Analogues of thiolactomycin as potential anti-malarial and anti-trypanosomal agents

Article abstract of DOI:10.1016/j.bmc.2003.11.023

A series of analogues of the naturally occurring antibiotic thiolactomycin (TLM) have been synthesised and evaluated for their ability to inhibit the growth of the malaria parasite, Plasmodium falciparum. Thiolactomycin is an inhibitor of Type II fatty acid synthase which is found in plants and most prokaryotes, but not an inhibitor of Type I fatty acid synthase in mammals. A number of the analogues showed inhibition equal to or greater than TLM. The introduction of hydrophobic alkyl groups at the C3 and C5 positions of the thiolactone ring lead to increased inhibition, the best showing a fourteenfold increase in activity over TLM. In addition, some of the analogues showed activity when assayed against the parasitic protozoa, Trypanosoma cruzi and Trypanosoma brucei.

Full text of DOI:10.1016/j.bmc.2003.11.023

Bioorganic & Medicinal Chemistry 12 (2004) 683–692
Analogues of thiolactomycin as potential anti-malarial and
anti-trypanosomal agents
Simon M. Jones,a Jonathan E. Urch,b Reto Brun,c John L. Harwood,b Colin Berryb
and Ian H. Gilberta,*
aWelsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3XF, UK
bCardiff School of Biosciences, Biomedical Building, Museum Avenue, Cardiff CF10 3US, UK
cSwiss Tropical Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
Received 10 July 2003; accepted 21 November 2003
Abstract—A series of analogues of the naturally occurring antibiotic thiolactomycin (TLM) have been synthesised and evaluated
for their ability to inhibit the growth of the malaria parasite, Plasmodium falciparum. Thiolactomycin is an inhibitor of Type II fatty
acid synthase which is found in plants and most prokaryotes, but not an inhibitor of Type I fatty acid synthase in mammals. A
number of the analogues showed inhibition equal to or greater than TLM. The introduction of hydrophobic alkyl groups at the C3
and C5 positions of the thiolactone ring lead to increased inhibition, the best showing a fourteenfold increase in activity over TLM.
In addition, some of the analogues showed activity when assayed against the parasitic protozoa, Trypanosoma cruzi and Trypano-
soma brucei.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
Fatty acid synthesis is a crucial function of living cells.
The main steps in this process in animals are carried out
by a single, multifunctional polypeptide fatty acid syn-
thase (Type I FAS). In contrast, plants and bacteria
utilise a dissociable multienzyme system (Type II FAS).5
The structural differences between these systems are
sufficient for the development of selective antibiotics
targeted against the b-ketoacyl-acyl carrier protein syn-
thase (KAS) and other individual enzymes of the Type
II FAS.6,7
Malaria is by far the world’s most important tropical
disease. In many developing countries and in Africa
especially, malaria exacts an enormous toll in lives, in
medical costs, and in days of labour lost. At present, at
least 300 million people are affected by malaria globally,
and there are between 1 and 1.5 million malaria deaths
annually.1,2 Over the last several decades, the rapid
development of drug-resistant strains has compounded
the already serious health problems. Of the four known
human malaria parasites, Plasmodium falciparum is the
predominant cause of mortality, with 120 million new
cases and 1 million deaths per year globally. It is this
particular species, which has given rise to formidable
drug-resistant strains, resulting in the urgent need for
new chemotherapeutic agents. The search for new
agents has recently been aided with the completion of
the P. falciparum genome.3 Detailed studies4 of this
genome have identified new potential drug and vaccine
targets. One such target appears to be fatty acid bio-
synthesis of P. falciparum.
In plants and bacteria, there appear to be three KAS
enzymes, denoted KAS I, KAS II and KAS III. The
initial C2-C4 step is catalysed by KAS III; thereafter
KAS I and KAS II are involved in chain elongation.8 In
most organisms with a Type II fatty acid synthase, KAS
I and KAS II show very high similarity at the protein
level. Recent discoveries acknowledge that Plasmodium
synthesises fatty acids in the apicoplast which is a vestigial
organelle thought to be derived from a chloroplast.9ꢀ12
Not surprisingly, Plasmodium fatty acid synthase (FAS)
is a Type II enzyme complex and, thus, differs markedly
from human Type I FAS. Analysis of the recently pub-
lished P. falciparum genome3 reveals just two different
KAS enzymes, with the KAS I and KAS II of typical
Type II systems replaced by a single enzyme, that we and
others, have denoted KAS1/2, and a separate KAS III.8
*Corresponding author. Fax: +44-29-2087-4149; e-mail: gilbertih@
cf.ac.uk
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.11.023

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S. M. Jones et al. / Bioorg. Med. Chem. 12 (2004) 683–692
2. Chemistry
The first racemic total synthesis of TLM 1 was reported
by Salvino et al. and involved the alkylation of a thio-
tetronic acid dianion with an isoprene cation equiv-
alent.21 A subsequent Wittig condensation with the
resulting aldehyde afforded TLM 1. The intermediate
thiotetronic acid was prepared according to methodol-
ogy developed by Benary.22 Using a modification of this
methodology we were able to synthesise TLM and a
range of TLM analogues with varying chain length and
saturation at the C5 and C3 positions of the ring
(Scheme 1). Methyl propionylacetate 2 was treated with
alkyl and benzyl halides in the presence of potassium
carbonate as base to provide the alkylated products 3.
Treatment of the resulting b-ketoesters 3 with pyri-
dinium tribromide in acetic acid afforded the bromides 4
as a mixture of diastereoisomers. Displacement of the
bromides 4 proceeded smoothly using thiolacetic acid
under basic conditions forming the thioacetates 5.
Cyclisation to the desired thiolactones 6 was accom-
plished upon treatment with aqueous potassium
hydroxide solution. Isolated yields following flash chro-
matography were varied (30–93%). These derivatives
were converted into the final compounds 7 after expo-
sure to sodium hydride, butyllithium and the appro-
priate alkyl halides in dry THF at ꢀ78 ꢁC. The
alkylations tended to be generally low yielding; these
findings are in agreement with previous publications in
this area.19 Attempts to protect the hydroxy function-
ality, in the hope that tying up of the free hydroxy
group may improve the yields for the alkylation step,
proved unsuccessful.
Figure 1. Structure of thiolactomycin.
