Synthesis, in vitro and in vivo antimalarial assessment of sulfide, sulfone and vinyl amide-substituted 1,2,4-trioxanes prepared via thiol-olefin co-oxygenation (TOCO) of allylic alcohols

Article abstract of DOI:10.1039/b924319d

Thiol-Olefin Co-Oxygenation (TOCO) methodology has been applied to the synthesis of a small library of weak base and polar 1,2,4-trioxanes. The 1,2,4-trioxane units synthesised exhibit remarkable stability as they survive base catalysed hydrolysis and mixed anhydride/amine coupling reactions. This unique stability feature has enabled a range of novel substitution patterns to be incorporated within the spiro 1,2,4-trioxane unit. Selected analogues express potent in vitro nM antimalarial activity, low cytotoxicity and oral activity in the Plasmodium berghei mouse model of malaria.

Full text of DOI:10.1039/b924319d

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Synthesis, in vitro and in vivo antimalarial assessment of sulfide, sulfone and
vinyl amide-substituted 1,2,4-trioxanes prepared via thiol-olefin
co-oxygenation (TOCO) of allylic alcohols†
Richard Amewu,a Peter Gibbons,a Amira Mukhtar,a Andrew V. Stachulski,a Stephen A. Ward,b Charlotte Hall,b
Karen Rimmer,b Jill Davies,b Livia Vivas,d John Bacsa,a Amy E. Mercer,c Gemma Nixon,b Paul A. Stocksb and
Paul M. O’Neill*a
Received 19th November 2009, Accepted 4th February 2010
First published as an Advance Article on the web 3rd March 2010
DOI: 10.1039/b924319d
Thiol-Olefin Co-Oxygenation (TOCO) methodology has been applied to the synthesis of a small library
of weak base and polar 1,2,4-trioxanes. The 1,2,4-trioxane units synthesised exhibit remarkable stability
as they survive base catalysed hydrolysis and mixed anhydride/amine coupling reactions. This unique
stability feature has enabled a range of novel substitution patterns to be incorporated within the spiro
1,2,4-trioxane unit. Selected analogues express potent in vitro nM antimalarial activity, low cytotoxicity
and oral activity in the Plasmodium berghei mouse model of malaria.
Introduction
Malaria is one of the world’s most prevalent infectious diseases,
remaining a major health problem and, therefore, a developmental
barrier for many countries. It is estimated that about 40% of
the world’s population is at risk of contracting malaria, leading
to around 350–500 million infections coupled with a staggering
million deaths per annum. The widely affordable antimalarial
drugs such as chloroquine1 (1) and sulfadoxine (2),2 either as
monotherapies or in combination, are now largely ineffective due
to drug resistance.
The potential impact of artemisinin (3), a Chinese herbal
extract, on malaria chemotherapy is hampered by supply and
Although the actual mechanism of action of artemisinin and
cost; the majority of infected people needing treatment for
related compounds remains a topic of intense debate,6 SAR
malaria cannot afford drugs containing an artemisinin derivative.3
studies have revealed that the 1,2,4-trioxane heterocycle is the
Nevertheless, artemisinin and its derivatives remain the most
key pharmacophore.7 In line with this, various groups have
effective antimalarials and are currently used in combination with
designed and synthesized a variety of compounds containing this
other drugs recommended by the World Health Organization.4
functional group, some of which show promising activity towards
R
As an example, Coartemꢀ, a combination of Lumefantrine–
the malaria parasite. Several methodologies have been developed
for the synthesis of the 1,2,4-trioxane pharmocophore, and many
of these involve the preparation of an a-peroxy alcohol followed
Artemether (Novartis), has been registered and is currently used
for the treatment of uncomplicated malaria.5
by in situ reaction with a carbonyl component in the presence
of an acid catalyst.7–10 Other approaches include synthesis from
nitriles,11 reaction of dioxetanes with carbonyls in the presence of
Lewis acids,12 acid-catalyzed cyclization of hydroxyperoxyacetals
with olefins13 and reactions of a-peroxy aldehydes with carbonyl
compounds.14
aDepartment of Chemistry, University of Liverpool, P.O. Box 147, Liverpool,
UK L69 3BX. E-mail: P.M.oneill01@liv.ac.uk; Fax: (+44) 151 794 3588;
Tel: (+44) 151 794 3553
bLiverpool School of Tropical Medicine, Pembroke Place, Liverpool,
UK L3 5QA
cMRC Centre for Drug Safety Science, Dept of Pharmacology and
Therapeutics, The University of Liverpool Sherrington Buildings, Ashton
Street, L69 3GE
An alternative method for preparing 1,2,4-trioxanes involves
the free-radical sequential thiol-olefin co-oxygenation (TOCO)
reaction. TOCO chemistry is particularly attractive because it
allows functional group manipulation at various stages. In terms
of versatility, there are no available methods in the literature for
the preparation of sulfide- or sulfone-functionalised spiro 1,2,4-
trioxanes in just two synthetic steps from readily available building
blocks. Furthermore, this chemistry enables rapid access to 1,2,4-
trioxane aldehydes in only three synthetic steps; such targets are
not accessible by alternative published synthetic methodologies.
dDepartment of Infectious and Tropical Diseases, London School of Hygiene
and Tropical Medicine, London, UK WC1E 7HT
† Electronic supplementary information (ESI) available: Spectroscopic and
analytical data for compounds 16–30 (sulfide trioxanes), 32–42 (trioxane
sulfones), 50–54 (trioxane carbaldehydes), 64–65 (trioxane acrylamides),
67–73 (trioxane acrylic acid methyl esters), 75–76 (trioxane acrylic acids)
and 79–85 (trioxane amides). CCDC reference numbers 680267, 680268.
