-[1,2,4,5]tetroxane-60,20’-tricyclo [3.3.1.13,7]decan]-4-yl]
benzyl}morpholine Mesylate
0.3 mmol of 10a was dissolved in 2 mL of anhydrous diethyl
ether and 1.5 mmol of 100 mM methane sulfonic acid stock solu-
tion was added. The precipitate formed was collected, washed with
diethyl ether and air dried to give the titled salt. White powder
(Yield 70%). Mpt 158–160 °C 1H NMR (400 MHz, DMSO d6) dH
9.76 (s, 1H, NH), 7.44 (d, 2H, J = 8.2 Hz, Ar), 7.37 (d, 2H, J = 8.1 Hz,
Ar), 4.31 (d, 2H, J = 5.0 Hz, CH2), 3.96 (d, 2H, J = 10.3 Hz, CH2),
3.62 (t, 2H, J = 11.6 Hz, CH2), 3.26 (d, 2H, J = 11.9 Hz, CH2), 3.18–
Please cite this article in press as: O’ Neill P.M., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.05.006

10
P.M. O’ Neill et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
2. Dondorp AM, Nosten F, Yi P, et al. Artemisinin resistance in Plasmodium
falciparum malaria. New Engl J Med. 2009;361:455–467.
3. Leang R, Taylor WR, Bouth DM, et al. Evidence of Plasmodium falciparum
Malaria Multidrug Resistance to Artemisinin and Piperaquine in Western
Cambodia: Dihydroartemisinin-Piperaquine Open-Label Multicenter Clinical
Assessment. Antimicrob Agents Chemotherapy. 2015;59:4719–4726.
3.03 (m, 4H, CH2), 2.78–2.69 (m, 1H, CH), 2.35 (s, 3H, SO2CH3),
1.96–1.51 (m, 22H, CH/CH2) MS (ES+), [M + H] + (1 0 0) 456.3 HRMS
calculated for 456.2750 C27H38NO5, found 456.2762.% C, H, N cal-
culated; C = 60.96, H = 7.49, N = 2.54; found C = 60.88, H = 7.40,
N = 2.21
4. Noedl H, Se Y, Schaecher K, et al. Resistance in Cambodia 1 Study, Evidence of
artemisinin-resistant malaria in western Cambodia. New Engl
2008;359:2619–2620.
J Med.
3.3.23. Preparation of 1-{4-[(10’r,30’r,50’R,70’R)-dispiro[cyclohexane-
1,30-[1,2,4,5]tetroxane-60,20’-tricyclo[3.3.1.13,7]decan]-4-yl]benzyl}-4-
fluoropiperidine mesylate
0.3 mmol 10b was dissolved in 2 mL of anhydrous diethyl ether
and 1.5 mmol of methane sulfonic acid stock solution was added.
The precipitate formed was collected, washed with diethyl ether
and air dried to give the titled salt. White powder (Yield 68%).
Mpt 108–110 °C 1H NMR (400 MHz, DMSO d6) dH 9.41 (s, 1H,
NH), 7.44 (d, 2H, J = 8.1 Hz, Ar), 7.36 (d, 2H, J = 8.1 Hz, Ar), 5.07–
4.70 (m, 1H, CHF), 4.32 (d, 2H, J = 5.1 Hz, CH2), 3.27 (d, 2H, J =
11.1 Hz, CH2), 3.19–2.96 (m, 4H, CH2), 2.79–2.66 (m, 1H,CH), 2.38
5. Dong Y, Wittlin S, Sriraghavan K, et al. The Structure-Activity Relationship of
the Antimalarial Ozonide Arterolane (OZ277). J Med Chem. 2010;53:481–491.