A known inhibitor of the dissociable Type II FAS
enzymes is the naturally occurring thiolactone anti-
biotic, thiolactomycin (TLM) 113,14 (Fig. 1). Originally
reported in 1982,15 TLM has since become the most
well-studied of the naturally occurring thiotetronic
acids. The thiolactone has shown moderate in vitro
activity against a broad spectrum of pathogens, includ-
ing Gram-positive and Gram-negative16 bacteria and
Mycobacterium tuberculosis.17 Furthermore, TLM ana-
logues showed enhanced inhibition of pea (Pisum sativum)
FAS,18 and effective activity against Staphylococcus
aureus and Pasteurella multocida.19 Because of its negli-
gible toxicity towards mammals,15 TLM or its analo-
gues are obvious candidates as new drugs for problem
diseases such as malaria. In fact, recently Waller et al.
have reported encouraging anti-malarial activity for
TLM and various analogues.20 Furthermore, because
there are KAS isoforms in target organisms and each
KAS isoform of Type II FAS complexes has been
shown to be sensitive to TLM, then use of such inhibi-
tors offers therapies with less chance of target-site resis-
tance due to point mutations occurring readily. In this
paper we present synthetic studies and findings in our
attempts to improve the overall activity of TLM against
P. falciparum cultured in red blood cells. Also studies
against Trypanosoma brucei rhodesiense trypomastigotes
and intracellular Trypanosoma cruzi amastigotes are
reported.
Derivatisation of the methyl substituent at the C5 posi-
tion was accomplished as shown in Scheme 2. Following
a
literature procedure,23 the b-ketoesters
9 were
Scheme 1. Reagents and conditions: (a) R1X, K2CO3 (4 equiv), THF, reflux; (b) PyBr3, AcOH, rt; (c) AcSH, Et3N, EtOAc, rt; (d) KOH (2 equiv),
H2O/EtOH (1:1), 45 ꢁC; (e) NaH (1.2 equiv), BuLi (1.1 equiv), R2X, THF/DMPU (1:1), rt to ꢀ78 ꢁC; (f) NaH, BuLi, C2H5OCHC(CH3)CHO (2
equiv), THF/Pyrimidone (1:1), rt to ꢀ78 ꢁC; (g) BuLi (2 equiv), Ph3P+–CH3Br,ꢀ THF, ꢀ78 ꢁC to rt.

S. M. Jones et al. / Bioorg. Med. Chem. 12 (2004) 683–692
685
Scheme 2. Reagents and conditions: (a) NaH (1.2 equiv), BuLi (1.1 equiv), R1I, 0 ꢁC to rt; (b) KOH (2 equiv), H2O/EtOH (1:1), 45 ꢁC; (c) NaH (1.2
equiv), BuLi (1.1 equiv), R3X, rt to ꢀ78 ꢁC.
Scheme 3. Reagents and conditions: (a) NaH (1.2 equiv), BuLi (1.1 equiv), iododecane, 0 ꢁC to rt; (b) iodobutane, K2CO3 (4 equiv), THF, reflux;
(c) PyBr3, AcOH, rt; (d) AcSH, Et3N, EtOAc, rt; (e) KOH (2 equiv), H2O/EtOH (1:1), 45 ꢁC.
prepared in excellent yields by treatment of methyl
acetoacetate 8 with sodium hydride, butyllithium and
the relevant alkyl iodides. The corresponding b-keto-
esters 10 were synthesised using the same methodology
described in Scheme 1, and once formed were duly con-
verted into the desired thiolactones 11 and 12.
T. brucei and T. cruzi (Table 1). The screening proce-
dures are briefly described in the Experimental (Section 5).
The basic structure of the thiolactomycin ring was
retained with the analogues represented mainly by vari-
ations in the isoprenoid moiety and the methyl sub-
stituents at the C3 and C5 positions of the ring.
Recently, thiolactomycin analogues have been tested in
assays for fatty acid synthesis18,20 and activities of the
individual condensing enzymes.17 The findings from
these studies suggest that replacement of the isoprenoid
side chain with longer tethers leads to an overall
increase in inhibition of fatty acid synthesis. This nota-
ble trend is also evident in our own findings (Table 1).
The analogues 7a and 7b showed inhibition comparable
to that of thiolactomycin 1. In comparison to 1, the
geranyl analogue 7c showed almost a fourfold increase
in activity, perhaps indicating that unsaturation in the
hydrophobic chain enhances inhibition. Waller et al.20
have reported a sixfold increase in efficacy for the ger-
anyl analogue over TLM against the P. falciparum
multidrug-resistant strain W2mef. The longer side-chain
analogues 7d (decyl) and 7e (hexadecyl) also showed
much improved activity over 1, with 7e registering a
6-fold (25 mM) increase in inhibition. The influence of
the longer hydrophobic chain on activity is further
Using the same methodology described in Scheme 2, we
were able to avoid the low yielding alkylation reaction
at the C5 position with the construction of the desired
carbon skeleton prior to cyclisation (Scheme 3). Sub-
sequent alkylations of methyl propionylacetate 2 at the
C4 and C2 positions respectively yielded the b-ketoester
13. Bromination at the tertiary C4 position proceeded in
reasonable yield (53%) forming the bromide 14, which
was duly converted to the thioacetate 15 in 48% yield.
Treatment of the thioacetate 15 with potassium
hydroxide delivered the derivitised thiolactone 16
(20%).
3. Results and discussion
All compounds were assayed in vitro for their activity
against the malarial parasite, P. falciparum (Table 1).
Furthermore, the compounds were also assayed against

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S. M. Jones et al. / Bioorg. Med. Chem. 12 (2004) 683–692
Table 1. Activities of compounds against P. falciparum cultured in red blood cells; T. brucei trypomastigotes; and T. cruzi amastigotes cultured in
rat skeletal myoblasts
Compd
R1
R2
R3
P. falciparum
IC50 (mM)
T. cruzi
IC50 (mM)
T. brucei
IC50 (mM)
1
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
Me
Me
Et
Isoprenoid
H
143
>347
233
>290
195
139
153
40
>427
>624
568
>522
>408
127
>350
68
68
28
195
165
54
256
>624
357
288
408
112
194
190
171
62
71
157
97
47
21
132
>568
6
6a
6b
6c
6d
7a
7b
7c
7d
7e
7f
H
H
H
Pr
Bn
Me
Me
Me
Me
Me
Et
Et
Et
Et
Pr
Bn
Me
Et
Me
Me
Bu
Hexyl
Octyl
Geranyl
Decyl
Hexadecyl
Hexyl
Octyl
Decyl
Hexadecyl
Decyl
Decyl
H
36
25
61
54
15
19
10
50
7g
7h
7i
43
56
64
7j
7k
11a
11b
12a
12b
16
>316
72
65
>568
164
72
Decyl
Et
Et
H
Decyl
Hexadecyl
Decyl
153
130
8
35
71
49
77
Me
Standards: For P. falciparum, chloroquine, IC50=0.126 mM; artemisinin, IC50=0.005 mM ; For T. brucei, melarsoprol, IC50=0.005 mM ; For T.
cruzi, benznidazole, IC50=1.68 mM. The IC50 values are the means of four values of two independent assays done in duplicate. They were deter-
mined by linear interpolation between the two adjacent drug concentrations above and below the 50% incorporation line.
emphasised by considering the intermediate compounds
6a–d and 11a, all of which showed a marked decrease in
inhibition (195 to >347) in comparison to 1.