For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/b924319d
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TOCO chemistry has been used by various groups15–19 for the
synthesis of a wide range of cyclic peroxides, including Yingzhaosu
A (4), a potent series of bicyclic yingzhaosu A analogues and a
series of endoperoxide chalcone pro-drugs.20 The reactions are
usually initiated by AIBN and ultra violet (UV) irradiation or by
di-tert-butylperoxalate (DBPO). We and others have previously
used the TOCO methodology to prepare endoperoxides from
terpene derivatives (Scheme 1A).18 Replacement of the terpene
with an allylic alcohol (7 and 8) led to the synthesis of 1,2,4-
trioxane derivatives (Scheme 1B).19 By using the corresponding
homo-allylic alcohol analogue of 7, 1,2,4-trioxepanes can also be
readily prepared.21
of hydrogen by 11 from the 4-chlorothiophenol species produces a
racemic mixture of a-hydroperoxyl intermediate 13 and liberates
a second thiyl radical to propagate the reaction (Scheme 2). It
was anticipated that stabilizing the carbon-centred radical could
improve the reaction; hence the commercially available 2-methyl-2-
propen-1-ol (7) was replaced with 2-phenyl-2-propen-1-ol (8). The
reaction of the allylic alcohol 8 occurs via the same mechanism
to produce the peroxy radical 12 and a racemic mixture of the
hydroperoxy intermediate 14. Intermediate 14 undergoes smooth
condensation with a wide range of ketones in the presence of tosic
acid catalyst to afford the 1,2,4-trioxanes 15–30 (Table 1) in good
yields.
Scheme 1 A) (i) AIBN, ArSH, O2, hv. (ii)◦PPh3, CH2Cl2, 0–5 ◦C, 2 h then
1
2
=
r.t. 1 h. B) (i) AIBN, ArSH, CH3CN, O2, 0 C, 4–6 h (ii) R R C O, TsOH,
CH2Cl2, r.t.
We report herein the application of the thiol-olefin co-
oxygenation to the synthesis of a library of novel 1,2,4-trioxane
analogues. Subsequent conversion of the newly incorporated
sulfide functional group into more polar functional groups has
been performed in a systematic fashion to reduce the ClogP and
improve the aqueous solubility profiles of hit molecules (vide infra).
Scheme 2
Replacement of the allylic alcohol 7 with 8 had marginal effect
on the yield but purification was much easier. Considering the
multi-step nature of the reaction, and the sequence of events
leading to the formation of several new bonds, the TOCO reactions
proceed in very good yield.
Room temperature 1H NMR analysis of the sulfides 15–30
revealed the OCH2 and SCH2 protons as broad singlets. This
results from interconversion of different conformations due to
ring flipping of the six-membered trioxane ring system. The peaks
were resolved into two doublets (d) for 15 and 18 by performing
variable temperature analysis. While 18 was resolved at 0 ◦C; 3.49
(d, 1H, J = 13.1 Hz, SCH2), 3.61 (d, 1H, J = 13.1 Hz, SCH2), 3.68
(d, 1H, J = 12.0 Hz, OCH2), 3.82 (d, 1H, J = 12.0 Hz, OCH2),
Results and discussion
With AIBN as a radical initiator and UV irradiation, we per-
formed a one-pot reaction of the allylic alcohol (7 or 8) and 4-
chlorothiophenol in acetonitrile. In this process, a thiyl radical
is generated which subsequently adds onto the double bond of
the allylic alcohol in a Markovnikov fashion to form a tertiary
carbon-centred radical 9. The carbon-centered radical then reacts
with molecular oxygen to form a peroxy radical 11. Abstraction
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Table 1 Trioxanes synthesized via the TOCO reaction with cyclic ketones
Compound
R1 and R2
R3 and R4
Yield (%)
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
(CH2)5
(CH2)4
(CH2)11
Adamantylidene
(CH2)5
(CH2)11
(CH2)5
(CH2)4
(CH2)5
R3 = Me; R4 = p-Cl-Ph-S-
R3 = Me; R4 = p-Cl-Ph-S-
R3 = Me; R4 = p-Cl-Ph-S
R3 = Me; R4 = p-Cl- Ph-S-
R3 = Ph; R4 = p-Cl-Ph-S-
R3 = Ph; R4 = p-Cl-Ph-S-
R3 = Ph; R4 = Ph-S-
64
78
50
70
80
46
68
54
53
61
42
80
68
64
65
25
R3 = Ph; R4 = Ph-S-
R3 = Me; R4 = Ph-S-
(CH2)3
R3 = Me; R4 = Ph-S-
Adamantylidene
4-t-Bu-cyclohexylidene
(CH2)11
R3 = Me; R4 = Ph-S-
R3 = Me; R4 = p-Cl-Ph-S-
R3 = Me; R4 = Ph-S-
(CH2)11
R3 = Me; R4 = 2-naphthalenethiol
R3 = Me; R4 = p-Cl-Ph-S-
R3 = Me; R4 = Ph-S-
cis-Bicyclo[3.3.0]octane-3,7dione
1,4-Cyclohexanedione
15 required further cooling to -20 ◦C; 3.48 (d, 1H, J = 13.1 Hz,
SCH2), 3.62 (d, 1H, J = 13.1 Hz, SCH2), 3.69 (d, 1H, J = 12.1 Hz,
OCH2), 3.88 (d, 1H, J = 12.1 Hz, OCH2). This suggests that the
cut off temperature at which the flipping begins is governed by the
nature of the groups attached to the ring.
Table 2 Trioxanes sulfones synthesized
Compound
R
R1 and R2
Ar
Yield (%)
31
32
33
34
35
36
37
38
39
40
41
42
Me
Me
Me
Me
Ph
(CH2)4
(CH2)5
(CH2)11
Adamantylidene
(CH2)5
(CH2)11
(CH2)5
(CH2)3
Adamantylidene
4-t-Butylcyclohexyl
1,4-Cyclohexanedione
(CH2)11
p-Cl-Ph
p-Cl-Ph
p-Cl-Ph
p-Cl-Ph
p-Cl-Ph
p-Cl-Ph
Ph
Ph
Ph
p-Cl-Ph
Ph
76
80
80
80
77
79
75
91
56
92
82
93
Posner and co-workers observed that within a series of tricyclic
1,2,4-trioxanes, the sulfone group imparted high antimalarial po-
tency even in instances where the corresponding sulfide displayed
Ph
no activity.22,23 Thus, we prepared the sulfones 31–42 from the
Me
Me
Me
Me
Me
Me
24
sulfides 15–30 using 2.2 equivalents of mCPBA
(Scheme 3,
Table 2) in high yields. For compound 35, a single X-ray crystal
structure (as dimethyl formamide solvate) was solved and is
depicted in Fig. 1.