6. Valecha Neena, Krudsood Srivicha, Tangpukdee Noppadon, Mohanty Sanjib,
Sharma SK, Tyagi PK, Anvikar Anupkumar, Mohanty Rajesh, Rao BS, Jha AC,
Shahi B, Narayan Singh Jai Prakash, Roy Arjun, Kaur Pawandeep, Kothari
Shantanu, Mehta Shantanu, Gautam Anirudh, Paliwal Jyoti K, Arora Sudershan,
Saha Nilanjan. Arterolane maleate plus piperaquine phosphate for treatment of
uncomplicated plasmodium falciparum malaria: A comparative, multicenter,
randomized clinical trial. Clin Infect Dis. 2012;55(5):663–671. https://doi.org/
10.1093/cid/cis475.
7. Charman SA, Arbe-Barnes S, Bathurst IC, et al. Synthetic ozonide drug candidate
OZ439 offers new hope for a single-dose cure of uncomplicated malaria. Proc
Natl Acad Sci USA. 2011;108:4400–4405.
8. Dong Y, Wang X, Kamaraj S, et al. Structure-Activity Relationship of the
Antimalarial Ozonide Artefenomel (OZ439). J Med Chem. 2017;60:2654–2668.
9. Kim HS, Hammill JT, Guy RK. Seeking the Elusive Long-Acting Ozonide:
Discovery of Artefenomel (OZ439). J Med Chem. 2017;60:2651–2653.
10. Ellis GL, Amewu R, Sabbani S, et al. Two-Step Synthesis of Achiral Dispiro-
1,2,4,5-tetraoxanes with Outstanding Antimalarial Activity, Low Toxicity, and
High-Stability Profiles. J Med Chem. 2008;51:2170–2177.
(a, 3H, SO2CH3), 2.15–1.96 (m, 2H, CH2), 1.95–1.50 (m, 22H, CH/
+
CH2) MS (ES+), [M + H]
(1 0 0) 472.3 HRMS calculated for
472.2863 C28H39NO4F, found 472.2859.% C, H, N Calculated; C =
71.31, H = 8.12, N = 2.97; found C = 71.02, H = 8.01, N = 2.68
11. Fisher LC, Blackie MAL. Tetraoxanes as Antimalarials: Harnessing the
Endoperoxide. Mini-Rev Med Chem. 2014;14:123–135.
4. Conclusion
12. Kumar N, Khan SI, Atheaya H, Mamgain R, Rawat DS. Synthesis and in vitro
antimalarial activity of tetraoxane-amine/amide conjugates. Eur J Med Chem.
2011;46:2816–2827.
13. Kumar N, Khan SI, Rawat DS. Synthesis and Antimalarial-Activity Evaluation of
Tetraoxane-Triazine Hybrids and Spiro[piperidine-4,3’-tetraoxanes]. Helv Chim
Acta. 2012;95:1181–1197.
N205 represents a molecule with a vastly improved overall pro-
file compared to the first generation tetraoxane RKA182 with com-
parable antimalarial potency compared to OZ439. Whilst data with
human liver microsomes and human blood indicate similar levels
of stability compared to OZ439, the latter has superior in rodent
in vitro microsomal and blood stability and in vivo rat PK profiles.
Data obtained in the Pf SCID mouse model for N205 were extre-
mely encouraging and suggest that the benzylamino tetraoxane
template 2 should be explored further to enhance the solubility,
metabolic and blood stability even further. A recent paper by Ven-
nerstom et al. has examined a series of analogues of OZ439 to
determine the key features that impart in vivo potency in the
mouse model of malaria; the conclusion from this work is that
whilst prolonged plasma exposure is important for curative activ-
ity, there are other factors involved in imparting high efficacy in
rodent models.8 This observation provides an additional key chal-
lenge in understanding the dynamics of this class of drug.
14. Kumar N, Singh R, Rawat DS. Tetraoxanes: Synthetic and Medicinal Chemistry
Perspective. Med Res Rev. 2012;32:581–610.
15. Opsenica D, Pocsfalvi G, Milhous WK, Solaja BA. Antimalarial peroxides: the
first intramolecular 1,2,4,5-tetraoxane. J Serb Chem Soc. 2002;67:465–471.