analogues 7h and 7i gave the best inhibition, showing 10
and 8-fold increases in inhibition over TLM respec-
tively. A further trend was also observed, analogues 7f–i
all displayed enhanced inhibition towards the parasite in
comparison to analogues 7a–e bearing a methyl sub-
stituent at C3 of the ring. This trend is highlighted when
comparing the activities of analogues 7b (153 mM) and
7g (54 mM). Compound 7g shows a 3-fold improve-
ment in inhibition over 7b. However with the more
active compounds, there is a much smaller improvement
in activity on addition of an ethyl group at the C3
position [compare 7h (15 mM) with 7d (36 mM); and 7i
(19 mM) with 7e (25 mM)]. Clearly, the ethyl substituent
is having a positive influence on the overall activity of
the inhibitors. This led us to probe the C3 position fur-
ther. Indeed, by extending the ethyl chain to a propyl
group, and placing a decyl side chain at the C5 position
(analogue 7j) an IC50 of 10 mM was achieved, thus giv-
ing a 14-fold increase in activity over TLM. Increasing
the propyl tether to a butyl derivative 16 resulted in a
decrease in activity. A similar result was also obtained
with the benzyl analogue 7k, which showed an IC50 of
50 mM. Seemingly, it is plausible that we have reached
an optimal chain length of three carbons at the C3
position, beyond which activity is diminished. To lend
support to this assertion we were able to dock analogue
7j into the E. coli KAS I using FlexX software (Figs 2
and 3). The propyl group appears to fit snugly into the
hydrophobic pocket with the terminus of the propyl
chain in close proximity to the Cys 163. This same
amino acid is also present in the P. falciparum KAS1/
2.25
Recently, Price et al. have solved the crystal structure of
TLM bound to Escherichia coli KAS I.24,25 From this
study a number of key interactions have been identified
involving the thiolactone ring with the protein’s active
site. Specifically, the carbonyl oxygen at the C2 position
binds with two histidines (His 298 and His 333). The
isoprenoid moiety appears to occupy a hydrophobic
crevice and is sandwiched between two pairs of amino
acids (Gly 391 and Phe 392; Ala 271 and Pro 272).
Interestingly, alignment of P. falciparum KAS1/2 with
the E. coli KAS I indicates that these four amino acids
are retained in the P. falciparum sequence (Gly 403 and
Phe 404; Ala 274 and Pro 275).25 Price et al.,25 further
observed that this crevice was not optimally filled by the
isoprenoid side chain. Our findings lend support to this
observation, and would suggest that alkyl chains of ten
carbons and above better occupy this cleft, therefore
increasing inhibition.
Price et al.25 made a detailed study of the crystal struc-
ture of E. coli KAS I, and found that the two ring
methyl substituents, at the C3 and C5 positions, are
accommodated in hydrophobic pockets (the C3 methyl
in a pocket bounded by Phe 229, Phe 392 and Thr 300;
the C5 pocket by Pro 272 and Gly 305). The analogues
7f–i, all possessing an ethyl substituent at the C3 posi-
tion of the ring, all showed greater inhibition than TLM
against P. falciparum. Once again, the longer chained

S. M. Jones et al. / Bioorg. Med. Chem. 12 (2004) 683–692
687
activities for analogues 11b (72 mM) and 7h (15 mM).
Clearly, modification of the methyl substituent at the C5
position does not appear as influential to the overall
activity of the analogues as modification at the C3
position.
All our compounds were also assayed in vitro against
the T. brucei parasite, which is the cause of human
sleeping sickness and livestock disease in Africa.
Recently, Englund26 identified thiolactomycin as a pro-
mising lead for antitrypanosomal drug development. T.
brucei requires large amounts of myristate for synthesis
of glycosyl phosphatidylinositol anchored variable sur-
face glycoprotein. Thiolactomycin was shown to effec-
tively inhibit trypanosomal myristate synthesis in vitro
with a reported IC50 of ꢂ150 mM.26 A number of our
compounds showed inhibition greater than thiolacto-
mycin 1 (IC50 256 mM in our assays), with the best two
[analogues 11b (6 mM) and 16 (8 mM)] showing a 42-fold
improvement in activity (Table 1). The most active
analogue (7j) against the malaria parasite also showed
improved inhibition against T. brucei, registering an
IC50 of 21 mM.
Figure 2. Compound 7j docked into the E. coli KAS I. Part of the
3-propyl group is obscured.
In vitro assays against T. cruzi, the causative organism
of Chagas disease, showed that thiolactomycin 1 was
inactive with an IC50 of >427 mM (Table 1). Several of
the analogues tended to show enhanced activity towards
the T. cruzi parasite over thiolactomycin. Analogue 7e
showed the greatest inhibition with an IC50 of 28 mM.
Finally, none of the compounds showed any appreci-
able activity against the Leishmania donovani parasite.
The lack of activity against Leishmania may be due to
the parasite being an intracellular parasite dwelling
within a parasitophorous vacuole within the host cell,
significantly reducing accessibility of compounds.
4. Conclusion
We have reported the design and synthesis of a number
of thiolactomycin analogues as agents against P. falci-
parum, a causative organism of malaria. The com-
pounds were designed with the intention of targeting the
Type II FAS utilised for fatty acid biosynthesis. Several
analogues showed inhibitory effects greater than thio-
lactomycin 1, with the best compound (7j) showing a
14-fold increase in activity (10 mM). Furthermore, all
compounds were assayed against three other parasitic
protozoa, T. cruzi, T. brucei and L. donovani. Varied
activities were found against the T. brucei and T. cruzi
parasites. However, the analogues showed no appreci-
able activity against L. donovani. Although the inhibi-
tory effects of these analogues are only in the
micromolar range, these show significantly better activ-
ity than thiolactomycin. This study has demonstrated
the scope that exists for the design of new fatty acid
biosynthesis inhibitors with enhanced activity against
the parasites which cause malaria, African trypanoso-
miasis and Chagas disease. Significantly improved inhi-
bitors could offer a valuable new way of controlling
these important diseases.
Figure 3. Inhibitor 7j in relation to the surface of E. coli KAS I.
In an attempt to improve the hydrophobic interactions
of the methyl substituent at the C5 position, an ethyl
substituent was introduced in place of the methyl group.
Both analogues 12a (65 mM) and 12b (35 mM) showed
improved inhibition over TLM (Table 1). However, a
direct comparison of these analogues with there methyl
counterparts [compare 12a (65 mM) with 7d (36 mM);
and 12b (35 mM) with 7e (25 mM)] suggests that the
presence of the ethyl substituent at C5 serves only to
reduce the activity of the inhibitors. Likewise, removal
of the C5 methyl substituent (analogue 11b) also
appears to reduce the inhibitory effects of the analogue.