Ph
the 1,2,4-trioxanes more polar by incorporation of amide side
chains. First, the sulfides 15–20 were oxidised with a stoichiometric
amount of mCPBA to generate the corresponding sulfoxides
43–48 (Scheme 4). Secondly, 43–48 were exposed to Pummerer
conditions according to the method reported by Arroyo-Gomez
and co-workers25,26 to give the aldehydes 49–54 in high yield.
Manipulation of the resulting aldehydes led to mixed results;
oxidation of aldehyde 5227,28 to the corresponding carboxylic
acid 55 failed, as did reductive amination29,30 with morpholine
((Scheme 5) to give amine 57). For substrates 49–54, it appears
Scheme 3 (i) mCPBA (2.2 equiv.), CH2Cl2, 4–6 h, r.t.
A major limitation with many synthetic endoperoxide anti-
malarials is their poor oral bioavailability which stems in part
from limited aqueous solubility. Our next focus was to make
Fig. 1 Single-crystal X-ray structure of the trioxane sulfone 35 (CCDC 680267). Non-H atoms are represented by thermal ellipsoids at the 50%
probability level and H-atoms are shown as circles with arbitrary radii.
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Scheme 4 (i) mCPBA (1 equiv.), CH2Cl2, 4–6 h, r.t., (ii) TFAA, CH2Cl2, 2,6-lutidine, 3 h, r.t.
Scheme 5 (i) NaBH(OAc)3, CH2Cl2, 18 h, r.t. (ii) tBuOH, H2O, 2-methylbutene, NaH2PO4, NaClO2, 2 h, r.t.
that the close proximity of the aldehyde to the endoperoxide
bridge results in decomposition in the presence of tertiary butoxide
(t-BuO-) or cyclic amines.
While the oxidation led to a complex mixture of products,
none of which were the desired carboxylic acid 55, the reductive
amination reaction led to the formation of 59 (via reductive
amination of 58), presumably generated from the breakdown of
52. A series of vinyl-substituted trioxanes 60–73 was prepared
from the aldehydes 49–54 via Wittig reactions (Scheme 6)31
with various ylides in very good yield, with the exception of
for 65 gave their chemical shifts as 6.66 (d, 1H, J = 15.4 Hz, CH)
and 6.89 (d, 1H, J = 15.4 Hz, CH) for the major fraction, and
5.52 (d, 1H, J = 13.1 Hz, CH) and 6.07 (d, 1H, J = 13.1 Hz, CH)
for the minor fraction.
The molecules 60, 61 and 62 have the capacity to release
chalcone derivatives, while analogues 63–73 can release fumarate-
like structures following reductive cleavage of the endoperoxide
bridge with Fe(II). Part of our future investigations will focus
on the mechanistic studies of the iron-mediated degradation
chemistry of this class of compounds.
=
=
Ph3P CHCON(C2H5)2. (Ph3P CHCON(C2H5)2 was prepared by
refluxing N,N-diethylchloroacetamide with triphenylphosphine in
nitromethane.32
Further manipulation of the ester functionality of 63–73 was
also attempted. The most important observation was that the
vinyl esters readily undergo base-catalysed hydrolysis to the
corresponding carboxylic acids (Scheme 7).33,34 The reactions were
surprisingly successful despite the use of potassium hydroxide
(KOH) at high temperatures indicating the remarkable stability
of the 1,2,4-trioxane within this series of trioxanes.
Reaction of a range of aldehydes with the methyl and ethyl
ester Wittig reagent gave 66–73 predominantly as trans-geometric
isomers (J > 15 Hz). However, in contrast, the reaction with
=
Ph3P CHCON(C2H5)2 resulted in 63–65 as a mixture of geo-
metric isomers with the trans-isomer being the major product.
The ratio of the trans : cis, determined from the fraction of major
and minor isolated products, is 9 : 1, 4 : 1, 4 : 1 for 63, 64 and 65,
respectively. The cis- and trans-geometric isomers were assigned
based on the coupling constants of the vinyl protons (J ~ 13 and
16 Hz, respectively). Broadening of proton signals observed with
the OCH2 and CH2S of 15–30 was encountered with the vinyl
protons of the amides 63–65. The broadening was resolved by
Opsenica and co-workers observed that inclusion of an amide
moiety as an auxiliary functional group increases their anti-
malarial activity.35 This observation was made during their work
exploring the influence of steroid carriers on the antimalarial
and antiproliferative activity of cholic acid-derived tetraoxanes.
Our recent work on 1,2,4,5-tetraoxanes also demonstrated that
the presence of an amide bond and basic amine side-chains
significantly improves antimalarial activity and disposition by
enhancing aqueous solubility.36 Based on this principle, a series
◦
1
running the H NMR at -20 C. For example, the vinyl protons
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+
-
+
-
3
=
Scheme 6 (i) ArCH = P(Ph)3Br , NHMDS, THF, 75 min, r.t. (ii) Ph3P CH2CON(C2H5)2Br , CHCl3, H2O, NaOH, r.t., 1 h. (iii) Ph3P CHCOR ,
CH2Cl2, 3 h, r.t.
Scheme 7 (i) KOH, MeOH, 70 ◦C, 1 h. (ii) CH2Cl2, H2O, HCl, r.t. (iii) (C2H5)3N, ClCO2C2H5, CH2Cl2, 0 ◦C, 1 h. (iv) NHR1R2, 0 ◦C, 30 min then r.t.,
90 min.
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of amides were prepared by reacting carboxylic acids 74–76 with
ethyl chloroformate and Et3N in dichloromethane to give mixed
anhydrides 77, which were coupled with a selection of amines to
give amides 78–85 (Scheme 7).35,37 Both the acid hydrolysis and the
amide couplings proceeded in high yields, typically 75–86%.
Room temperature 1H NMR analysis of the amides 78–85
revealed the vinyl protons mostly as broad singlets, a similar
phenomenon observed with the sulfide OCH2 and SCH2, due to
interconversion of different conformations of the six-membered
(Table 3).38 Artemisinin and chloroquine were used as standard
positive controls.
From the large volume of data recorded against the 3D7 strain
of Plasmodium falciparum, several SAR trends emerge. For spiro
1,2,4-trioxane sulfides 15–18, the best activity is conferred by
fusion to a cyclopentyl or adamantyl ring system. Replacement of
the methyl function with a phenyl ring system reduces activity for
most analogues prepared (see, for example, 19). An unexpected
result was the high potency conferred by fusion of the 1,2,4-
trioxane heterocycle to a cis-bicyclo[3.3.0]octane: this molecule
(29) had excellent activity of 24 nM. In spite of this observation,
further studies were not performed due to the expense of the cis-
bicyclo[3.3.0]octane-3,7-dione starting material.