16. O’Neill PM, Amewu RK, Nixon GL, et al. Identification of a 1,2,4,5-tetraoxane
antimalarial drug-development candidate (RKA 182) with superior properties
to the semisynthetic artemisinins. Angew Chem Int Ed Engl.
2010;49:5693–5697.
17. O’Neill PM, Sabbani S, Nixon GL, et al. Optimisation of the synthesis of second
generation 1,2,4,5 tetraoxane antimalarials. Tetrahedron. 2016;72:6118–6126.
18. O’Neill PM, Amewu RK, Sabbani S, et al. A tetraoxane-based antimalarial drug
candidate that overcomes PfK13-C580Y dependent artemisinin resistance. Nat
Commun. 2017;8:15159.
19. Ghorai P, Dussault PH.
A new peroxide fragmentation: efficient chemical
generation of (1O2) in organic media. Org Lett. 2009;11:4572–4575.
20. Smilkstein M, Sriwilaijaroen N, Kelly JX, Wilairat P, Riscoe M. Simple and
inexpensive fluorescence-based technique for high-throughput antimalarial
drug screening. Antimicrob Agents Chemotherapy. 2004;48:1803–1806.
21. Wang XF, Dong YX, Wittlin S, et al. Comparative Antimalarial Activities and
ADME Profiles of Ozonides (1,2,4-trioxolanes) OZ277, OZ439, and Their 1,2-
Acknowledgements
Dioxolane, 1,2,4-Trioxane, and 1,2,4,5-Tetraoxane Isosteres.
2013;56:2547–2555.
J Med Chem.
Funding: This work was supported by grants from the European
Union (Antimal, SAW, PON, EU FP6), the Medicines for Malaria
Venture (SAW, PMO, GB). The authors would also like to thank
Dr Sergio Wittlin and Christoph Fischli (Swiss Tropical and Public
Health Institute Socinstrasse 57, 4051 Basel, Switzerland) for the
data recorded in Table 2 (P. berghei mouse model).
22. Neely MN, van Guilder MG, Yamada WM, Schumitzky A, Jelliffe RW. Accurate
detection of outliers and subpopulations with Pmetrics, a nonparametric and
parametric pharmacometric modeling and simulation package for R. Ther Drug
Monit. 2012;34:467–476.
23. Angulo-Barturen I, Jimenez-Diaz MB, Mulet T, et al.
A murine model of
falciparum-malaria by in vivo selection of competent strains in non-
myelodepleted mice engrafted with human erythrocytes. PloS one. 2008;3:
e2252.
24. Jimenez-Diaz MB, Mulet T, Viera S, et al. Improved Murine Model of Malaria
Using Plasmodium falciparum Competent Strains and Non-Myelodepleted
NOD-scid IL2R gamma(null) Mice Engrafted with Human Erythrocytes.
Antimicrob Agents Chemotherapy. 2009;53:4533–4536.
25. Younis Y, Douelle F, Feng TS, et al. 3,5-Diaryl-2-aminopyridines as a Novel Class
of Orally Active Antimalarials Demonstrating Single Dose Cure in Mice and
Clinical Candidate Potential. J Med Chem. 2012;55:3479–3487.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmc.2018.05.006.
26. Tan JQ, Kim RM, Mirc JW. Preparation of biphenylpyridine compounds as soluble
guanylate cyclase activators. USA: Merck Sharp & Dohme Corp; 2012. 115.
27. DeGraw JI, Christie PH, Colwell WT, Sirotnak FM. Synthesis and antifolate
References
properties
1992;35:320–324.
of
5,10-ethano-5,10-dideazaaminopterin.
J
Med
Chem.
1. WHO, Global report on antimalarial drug efficacy and drug resistance: 2000–
2010, 2010.
Please cite this article in press as: O’ Neill P.M., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.05.006