This is highlighted with a direct comparison of the

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S. M. Jones et al. / Bioorg. Med. Chem. 12 (2004) 683–692
5. Experimental
15.17 mmol), 6b was obtained as a white solid
(1.07 g, 89%); mp 75–76 ꢁC, nMAX/cmꢀ1 3188, 2923,
2869, 1563, 1291 and 1184; dH (acetone-d6) 1.94 (3H, t,
J=7.2, 20-CH3), 1.53 (3H, d, J=7.0, CH3), 2.18 (2H, q,
J=7.2, 10-CH2), 4.21 (1H, q, J=7.0, 5-CH) and
10.11 (1H, br s, OH); dC (acetone-d6) 13.4 (20-CH3),
17.1 (10-CH2), 20.2 (5-CH3), 43.4 (5-C), 117.1 (3-C),
179.2 (4-C) and 194.8 (CO); MS (ESꢀ) m/z 156.7
(MꢀH,ꢀ 100%); HRMS (ESꢀ) (MꢀH)ꢀ C7H9O2S
requires 157.0324, found 157.0323. Anal. calcd for
C7H10O2S.0.1H2O: C, 52.5; H, 6.4. Found: C, 52.6; H,
6.4%.
Melting points were determined using a Gallenkamp
melting point apparatus and are uncorrected. Infrared
spectra were recorded as thin films for liquid samples, or
as Nujol mulls for solid samples, on a Perkin–Elmer
1600 series FTIR spectrophotometer using sodium
chloride plates. 1H and 13C NMR spectra were recorded
on a Bruker Advance DPX300 spectrometer operating
at 300 and 75 M Hz, respectively, with tetramethylsilane
as internal standard, using deuterated chloroform pur-
chased from Goss unless stated otherwise. Low-resolution
mass spectra, that is, electrospray [ES], were recorded
using a fisons VG Platform II spectrometer. High-reso-
lution spectra were obtained on a VG ZAB spectro-
meter from the EPSRC Mass Spectrometry Service at
Swansea University, UK. Microanalyses were obtained
from the analytical and chemical consultancy services
MEDAC LTD. All reactions were performed in pre-
dried apparatus under an atmosphere of nitrogen unless
otherwise stated. Solvents and reagents were purchased
from chemical companies and used without further
purification. Dry solvents were generally purchased in
sure sealed bottles stored over molecular sieves. Thin-
layer chromatography (tlc) was performed on Merck
silica gel 60F254 plates. Column chromatography was
carried out using Fisons matrix silica 60 (35–70 micron).
5.4. 4-Hydroxy-5-methyl-3-propyl-2,5-dihydro-2-thiophene
(6c)
As described in procedure A, starting from 5c (3.50 g,
14.22 mmol) and potassium hydroxide (1.59 g, 28.44
mmol), 6c was obtained as a colourless solid (1.04 g,
42%); mp 49–50 ꢁC, dH (acetone-d6) 0.91 (3H, t, J=7.4,
30-CH3), 1.41–1.54 (2H, m, 20-CH2), 1.65 (3H, d, J=7.0,
CH3), 2.22 (2H, t, J=7.5, 10-CH2) and 4.29 (1H, q,
J=7.0, 5-CH); dC (acetone-d6) 18.5 (30-CH3), 24.5
(CH3), 26.6 (20-CH2), 29.8 (10-CH2), 47.6 (5-CH), 119.8
(3-C), 183.9 (4-C) and 199.1 (2-CO); MS (ES00) m/z
170.9 (MꢀH,ꢀ 100%); HRMS (ES+) (M+NH4)+
C8H12O2S requires 190.0896, found 190.0895. Anal.
calcd for C8H12O2S: C, 55.8; H, 7.0. Found: C, 55.4; H,
7.1%.
5.1. Cyclisation, general procedure A
Potassium hydroxide (2.0 equiv) in water (10 mL) was
added dropwise to a stirred solution of the thioacetate
(1.0 equiv) in ethanol (20 mL) at ambient temperature.
The resulting solution was warmed to 40 ꢁC and stirred
for 3 h. The ethanol was then evaporated and water (ꢂ10
mL) added. The aqueous layer was washed with diethyl
ether (2ꢃ10 mL) and acidified to ph 1 with the addition
of 2 M HCl (ꢂ10 mL). The aqueous layer was extracted
with diethyl ether. The organic layers were washed with
brine, dried over anhydrous magnesium sulfate and the
solvent removed in vacuo. The crude residue was pur-
ified by flash column chromatography (5–20% ethyl
acetate in hexanes). The coupling constants (J) are in Hz.
5.5. 3-Benzyl-4-hydroxy-5-methyl-2,5-dihydro-2-thiophene
(6d)
As described in procedure A, starting from 5d (6.00 g,
20.40 mmol) and potassium hydroxide (2.69 g, 40.80
mmol), 6d was obtained as a white powder (1.32 g,
29%); mp 139–141 ꢁC, dH (acetone-d6) 1.59 (3H, d,
J=6.9, CH3), 3.50 (2H, s, CH2Ph), 4.28 (1H, q, J=6.9,
5-CH), 7.08–7.21 (5H, m, ArH) and 10.33 (1H, br s,
OH); dC (acetone-d6) 20.2 (CH3), 29.3 (CH2Ph), 43.6 (5-
CH), 115.0 (3-C), 127.1 (ArCH), 129.4 (ArCH), 129.5
(ArCH), 140.9 (ArC), 180.2 (4-C) and 194.6 (CO); MS
(ESꢀ) m/z 219.5 (MꢀH,ꢀ 100%); HRMS (ESꢀ)
(MꢀH)ꢀ C12H11O2S requires 219.0480, found 219.0484.
Anal. calcd for C12H12O2S.0.1H2O: C, 64.9; H, 5.5.
Found: C, 64.7; H, 5.5%.
5.2. 4-Hydroxy-3,5-dimethyl-2(5H)-thiophenone (6a)
As described in procedure A, starting from 5a (1.0 g,
4.58 mmol) and potassium hydroxide (0.5 g, 9.16 mmol),
6a was obtained as a white solid (0.60 g, 90%); mp 131–
132 ꢁC, nMAX/cmꢀ1 3197, 2923, 2878, 1555, 1291 and
1186; dH (acetone-d6) 1.66 (3H, d, J=7.0, 5-CH3), 1.74
(3H, s, 3-CH3), 4.32 (1H, q, J=7.0, 5-H) and 10.22 (1H,
br s, OH); dC (acetone-d6) 12.6 (3-CH3), 24.5 (5-CH3),
47.9 (4-CH), 115.5 (3-C), 183.6 (4-C) and 199.3 (CO);
MS (ES-) m/z 142.6 (MꢀH,ꢀ 100%); HRMS (ES+)
(M+H)+ C6H9O2S requires 145.0323, found 145.0321.
Anal. calcd for C6H8O2S.0.1H2O: C, 49.4; H, 5.7.