In general, the sulfones expressed reduced activity when com-
pared with the corresponding sulfides (see 16 versus 31, and
15 versus 34). For the vinyl-substituted dispiro 1,2,4-trioxanes,
excellent activity was noted for diethyl amides 63, 64 and 65.
Additional analogues with low nanomolar activity include the
morpholine analogues 81 and 82, and alkyl amine-linked deriva-
tive 83. As noted elsewhere,10 for the polar vinyl 1,2,4-trioxane
series, the adamantylidene fused derivatives express the best level
of antimalarial activity.
◦
endoperoxide ring system. As before, cooling to -20 C revealed
the vinyl protons as the expected doublets.
A crystal was grown for compound 83 by slow evaporation of
a CHCl3–hexane mixture and the single-crystal X-ray structure
is depicted in Fig. 2 below. The X-ray crystal structure confirms
our 1H NMR assignment of trans geometry for the double bond
in trioxane 83. (As clearly shown in the X-ray crystal structure,
recrystallisation of 83 from chloroform–hexane has produced
the hydrochloride hydrate, and a plausible explanation for this
observation is the presence of HCl in the chloroform solvent.)
Antimalarial properties
From the calculated ClogP and aqueous solubilities it is clear
that analogues 63–65 and 79–85 have significantly reduced lipid
In vitro analysis on 36 selected trioxanes was performed on the 3D7
chloroquine-sensitive parasite isolate of Plasmodium falciparum
Fig. 2 The asymmetric unit of the single-crystal X-ray structures of the trioxane amide 83 (HCl·H2O) (CCDC 680268). Non-H atoms are represented
by thermal ellipsoids at the 50% probability level and H-atoms are shown as circles with arbitrary radii.
Table 3 In vitro antimalarial activity versus the chloroquine sensitive 3D7 strain of Plasmodium falciparum and calculated logP and aqueous solubilities
(LogS). Lead compounds are in italics
Comp.
IC50/nMa
LogP
Solubility/mg mL-1
Comp.
IC50/nMa
LogP
Solubility/mg mL-1
15
16
17
18
19
23
24
26
29
30
31
32
34
37
39
40
41
60
61
110.50
37.91
388.99
59.07
361.50
113.91
314.00
88.18
4.90
4.46
7.71
5.68
6.01
4.25
3.41
6.41
3.77
2.73
3.07
3.51
4.30
2.89
3.70
4.38
1.37
8.21
5.09
5.72
8.47
0.40
0.50
0.86
12.85
33.54
0.58
63
64
65
66
67
68
69
70
71
78
79
80
81
82
83
84
85
1
48.32
38.82
19.41
79.21
129.22
68.29
124.88
112.35
79.21
250.80
111.75
37.03
27.95
37.80
36.70
121.08
71.70
19.20
10.20
2.76
2.29
3.57
1.98
2.42
3.20
2.39
2.84
3.61
1.40
0.94
2.03
2.60
2.19
3.31
3.03
2.56
4.57
2.72
553.86
1356.48
18.34
1057.68
499.80
31.56
829.68
390.79
20.82
3920.36
4188.70
1636.33
85.21
24.63
154.00
92.90
7.36
47.77
37.17
25.55
2.73
136.90
131.80
108.88
72.00
140.06
>1000
103.19
101.90
87.87
8.28
13.27
348.26
0.41
131.57
36.64
100.53
194.84
44.16
1.13
3
39.89
a See the Experimental section for the description of the assays. IC50 values were averaged values determined from three independent experiments. The
logP and solubilities were calculated using Virtual Computational Chemistry Laboratory (VCCLAB), http://www.vcclab.org, 2005.
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Table 4 In vitro cytotoxicity of selected analoguesa
poor oral antimalarial activity as did their sulfone counterparts.
Further medicinal chemistry optimisation of the side chain was
successful in terms of reducing the ClogP, with analogues 63–65
having significantly reduced lipophilicity compared with initial hit
molecule 16. Additional manipulation of the side-chain to enhance
polarity (79–85) provided analogues with acceptable ClogP values,
low nanomolar activity, low cytotoxicity and, importantly for
trioxane 82, good oral activity in the Plasmodium yoelli model
of malaria. Based upon the synthetic chemistry reported here,
further optimisation of this template should now be possible in
order to enhance the oral activity of these molecules further.
Compound
IC50/mM
16
29
31
82
83
>100
>100
>100
>100
>100
a Freshly isolated PBMC, 72 h (n = 3), cytotoxicity assessed by MTT assay.
Table 5 Peter’s suppressive test results versus Plasmodium yoelli in micea
Compound
% of inhibition at 30 mg kg-1
Experimental
16
5
All reagents were obtained from commercial suppliers including
Aldrich Chemical Co. and Lancaster. Dichloromethane (DCM),
triethylamine (Et3N) and tetrahydrofuran (THF) were freshly
distilled before use. The analytical thin layer chromatography
was performed on pre-coated silica gel (0.25 mm layer of silica
gel F254) aluminium sheets. UV light (254 nm) was used for all
visualizations and flash column chromatography was performed
using Merck 938S Kieselgel 60 Silica gel. All of the IR spectra
were run using the Perkin-Elmer 298 infrared spectrophotometer.
Solid samples were dissolved in CHCl3 and liquids/oils neat on
sodium chloride discs.
31
12.0
25.1
65
50
80
100
100
34
65
81
82
Artesunate
Artemether
a Parasitaemia was determined by microscopic examination of Giemsa-
stained blood films taken on day 4. Microscopic counts of blood films
from each mouse were processed using spreadsheet (Microsoft Corp.)
and expressed as percentage of inhibition from the arithmetic mean
parasitaemia of each group in relation to the untreated group.
1H NMR spectra were recorded using the Bruker (400, 250, and
200 MHz) NMR spectrophotometers. Spectra were referenced to
the residual solvent peak and chemical shifts expressed in ppm
from TMS as the internal reference peak. With exception of those
stated, all of the NMR was performed at room temperature.
The following notations were used to describe the multiplicity; b,
broad, s, singlet, bs, broad-singlet, d, doublet, dd, double-doublet,
t, triplet, q, quartet, m, multiplet, and coupling constants were
recorded in hertz.
Mass spectra were recorded between 20–70 eV using a VG7070E
and/or Micromass LCT mass spectrometers. The molecular
ion M+ with intensities in parenthesis is given followed by
peaks corresponding to major fragment losses. Melting points
are expressed in degree Celsius (◦C) and performed using the
Gallemkamp melting point apparatus and capillary tubes. The
antiparasitic properties (IC50, 4 day Peters’ experiment) were
performed at the University of Liverpool and London Schools
of Tropical Medicine. X-ray crystal structure measurements were
performed at the Department of Chemistry the University of
Liverpool.
solubility profiles compared with the initial hit molecule 16. The
concept of reducing ClogP in antimalarial endoperoxide drug
design has been discussed,39 and here the benefits are evident in
terms of producing improved lead compounds with encouraging
in vitro and in vivo (vide infra) antimalarial activity profiles.
Table 4 lists the in vitro cytotoxicity of analogues 16, 31, 82 and
83 versus human peripheral mono-nuclear type B cells (n = 3),
where the IC50 values were all greater than 100 mM. The MTT
assays were carried out after 72 h of drug treatment and it is clear
that representative molecules from the sulfide (16), sulfone (31)
and vinyl amides (82 and 83) have very high therapeutic indices.
A 4-day Peters’40 test was performed on 16, 31, 34, 81 and 82, and
the results are summarized in Table 5 below. The compounds were
administered orally by dissolving in standard suspending formula
(SSV) (0.5% sodium carboxymethylcellulose, 0.5% benzyl alcohol,
0.4% Tween 80, 0.9% NaCl), followed by treatment with 0.2 mL
of a solution of the test compound two hours (day 0) and on days
1, 2, and 3 post infections.
The sulfides 15–18 were shown to have poor oral antimalarial
activity following administration to mice (15, 17 and 18 <5%
inhibition (data not shown)). Since oxidative metabolism may
have been an issue with this series, we next examined the sulfone
derivatives 31 and 34. Although activity was improved, maximum
inhibition was never above 20–25%. The vinyl amide adaman-
tanone analogues 65, 81 and 82 performed somewhat better, with
analogue 82 expressing activity approaching the semi-synthetic
derivatives artesunate and artemether by oral administration.
In vitro sensitivity assays
Drug susceptibilities were assessed by the measurement of flu-
orescence after the addition of SYBR Green I as previously
described by Smilkstein et al.41 Drug IC50s were calculated from
the log of the dose/response relationship, as fitted with Grafit
software (Erithacus Software, 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 mL of SYBR
Green I in lysis buffer (0.2 mL of SYBR Green I/mL of lysis
buffer) was 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
Conclusions
This study exemplifies the value of TOCO chemistry for the syn-
thesis of a diversely functionalised set of 1,2,4-trioxane derivatives.
The initial hit sulfide series, although active in vitro, proved to have
2074 | Org. Biomol. Chem., 2010, 8, 2068–2077
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a Varioskan fluorescence multiwell plate reader from Thermo
Electron Corporation with excitation and emission wavelengths
of 485 and 530 nm, respectively.
13C NMR (100 MHz, CDCl3), dC 20.2, 23.5, 24.9, 30.1, 58.9, 67.6,
114.9, 129.8, 139.9, 140.8. MS (ES+) [M + Na]+ (100) 369, [2M +
Na]+ 715/717; HRMS calculated for 369.0539 C15H19O5NaSCl,
found 369.0532. Elemental analysis C: 51.96, H: 5.48 (required
values C: 51.95, H: 5.52).
Representative procedure for the preparation of trioxane sulfides
15–30
General procedure for the preparation of sulfoxides 43–48
Preparation of 3-(4-chloro-phenylsulfanylmethyl)-3-methyl-
1,2,4-trioxa-spiro[5.5]undecane (15). A 2-necked 500 mL round
bottom flask was charged with a solution of 2-methyl-2-propenol
(500 mg, 0.58 mL, 0.69 mmol) and AIBN (77.5mg, 4.72mmol) in
CH3CN (115 mL). The reaction vessel was flushed with oxygen for
several minutes at 0 ◦C then stoppered and kept under a positive
pressure of pure oxygen, with the aid of two oxygen balloons.
The reaction mixture was vigorously stirred and UV irradiated
at 0 ◦C using an externally mounted 100 W BLACK-RAY UV
lamp at a distance of 5–7 cm, with the simultaneous addition
of 4-chlorothiophenol (1250 mg, 8.64 mol, 3.13 equiv.) solution
in CH3CN (65 mL) over a period of 30 min. After completion
of the addition, the reaction was stirred at 0 ◦C for 4–6 h or
until consumption of starting materials (monitored by tlc). The
A solution of the sulfide (1 equiv.) and mCPBA (1 equiv.) in
DCM was stirred for 4–6 h at room temperature. The mixture
was poured into a saturated solution of 5% K2CO3 and extracted
with DCM. The organic layer was separated, dried over MgSO4
and evaporated. The mixtures of the two enantiomers were used
without purification for the Pummerer reaction.