Found: C, 49.3; H, 5.4%.
5.6. 5-Ethyl-4-hydroxy-3-methyl-2,5-dihydro-2-thiophene
(11a)
As described in procedure A, starting from 10a (5.36 g,
23.10 mmol) and potassium hydroxide (2.59 g, 46.20
mmol), 11a was obtained as a pale yellow solid (1.15 g,
32%); mp 95–96 ꢁC, dH (acetone-d6) 0.89 (3H, t, J=7.4,
200-CH3), 1.58 (3H, s, 10-CH3), 1.61–1.68 (1H, m, 100-
CHAHB), 2.07–2.17 (1H, m, 100-CHAHB) and 4.14 (1H,
dd, J=6.2 and 1.1, 5-CH); dC (acetone-d6) 8.1 (7-CH3),
11.4 (3-CH3), 27.0 (6-CH2), 50.8 (5-CH), 112.0 (3-C),
178.0 (4-C) and 195.2 (2-CO); MS (ESꢀ) m/z 156.8
(MꢀH,ꢀ 100%); HRMS (ES+) (M+NH4)+
C7H14NO2S requires 176.0740; found 176.0740. Anal.
calcd for C7H10O2S: C, 53.1; H, 6.4. Found: C, 52.7; H,
6.4%.
5.3. 3-Ethyl-4-hydroxy-5-methyl-2(5H)-thiophenone (6b)
As described in procedure A, starting from 5b
(3.52 g, 7.58 mmol) and potassium hydroxide (1.70 g,

S. M. Jones et al. / Bioorg. Med. Chem. 12 (2004) 683–692
689
5.7. 5-Decyl-3-ethyl-4-hydroxy-2(5H)-thiophenone (11b)
to warm to ambient temperature and stirred overnight.
2 M HCl (10 mL) was added and the organic layer
separated. The aqueous layer was extracted with ethyl
acetate (3ꢃ10 mL). The combined organic solutions
were washed with water (2ꢃ20 mL) and brine (2ꢃ20
mL), then dried and evaporated. Purification by column
chromatography (20% ethyl acetate in hexanes) pro-
vided thiolactomycin 1 as a pale yellow oil (12 mg, 29%
yield), nMAX/cmꢀ1 3600, 1700, 1630, 1450, 1380, 1325,
1280 and 1100; 1H NMR d 1.79 (3H, s, CH3), 1.83 (3H,
s, CH3), 1.91 (3H, s, CH3), 5.15 (1H, d, J=11.1, 40-Ha),
5.33 (1H, d, J=17.2, 40-Hb), 5.62 (1H, s, 10-H) and 6.37
(1H, dd, J=17.2 and 11.1, 30-H); dC 7.6 (3-CH3), 12.1
(CH3), 29.9 (5-CH3), 58.6 (5-C), 110.8 (3-C), 114.0
(CH2), 129.8, 140.6, 141.1, 181.3 (4-C) and 198.6 (CO).
Spectroscopic data was identical to those recorded in
the literature.21
As described in procedure A, starting from 10b (870 mg,
2.43 mmol) and potassium hydroxide (270 mg, 4.86
mmol), 11b was obtained as a pale yellow solid (74 mg,
11%), mp 35–36 ꢁC, dH 0.88 (3H, t, J=6.8, 15-CH3),
1.04 (3H, t, J=7.4, 20-CH3), 1.26 (14H, m, 7ꢃCH2),
1.67–1.73 (2H, m, CH2), 2.24–2.31 (4H, m, 6-CH2 and
10-CH2) and 4.18 (1H, dd, J=9.3 and 2.6, 5-CH); dC
13.1 (15-CH3), 14.5 (20-CH3), 16.5, 23.1, 27.9, 29.6, 29.7,
29.8, 30.0, 32.3, 33.3 (all CH2), 50.1 (5-CH), 118.0 (3-C),
179.2 (4-C) and 199.9 (2-CO); MS (ESꢀ) m/z 283.1
(MꢀH,ꢀ 100%); HRMS (ES)+ (M+H)+ C16H29O2S
requires 285.1883, found 285.1892. Anal. calcd for
C16H28O2S.0.2H2O: C, 66.7; H, 9.9. Found: C, 66.8; H,
10.1%.
5.8. 3-Butyl-5-decyl-4-hydroxy-5-methyl-2(5H)-thiophe-
none (16)
5.11. 5-Hexyl-4-hydroxy-3,5-dimethyl-2(5H)-thiophenone
(7a)
As described in procedure A, starting from 15 (2.84 g,
7.39 mmol) and potassium hydroxide (0.82 g, 14.79
mmol), 16 was obtained as a pale yellow oil (0.47 g,
20%), dH 0.89–0.93 (6H, m, 15-CH3 and 40-CH3), 1.23–
1.37 (20H, m, 10ꢃCH2), 1.72 (3H, s, CH3), 1.93–1.96
(2H, m, 6-CH2) and 2.29 (2H, t, J=7.2, 10-CH2); dC
14.1, 14.3 (both CH3), 22.8, 22.9, 23.1, 24.4, 25.4, 25.5,
26.4, 26.6, 29.7, 29.8, 29.9, 30.0, 32.0, 38.7 (all CH2),
58.4 (5-C), 115.6 (3-C), 181.7 (4-C) and 198.6 (2-CO);
MS (ESꢀ) m/z 325.4 (MꢀH,ꢀ 100%); HRMS (ESꢀ)
(MꢀH)ꢀ C19H33O2S requires 325.2196, found 325.2199.
As described in procedure B, starting from 6a (500 mg,
3.46 mmol), sodium hydride (160 mg, 4.16 mmol),
butyllithium (1.52 mL, 3.80 mmol) and bromohexane
(0.48 mL, 3.46 mmol), 7a was obtained as a colourless
solid (80 mg, 10%); mp 62–64 ꢁC, nMAX/cmꢀ1 3145,
2929, 1600, 1454, 1247 and 1010; dH 0.87 (3H, t, J=6.5,
60-CH3), 1.26–1.34 (8H, m, 4ꢃCH2), 1.66 (3H, s, CH3),
1.74 (3H, s, CH3) and 1.85 (2H, t, J=8.0, 10-CH2); dC
7.92 (60-CH3), 14.4 (3-CH3), 22.9 (CH2), 25.5 (5-CH3),
26.4, 29.6, 32.0, 38.9 (all CH2), 58.0 (5-C), 110.9 (3-C),
183.6 (4-C) and 198.4 (CO); MS (ESꢀ) m/z 227.2
(MꢀH,ꢀ 100%); HRMS (ESꢀ) (MꢀH)ꢀ C12H19O2S
requires 227.1106, found 227.1106. Anal. calcd for
C12H20O2S.0.2H2O: C, 62.1; H, 8.9. Found: C, 61.9; H,
8.9%.
5.9. Alkylation, general procedure B
Thiophenone (1.0 equiv) was added portionwise to a
stirred suspension of sodium hydride (1.2 equiv) in dry
THF (4 mL) and DMPU (4 mL) at ambient tempera-
ture. The suspension was stirred for 0.5 h, then cooled
to ꢀ78 ꢁC and butyllithium (2.5 M solution in hexanes,
1.1 equiv) added dropwise. After stirring for 10 min, the
mixture was allowed to warm to ambient temperature
and stirred for 1 h. Once again the mixture was cooled
to ꢀ78ꢁ and the halide (1.0 equiv) added dropwise.
After stirring for 15 min, the mixture was allowed to
warm to ambient temperature and stirred overnight.
The reaction was quenched by the addition of 2 M HCl
(10 mL) and the organic layer separated. The aqueous
layer was then extracted with ethyl acetate (3ꢃ10 mL).
The organic layers were washed with brine, dried over
anhydrous magnesium sulfate and the solvent removed
in vacuo. The crude residue was purified by flash col-
umn chromatography (0–20% ethyl acetate in hexanes).
5.12. 4-Hydroxy-3,5-dimethyl-5-octyl-2(5H)-thiophenone
(7b)
As described in procedure B, starting from 6a (500 mg,
3.46 mmol), sodium hydride (160 mg, 4.16 mmol),
butyllithium (1.52 mL, 3.80 mmol) and 1-iodooctane
(1.05 mL, 3.46 mmol), 7b was obtained as a pale yellow
solid (108 mg, 12%); mp 81–83 ꢁC, nMAX/cmꢀ1 1611
and 1076; dH 0.97 (3H, t, J=6.9, 80-CH3), 1.31 (10H, m,
5ꢃCH2), 1.75 (3H, s, CH3), 1.83 (3H, s, CH3) and 1.89–
1.94 (2H, m, 10-CH2); dC 7.9 (80-CH3), 14.5 (3-CH3),
23.0 (CH2), 25.6 (5-CH3), 26.3, 29.6, 29.8, 29.9, 32.2,
38.8 (all CH2), 58.5 (5-C), 110.8 (3-C), 181.2 (4-C) and
198.3 (CO); MS (ESꢀ) m/z 255.2 (MꢀH,ꢀ 100%);
HRMS (ES+) (M+H)+ C14H25O2S requires 257.1570,
found 257.1568. Anal. calcd for C14H24O2S.0.1H2O: C,
65.1; H, 9.4. Found: C, 65.1; H, 9.6%. Spectroscopic
data was identical to those recorded in the literature.17
5.10. Thiolactomycin 1
Butyllithium (1.6 mL of a 2.5 M solution in hexanes,
4.01 mmol) was added dropwise to a stirred suspension
of the phosphonium salt (710 mg, 2.00 mmol) in dry
THF (8 mL) at ꢀ78 ꢁC. The resulting dark red solution
was allowed to warm to ambient temperature and stir-
red for 1 h. The solution was then cooled to ꢀ78 ꢁC and
the aldehyde (430 mg, 2.00 mmol) added dropwise. A
light brown suspension formed, which was then allowed
5.13. 5-[(2E)-3,7-Dimethyl-2,6-octadienyl]-4-hydroxy-3,5-
dimethyl-2(5H)-thiophenone (7c)
As described in procedure B, starting from 6a (500 mg,
3.46 mmol), sodium hydride (160 mg, 4.16 mmol),
butyllithium (1.52 mL, 3.80 mmol) and geranyl bromide
(0.69 g, 3.46 mmol), 7c was obtained as a thick yellow

690
S. M. Jones et al. / Bioorg. Med. Chem. 12 (2004) 683–692
oil (160 mg, 16%); nMAX/cmꢀ1 2972, 2927, 1600, 1450,
1383, 1280 and 969; dH 1.61 (3H, s, CH3), 1.64 (3H, s,
CH3), 1.66 (3H, s, CH3), 1.69 (3H, s, CH3), 1.75 (3H, s,
CH3), 2.04–2.12 (4H, m, 2ꢃCH2), 2.52–2.69 (2H, m,
CH2), 5.08 (1H, m, CH) and 5.18 (1H, m, CH); dC 7.9
(CH3), 16.9 (3-CH3), 18.1 (CH3), 25.2 (5-CH3), 26.1
(CH3), 26.8 (CH2), 37.7, 40.2 (both CH2), 58.2 (5-C),
110.7 (3-C), 119.2, 124.3 (both CH), 132.2, 140.5 (both
C), 180.2 (4-C) and 203.2 (CO); MS (ESꢀ) m/z 279.1
(MꢀH,ꢀ 100%); HRMS (ESꢀ) (MꢀH)ꢀ C16H23O2S
requires 279.1419, found 279.1416. Spectroscopic data
was identical to those recorded in the literature.17
29.5, 31.9 and 38.8 (all CH2), 58.1 (5-C), 116.9 (3-C),
180.4 (4-C) and 197.5 (2-CO); MS (ESꢀ) m/z 240.9
(MꢀH,ꢀ 100%). HRMS (ESꢀ) (MꢀH)ꢀ C13H21O2S
requires 241.1262, found 241.1259. Anal. calcd for
C13H22O2S.0.2H2O: C, 63.5; H, 9.2. Found: C, 63.6; H,
9.3%.
5.17. 3-Ethyl-4-hydroxy-5-methyl-5-octyl-2(5H)-thiophe-
none (7g)
As described in procedure B, starting from 6b (300 mg,
1.89 mmol), sodium hydride (91 mg, 2.27 mmol), butyl-
lithium (1.31 mL of a 1.57 M solution in hexanes, 2.07
mmol) and 1-iodooctane (0.31 mL, 1.89 mmol), 7g was
obtained as a pale yellow oil (40 mg, 18%); dH 0.92 (3H,
t, J=6.7, 800-CH3), 1.09 (3H, t, J=7.5, 200-CH3), 1.30
(12H, m, 6ꢃCH2), 1.72 (3H, s, CH3), 1.83–1.86 (2H, m,
10-CH2), 2.28 (2H, q, J=7.5, 10-CH2) and 7.68 (1H, br s,
OH); dC 13.3, 14.5 and 16.4 (all (CH3), 23.0, 25.4, 26.4,
29.5, 29.6, 29.9, 32.2 and 38.8 (all CH2), 57.8 (5-C),
116.9 (3-C), 179.9 (4-C) and 196.7 (5-C); MS (ESꢀ)
m/z 269.1 (MꢀH,ꢀ 100%); HRMS (ESꢀ) (MꢀH)ꢀ
C15H25O2S requires 269.1576, found 269.1578.