General procedure for the preparation of aldehydes 49–54
Preparation of 8-methyl-6,7,10-trioxa-spiro[4.5]decane-8-carbal-
dehyde (49). To a solution of the sulfoxide 44 (1 g, 3.02 mmol)
at 0 ◦C in CH3CN (5 mL), 2,6-lutidine (1.5 g, 12.8 mmol) and
trifluoroacetic anhydride (TFAA) (1.63 g, 11.65 mmol), in CH3CN
(12mL) were added. The mixture was stirred at room temperature
for 3 h and extracted with ethyl acetate (50 mL). The organic
layer was dried in MgSO4 and the solvent removed under reduced
pressure to give the aldehyde. Purification was achieved by flash
column chromatography using ethyl acetate–hexane (1 : 9, v/v, Rf
◦
reaction vessel was then cooled to -10 C, flushed with nitrogen
and a solution of cyclohexanone (1703 mg, 17.35 mmol, 1.63 mL)
in DCM (65 mL) was added, followed by a catalytic amount of
◦
tosic acid. The mixture was stirred at -10 C, and then allowed
to warm slowly to room temperature overnight. Concentration
in vacuo and purification by flash column chromatography using
ethyl acetate–hexane (1 : 5, v/v, Rf 0.44) gave 15 (1.46 g, 64%) as
a white powder. Mp 57–59. ◦C nmax (CHCl3)/cm-1 810.2, 920.1,
1
0.70) as eluent to give 49 (0.53 g, 89%) as a colourless oil. H
NMR (400 MHz, CDCl3) dH 1.07 (s, 3H, CH3), 1.60-1.82 (m,
6H, cyclohexyl), 1.86-1.96 (m, 1H, cyclopentyl), 2.41-2.51 (m, 1H,
cyclopentyl), 3.70 (d, 1H, J = 11.6 Hz, CH2O), 4.18 (d, 1H, J =
11.6 Hz, CH2O), 9.90 (s, 1H, CHO) 13C NMR (400 MHz, CDCl3),
dC 16.7, 23.5, 37.3, 64.3, 84.7, 114.7, 203.2 MS (ES+) [M + Na]+
(100) 209.1, HRMS calculated for 209.0814 C9H14O4Na, found
209.0817.
1
1007.2, 1090.5, 1446.6, 1473.1, 2855.3, 2931.1, 3006.8. H NMR
(400 MHz, CDCl3, 0 ◦C) dH 1.14 (s, 3H, CH3), 1.33-1.57 (m, 8H,
cyclohexyl), 1.90 (bs, 1H, cyclohexyl), 2.14 (bs, 1H, cyclohexyl),
3.48 (d, 1H, J = 13.1 Hz, SCH2), 3.62 (d, 1H, J = 13.1 Hz, SCH2),
3.69 (d, 1H, J = 12.1 Hz, OCH2), 3.88 (d, 1H, J = 12.1 Hz,
OCH2), 7.24 (d, 2H, J = 8.6 Hz, Ar), 7.35 (d, 2H, J = 8.6 Hz, Ar);
13C NMR (100 MHz, CDCl3). dC 20.6 0, 22.6 0, 22.7, 25.9, 33.8,
64.2, 79.4, 102.7, 129.4, 129.7, 131.0, 131.7. MS (ES+) [M + Na]+
(100) 367.1, [2M + Na]+ 679.2. HRMS calculated for 351.0798
C16H21O3NaS35Cl, found 351.0803. Elemental analysis C: 58.62,
H: 6.48 (required values C: 58.44, H:6.44).
Representative procedure for the preparation of 60–62
Preparation of 3-methyl-3-styryl-1,2,5-trioxa-spiro[5.11]hepta-
decane 60. To a stirred suspension of benzyltriphenylphospho-
nium bromide (0.51 g, 1.18 mmol) in THF (2 mL) was added
NHMDS (1.18 mL, 1.18 mmol, 1 M solution in THF) via
syringe. The reaction mixture was allowed to stir at room tem-
perature for 15 min, and then a solution of 3-methyl-1,2,5-trioxa-
spiro[5.11]heptadecane-3-carbaldehyde (0.21 g, 0.738 mmol) in
THF (2 mL) was added. After an additional 1 h, the reaction
was quenched with saturated aq. NaHCO3, extracted with ether,
washed with brine, dried (Na2SO4) and concentrated in vacuo. The
product was purified by flash chromatography to give the desired
compound as a white solid in 69% yield. The product was purified
by column chromatography using ethyl acetate–hexane (25 :◦75,
v/v, Rf 0.78) to give the product as a white solid. Mp 86–87 C.
1H NMR (400 MHz, CDCl3) dH 7.32 (m, 5H, aromatic), 7.11 (d,
Representative procedure for preparation of sulfones 31–42
Preparation of 8-(4-chloro-benzenesulfonylmethyl)-8-methyl-
6,7,10-trioxa-spiro[4.5]-decane (31). A solution of sulfide 16
(0.1 g, 0.32 mmol, 1 equiv.) and mCPBA (0.16 g, 0.95 mmol,
2.2 equiv.) in DCM (2 mL) was stirred for 4–6 h at room
temperature. After consumption of the more polar intermediate
(monitored by tlc), the mixture was poured into a saturated
solution of 5% K2CO3. The mixture was extracted with DCM and
the organic layer separated, dried over MgSO4 and evaporated.
Purification of the residue by flash column chromatography using
ethyl acetate–hexane (1 : 4, v/v, Rf 0.58) gave 31 in (0.08 g, 76%) as
white powder. Mp 114–116 ◦C. nmax (CHCl3)/cm-1820.0, 963.6,
1008.7, 1086.6, 1144.0, 1316.2, 1390.0, 1472.0, 1578.6, 2871.3,
2969.7, 3010.7.1H NMR (400 MHz, CDCl3) dH 1.46 (s, 3H, CH3),
1.52-1.84 (m, 8H, cyclopentyl), 3.67 (d, 2H, J = 11.9 Hz, OCH2),
3.90 (d, 1H, J = 14.1 Hz, SO2CH2), 4.00 (d, 1H, J = 12.1 Hz,
SO2CH2), 7.55 (d, 2H, J = 8.7 Hz, Ar), 7.90 (d, 2H, J = 8.7 Hz, Ar).
=
J = 12.67 Hz, 1H, trans olefin–C CH), 6.72 (d, J = 12.67 Hz,
trans olefin), 3.90 (bs, 1H, -OCH2), 3.61 (bs, 1H, -OCH2), 1.28
(m, 22H, cyclodeodecane moiety), 1.23 (s, 3H, -CH3). 13C NMR
(100 MHz,CDCl3) 134.17, 129.17, 128.90, 128.29, 126.92, 126.84,
106.55, 79.41, 60.71, 40.70, 31.94, 26.36, 22.99 and 21.34. MS
(ES+) m/z 381.3 [M + Na]+ (98), 413.3 [M + CH3OH + Na]+ (62).
HRMS m/z calcd for C23H34O3Na [M + Na]+ 381.2406 found,
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381.2419. Elemental analysis C: 77.03, H: 9.58 (required values C:
77.05, H: 9.56).
hydrolysed in MeOH (24 mL) at 70 ◦C with potassium hydroxide
(0.74 g, 13.1 mmol) and water (3 mL). After an hour heating, the
reaction mixture was cooled and diluted with DCM (30 mL) and
water (12 mL). The aqueous layer was acidified with concentrated
hydrochloric acid (3 mL) and further extracted with DCM. The
combined organic layers were washed with water, brine, dried over
Na2SO4 and evaporated to dryness. Purification by flash column
chromatography using ethyl acetate: dichoromethane (1 : 1, v/v,
Rf 0.2) as eluent gave 74 (0.46 g, 86%) as white powder. 1H NMR
(400 MHz, CDCl3) dH 1.22 (bs, 3H, CH3), 1.61-1.92 (m, 8H,
cyclopentyl), 3.70-3.92 (m, 2H, CH2O), 6.20 (bs, 1H, CH), 7.30
(bs, 1H, CH), 10.70 (bs, 1H, OH). 13C NMR (100 MHz, CDCl3),
dC 21.6, 23.6, 38.7, 67.7, 78.9, 114.9, 122.0, 151.4, 171.7. MS (ES-)
[M - H] -(100) 227.1.
Representative procedure for the preparation of acrylamides 63–65
Preparation of (E/Z)-N,N-diethyl-3-(3-methyl-1,2,5-trioxa-
spiro[5.5]undec-3-yl)-acrylamide (63). To
a
mixture of
Ph3P+=CHCON(C2H5)2Br- (0.37 g, 0.99 mmol) in water
(3 mL) and aldehyde 52 (0.2 g, 0.97 mmol) in DCM (10 mL),
was added drop-wise a solution of NaOH (0.18 g, 0.46 mmol)
in water (1 mL). The mixture was stirred for a few minutes and
the organic layer was separated, dried over MgSO4, filtered and
evaporated. The resulting residue was passed through a silica
gel column (EtOAc–Hex 1 : 1) to remove the triphenylphosphine
oxide. Evaporation of the solvent gave 63 as colourless oil.
Major fraction (trans-isomer). This fraction was collected in
40% as a colourless oil. nmax(neat)/cm-1 923.4, 1088.7, 1139.8,
1277.5, 1360.1, 1430.9, 1446.7, 1619.8, 1659.1, 2860.5, 2931.3,
2962.7. 1H NMR (400 MHz, CDCl3) dH 1.10-1.24 (m, 9H, CH3),
1.40-1.70 (m, 8H, cyclohexyl), 1.80-2.30 (m, 2H, cyclohexyl), 3.32-
3.52 (m, 4H, NCH2), 3.82 (bs, 2H, CH2O), 6.68 (bs, 1H, CH),
6.85 (bs, 1H, CH). 13C NMR (100 MHz, CDCl3), dC 13.5, 15.3,
22.7, 24.3, 41.3, 42.7, 65.4, 79.2, 102.7, 122.3, 141.9, 167.3. MS
(ES+), [M + Na]+ (100) 366.2; HRMS calculated for 320.1838
C16H27NO4Na, found 320.1824.
Representative procedure for the synthesis of the amides 78–85
Preparation of (E)-3-(8-methyl-6,7,10-trioxa-spiro[4.5]dec-8-yl)-
1-morpholin-4-yl-propenone (78). A solution of acid 77 (0.12 g,
0.53 mmol) in dry DCM (27 mL), with added triethylamine (0.06 g,
0.008 mL, 0.53 mmol) and ethylchloroformate (0.008 g, 0.07 mL,
0.7 mmol) was stirred for 60 min at 0 ◦C. Morpholine (0.09 g,
0.09 mL, 1.06 mmol) was added and the solution allowed to stir
for 30 min at 0 ◦C before being warmed to room temperature.
After 90 min, it was diluted with water and extracted with
DCM. The organic extract was washed with brine, and dried
over anhydrous Na2SO4. The crude product was purified by flash
chromatography using ethyl acetate–hexane (1 : 1, v/v, Rf 0.17)
as eluent to give 78 (0.13 g, 83%) as a white powder. Mp 68–
70 ◦C. nmax (CHCl3)/cm-1 1433.2, 1614.3, 1655.1, 2858.9, 2922.3,
2976.5. 1H NMR (400 MHz, CDCl3) dH 1.22 (bs, 3H, CH3), 1.59-
1.92 (m, 8H, cyclopentyl), 3.59 (bs, 2H, CH2O), 3.65-3.74 (m, 6H,
NCH2/CH2O), 3.83 (d, 1H, J = 15.5 Hz, CH), 6.50 (d, 1H, J =
15.5 Hz, CH), 6.80 (bs, 1H, CH). 13C NMR (100 MHz, CDCl3) dC
21.0, 24.5, 42.4, 46.4, 66.8, 67.5, 78.7, 114.3, 121.3, 144.7, 165.4.
MS (ES+) [M + Na]+ (100) 320.1, and [2M + Na]+ 617.2 HRMS
calculated for 320.1474 C15H23NO5Na, found 320.1480. Elemental
analysis C: 60.83, H: 7.82 (required values C: 60.59, H: 7.80).
Minor fraction (cis-isomer). This fraction was collected in 5%
as a colourless oil. This compound was purified by flash column
chromatography using ethyl acetate–hexane (1 : 1, v/v, Rf 0.44)
as eluent. nmax(neat)/cm-1 923.4, 1088.7, 1143.7, 1265.7, 1446.7,
1462.4, 1619.8, 2868.3, 2939.1, 2970.6. 1H NMR (400 MHz,
CDCl3) dH 1.12-1.23 (m, 9H, CH3), 1.30-1.65 (m, 10H, cyclohexyl),
3.31-3.51 (m, 4H, NCH2), 3.82 (bs, 1H, CH2O), 4.15 (d, 1H, J =
11.0 Hz, CH2O), 6.08 (d, 1H, J = 12.9 Hz, CH), 6.25 (bs, 1H,
CH). 13C NMR (100 MHz, CDCl3) dC 13.0, 14.3, 22.8, 25.9, 39.6,
43.1, 65.2, 81.0, 102.4, 123.7, 141.9, 167.3. MS (ES+) [M + K]+
(100) 366.2, HRMS calculated for 336.1553 C16H27NO4K, found
336.1574.