5.14. 5-Decyl-4-hydroxy-3,5-dimethyl-2(5H)-thiophenone
(7d)
As described in procedure B, starting from 6a (500 mg,
3.46 mmol), sodium hydride (160 mg, 4.16 mmol),
butyllithium (1.52 mL, 3.80 mmol) and 1-iodododecane
(0.73 mL, 3.46 mmol), 7d was obtained as a white solid
(110 mg, 10%); mp 74–75 ꢁC, nMAX/cmꢀ1 3130, 2947,
1592, 1486, 1270, 1170 and 989; dH 0.82 (3H, t, J=6.6,
100-CH3), 1.20 (16H, m, 8ꢃCH2), 1.62 (3H, s, CH3),
1.71 (3H, s, CH3) and 1.80–1.85 (2H, m, 10-CH2); dC 7.9
(100-CH3), 14.5 (3-CH3), 23.1 (CH2), 25.6 (5-CH3), 26.3,
29.7, 29.8, 29.9, 30.0, 32.3, 38.8 (all CH2), 58.5 (5-C),
110.8 (3-C), 181.1 (4-C) and 198.3 (2-CO); MS (ESꢀ)
m/z 283.0 (MꢀH,ꢀ 100%); HRMS (ES+) (M+NH4)+
C16H32NO2S requires 302.2154, found 302.2149. Anal.
calcd for C16H28O2S.0.2H2O: C, 66.7; H, 9.9. Found: C,
66.7; H, 9.9%.
5.18. 5-Decyl-3-ethyl-4-hydroxy-5-methyl-2(5H)-thiophene
(7h)
As described in procedure B, starting from 6b (300 mg,
1.89 mmol), sodium hydride (91 mg, 2.27 mmol), butyl-
lithium (1.31 mL of a 1.57 M solution in hexanes, 2.07
mmol) and 1-iododecane (0.40 mL, 1.89 mmol), 7h was
obtained as a colourless solid (125 mg, 22%), mp 36–
38 ꢁC, dH 0.93 (3H, t, J=6.8, 1000-CH3), 1.10 (3H, t,
J=7.5, 20-CH3), 1.30 (14H, m. 7ꢃCH2), 1.71 (3H, s,
OCH3), 1.89 (2H, m, CH2), 2.00 (2H, m, CH2), 2.25
(2H, q, J=7.5, 10-CH2) and 6.85 (1H, br s, OH); dC
13.3, 14.5 and 16.5 (all CH3), 23.1, 25.5, 26.4, 29.6, 29.7,
29.8, 29.9, 30.0, 32.3 and 38.8 (all CH2), 57.9 (5-C),
117.0 (3-C), 179.7 (4-C) and 198.2 (2-C); MS (ESꢀ)
m/z 297.3 (MꢀH,ꢀ 100%); HRMS (ESꢀ) (MꢀH)ꢀ
C17H29O2S requires 297.1889, found 297.1888. Anal.
calcd for C17H30O2S: C, 68.4; H, 10.1. Found: C, 68.1;
H, 10.3%.
5.15. 5-Hexadecyl-4-hydroxy-3,5-dimethyl-2(5H)-thiophe-
none (7e)
As described in procedure B, starting from 6a (500 mg,
3.46 mmol), sodium hydride (160 mg, 4.16 mmol),
butyllithium (1.52 mL, 3.80 mmol) and 1-bromohexa-
decane (1.05 mL, 3.46 mmol), 7e was obtained as a
white solid (87 mg, 7%), mp 56–57 ꢁC, nMAX/cmꢀ1
2917, 2848, 1573, 1284, 1226 and 1145; dH 0.98 (3H, t,
J=6.9, 160-CH3), 1.36 (28H, m, 14ꢃCH2), 1.77 (3H, s,
CH3), 1.85 (3H, s, CH3) and 1.95 (2H, t, J=7.9, 10-
CH2); dC 7.9 (160-CH3), 14.5 (3-CH3), 23.1 (CH2), 25.6
(5-CH3), 26.3, 29.7, 29.8, 30.0, 30.1, 32.3, 38.9 (all CH2),
58.2 (5-C), 110.9 (3-C), 180.1 (4-C) and 190.3 (2-CO);
MS (ESꢀ) m/z 367.2 (MꢀH,ꢀ 100%); HRMS (ESꢀ)
(MꢀH)ꢀ C22H39O2S requires 367.2671, found:
367.2666. Anal. calcd for C22H40O2S: C, 71.7; H, 10.9.
Found: C, 71.7; H, 11.2%.
5.19. 3-Ethyl-5-hexadecyl-4-hydroxy-5-methyl-2(5H)-thio-
phenone (7i)
As described in procedure B, starting from 6b (300 mg,
1.89 mmol), sodium hydride (91 mg, 2.27 mmol), butyl-
lithium (1.31 mL of a 1.57 M solution in hexanes, 2.07
mmol) and 1-bromohexadecane (0.31 mL, 1.89 mmol),
7i was obtained as a colourless solid (160 mg, 22%), mp
86–87 ꢁC, nMAX/cmꢀ1 3183, 2916, 2848, 1580, 1471,
1330, 1251 and 1145; dH 1.00 (3H, t, J=6.6, 1600-CH3),
1.16 (3H, t, J=7.5, 20-CH3), 1.37 (28H, m, 14ꢃCH2),
1.80 (3H, s, 5-CH3), 1.97–2.01 (2H, m, 100-CH2), 2.36
(1H, q, J=7.5, 10-CH2) and 8.04 (1H, br s, OH); dC
13.3, 14.5, 16.5, 20.7, 23.1, 25.5, 26.4, 29.8, 29.8, 29.9,
29.9, 30.0, 30.1, 32.3, 38.9, 57.9 (5-C), 117.1 (3-C), 179.5
(4-C) and 196.9 (2-CO); MS (ESꢀ) m/z 381.2 (MꢀH,ꢀ
100%); HRMS (ESꢀ) (MꢀH)ꢀ C23H41O2S requires
381.2828, found 381.2835. Anal. calcd for C23H42O2S:
C, 72.2; H, 11.1. Found: C, 72.3; H, 11.2%.