Representative procedure for the synthesis of compounds 66–73
Crystal data for 35 dimethylformamide solvate (CCDC 680267)
Preparation of (E)-3-(8-methyl-6,7,10-trioxa-spiro[4.5]dec-8-yl)-
acrylic acid methyl ester (66). To a solution of the aldehyde 49
C24H30ClNO6S, M = 496.00, colourless plate, 0.50 ¥ 0.20 ¥
=
(0.38 g, 2.04 mmol) in DCM (15 mL) was added Ph3P CHCO2Me
3
¯
0.04 mm , triclinic, space group P1 (No. 2), a = 5.6889(13), b =
(0.73 g, 2.19 mmol) at room temperature. The reaction was stirred
at this temperature for 3 h and was subsequently concentrated and
chromatographed using hexane–ethyl acetate (1 : 4, v/v, Rf 0.60)
as eluent on a silica gel to give 66 (0.31 g, 62%) as a colourless
oil. nmax(neat)/cm-1 861.0, 971.8, 1090.7, 1197.3, 1283.4, 1312.1,
1431.0, 1656.5, 1722.2, 2871.3, 2953.3. 1H NMR (400 MHz,
CDCl3) dH 1.15 (bs, 3H, CH3), 1.45-1.90 (m, 8H, cyclopentyl), 3.78
(s, 3H, CH3O), 3.85 (s, 2H, CH2O), 6.15 (bs, 1H, CH), 7.18 (bs,
1H, CH). 13C NMR (100 MHz, CDCl3) dC 21.8, 23.8, 37.4, 52.1,
67.8, 78.9, 114.7, 122.4, 149.1, 167.0. MS (ES+), [M + Na]+ (100)
265, HRMS calculated for 265.1052 C12H18O5Na, found 265.1058.
˚
13.461(3), c = 16.663(4) A, a = 67.393(4), b = 80.926(5), g =
◦
3
-3
˚
83.493(4) , V = 1161.3(5) A , Z = 2, Dc = 1.418 g cm , F000 = 524,
Bruker D8 diffractometer with APEX detector, Mo-Ka radiation,
l = 0.71073 A, T = 100(2) K, 2qmax = 53.5◦, 6737 reflections
˚
collected, 4786 unique (Rint = 0.0224). Final GooF = 1.068, R1 =
0.0457, wR2 = 0.0963, R indices based on 3862 reflections with
I > 2s(I) (refinement on F2), 300 parameters, 0 restraints. Lp and
absorption corrections applied, m = 0.296 mm-1.
Crystal data for 83 hydrochloride hydrate (CHCl3/hexane)
(CCDC 680268)
Representative procedure for the synthesis of carboxylic acids
74–76
C22H39ClN2O5, M = 447.00, colourless plate, 0.50 ¥ 0.40 ¥
0.10 mm3, monoclinic, space group P21/c (No. 14), a = 18.705(3),
◦
3
˚
˚
Preparation of (E)-3-(8-methyl-6,7,10-trioxa-spiro[4.5]dec-8-yl)-
acrylic acid (74). Ethyl ester 66 (0.6037 g, 2.4 mmol) was
b = 7.6876(13), c = 16.257(3) A, b = 93.288(3) , V = 2333.9(7) A ,
Z = 4, Dc = 1.272 g cm-3, F000 = 968, Bruker D8 diffractometer
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˚
20 P. M. O’Neill, P. A. Stocks, M. D. Pugh, N. C. Araujo, E. E. Korshin,
J. F. Bickley, S. A. Ward, P. G. Bray, E. Pasini, J. Davies, E. Verissimo
and M. D. Bachi, Angew. Chem., Int. Ed., 2004, 43, 4193–4197.
21 R. Amewu, A. V. Stachulski, N. G. Berry, S. A. Ward, J. Davies, G.
Labat, J. F. Rossignol and P. M. O’Neill, Bioorg. Med. Chem. Lett.,
2006, 16, 6124–6130.
with APEX detector, Mo-Ka radiation, l = 0.71073 A, T = 100(2)
K, 2qmax = 50.7◦, 14079 reflections collected, 4241 unique (Rint
=
0.0506). Final GooF = 1.143, R1 = 0.0730, wR2 = 0.1611, R indices
based on 3573 reflections with I > 2s(I) (refinement on F2), 293
parameters, 3 restraints. Lp and absorption corrections applied,
m = 0.198 mm-1.
22 G. H. Posner, H. O’Dowd, P. Ploypradith, J. N. Cumming, S. Xie and
T. A. Shapiro, J. Med. Chem., 1998, 41, 2164–2167.
23 G. H. Posner, J. P. Maxwell, H. O’Dowd, M. Krasavin, S. J. Xie and
T. A. Shapiro, Bioorg. Med. Chem., 2000, 8, 1361–1370.
24 G. H. Posner, H. O’Dowd, T. Caferro, J. N. Cumming, P. Ploypradith,
S. J. Xie and T. A. Shapiro, Tetrahedron Lett., 1998, 39, 2273–2276.
25 Y. Arroyo-Gomez, J. F. Rodriguez-Amo, M. Santos-Garcia and M. A.
Sanz-Tejedor, Tetrahedron: Asymmetry, 2000, 11, 789–796.
26 For use of the modified Pummerer reaction in the synthesis of bicylic
endoperoxide aldehydes see: M. D. Bachi, E. E. Korshin, R. Hoos and
A. M. Szpilman, J. Heterocycl. Chem., 2000, 37, 639–646.
27 Y. Watanabe, K. Miura, M. Shiozaki, S. Kanai, S. Kurakata and M.
Nishijima, Carbohydr. Res., 2003, 338, 47–54.
28 T. A. Johnson, D. O. Jang, B. W. Slafer, M. D. Curtis and P. Beak,
J. Am. Chem. Soc., 2002, 124, 11689–11698.
29 O. Dechy-Cabaret, F. Benoit-Vical, C. Loup, A. Robert, H. Gornitzka,
A. Bonhoure, H. Vial, J. F. Magnaval, J. P. Seguela and B. Meunier,
Chem.–Eur. J., 2004, 10, 1625–1636.
Acknowledgements
The authors thank the BBSRC (SAW, PON, PG Grants
BB/C006321/1 and 26/B13581), the EU (Antimal FP6 Malaria
Drugs Initiative) and Romark (RA) for generous funding of this
work.
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36 (a) For studies with weak base 1,2,4,5-tetraoxanes see: R. Amewu,
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