5.16. 3-Ethyl-5-hexyl-4-hydroxy-5-methyl-2(5H)-thiophe-
none (7f)
As described in procedure B, starting from 6b (400 mg,
2.53 mmol), sodium hydride (120 mg, 3.03 mmol),
butyllithium (1.77 mL of a 1.57 M solution in hexanes,
2.78 mmol) and 1-iodohexaane (0.37 mL, 2.53 mmol),
7f was obtained as a colourless oil (100 mg, 16%), dH
0.82 (3H, t, J=6.8, CH3), 0.98 (3H, t, J=7.5, 20-CH3),
1.21 (8H, m, 4ꢃCH2), 1.62 (3H, s, CH3), 1.80–1.83
(2H, m, CH2) and 2.19 (2H, q, J=7.5, 10-CH2); dC
13.3 (CH3), 14.4 (CH3), 16.5 (CH3), 23.0, 25.4, 26.3,

S. M. Jones et al. / Bioorg. Med. Chem. 12 (2004) 683–692
691
5.20. 5-Decyl-4-hydroxy-5-methyl-3-propyl-2,5-dihydro-
2-thiophene (7j)
5.24. Biological assays
5.24.1. Plasmodium falciparum. In vitro activity against
erythrocytic stages of P. falciparum was determined
As described in procedure B, starting from 6c (250 mg,
1.45 mmol), sodium hydride (69 mg, 1.74 mmol), butyl-
lithium (0.63 mL of a 2.5 M solution in hexanes, 1.59
mmol) and 1-iododecane (0.31 mL, 0.38 g, 1.45 mmol),
7j was obtained as a yellow oil (57 mg, 12%); dH 0.93
(6H, m, 2ꢃCH3), 1.29 (14H, m, 7ꢃCH2), 1.44–1.60 (2H,
m, CH2), 1.70 (3H, s, CH3), 1.92–1.94 (4H, m, 2ꢃCH2)
and 2.25 (2H, t, J=7.3, 10-CH2); dC 14.1, 14.5 (both
CH3), 21.8 (CH2), 23.0, 25.5, 26.0 (all CH2), 26.6 (CH3),
29.7, 29.8, 29.9, 30.0, 30.5, 34.5 and 38.8 (all CH2),
68.4 (5-C), 115.3 (3-C), 180.8 (4-C) and 196.8 (2-CO);
MS (ESꢀ) m/z 311.2 (MꢀH,ꢀ 100%); HRMS (ESꢀ)
(MꢀH)ꢀ C18H32O2S requires 313.2196, found 313.2195.
Anal. calcd for C18H32O2S: C, 69.2; H, 10.3. Found: C,
69.3; H, 10.7%.
3
using a H-hypoxanthine incorporation assay,27,28 using
the chloroquine and pyrimethamine resistant K1 strain
and the standard drugs chloroquine (Sigma C6628) and
artemisinin (Sigma 36,159-3). Compounds were dissolved
in DMSO at 10 mg/mL and added to parasite cultures
incubated in RPMI 1640 medium without hypoxanthine,
supplemented with HEPES (5.94 g/L), NaHCO3 (2.1 g/L),
neomycin (100 U/mL), AlbumaxR (5 g/L) and washed
human red cells A+ at 2.5% haematocrit (0.3% para-
sitaemia). Serial doubling dilutions of each drug were pre-
pared in 96-well microtiter plates and incubated in a
humidified atmosphere at 37 ꢁC; 4% CO2, 3% O2, 93% N2.
3
After 48 h 50 mL of H-hypoxanthine (=0.5 mCi) was
added to each well of the plate. The plates were incu-
bated for a further 24 h under the same conditions. The
plates were then harvested with a BetaplateTM cell har-
vester (Wallac, Zurich, Switzerland), and the red blood
cells transferred onto a glass fiber filter then washed
with distilled water. The dried filters were inserted into a
plastic foil with 10 mL of scintillation fluid, and counted
in a BetaplateTM liquid scintillation counter (Wallac,
Zurich, Switzerland). IC50 values were calculated from
sigmoidal inhibition curves using Microsoft Excel.
5.21. 3-Benzyl-5-decyl-4-hydroxy-5-methyl-2,5-dihydro-
2-thiophene (7k)
As described in procedure B, starting from 6d (250 mg,
1.13 mmol), sodium hydride (54 mg, 1.36 mmol), butyl-
lithium (0.49 mL of a 2.5 M solution in hexanes, 1.24
mmol) and 1-iododecane (0.24 mL, 0.30 g, 1.13 mmol),
7k was obtained as a yellow oil (16 mg, 4%); dH 0.85
(3H, t, J=6.8, 100-CH3), 1.20–1.21 (16H, m, 8ꢃCH2),
1.58 (3H, s, CH3), 1.79 (2H, t, J=7.8, 10-CH2), 3.52
(2H, s, CH2Ph) and 7.10–7.21 (5H, m, ArH); dC 14.5
(CH3), 23.1 (CH2), 25.5 (CH2), 26.4 (CH3), 29.3, 29.7,
29.8, 29.9, 30.0, 32.3 and 38.9 (all CH2), 57.9 (5-C),
114.0 (3-C), 127.3, 128.9 and 129.4 (all ArCH), 137.9
(ArC), 180.5 (4-C) and 196.1 (2-CO); MS (ESꢀ) m/z 359.3
(MꢀH,ꢀ 100%); HRMS (ESꢀ) (MꢀH)ꢀ C22H31O2S
requires 359.2045, found 359.2048.
5.24.2. Trypanosoma cruzi. Rat skeletal myoblasts (L-6
cells) were seeded in 96-well microtiter plates at 2000
cells/well/100 mL in RPMI 1640 medium with 10% FBS
and 2 mM l-glutamine. After 24 h, 5000 trypomasti-
gotes of T. cruzi [Tulahuen strain C2C4 containing the
b-galactosidase (Lac Z) gene] were added in 100 mL per
well with 2ꢃ of a serial drug dilution. The plates were
incubated at 37 ꢁC in 5% CO2 for 4 days. Then the
substrate CPRG/Nonidet was added to the wells. The
colour reaction, which developed during the following
2–4 h, was read photometrically at 540 nm. From the
sigmoidal inhibition curve IC50 values were calculated.
5.22. 5-Decyl-5-ethyl-4-hydroxy-3-methyl-2,5-dihydro-2-
thiophene (12a)
As described in procedure B, starting from 11a (250 mg,
1.58 mmol), sodium hydride (75 mg, 1.51 mmol), butyl-
lithium (0.69 mL of a 2.5 M solution in hexanes, 1.89
mmol) and 1-iododecane (0.33 mL, 0.38 g, 1.58 mmol),
12a was obtained as a yellow oil (34 mg, 14%); dH 0.90–
0.98 (6H, m, 2ꢃCH3), 1.29 (14H, m, 7ꢃCH2), 1.78 (3H,
s, CH3) and 1.89–2.00 (4H, m, 2ꢃCH2); dC 6.3 (CH3),
7.9 (CH3), 13.1 (CH3), 21.6, 23.6, 28.2, 28.3, 28.4, 28.5,
28.6, 29.7, 30.8, 36.6 (all CH2), 61.9 (5-C), 110.9 (3-C),
177.1 (4-C) and 191.2 (2-CO).
5.24.3. Trypanosoma brucei rhodesiense. Minimum
Essential Medium (50 mL) supplemented according to
Baltz et al.29 with 2-mercaptoethanol and 15% heat-
inactivated horse serum was added to each well of a 96-
well microtiter plate. Serial drug dilutions were added to
the wells. Then 50 mL of trypanosome suspension (T.b.
rhodesiense STIB 900) was added to each well and the
plate incubated at 37ꢁC under a 5% CO2 atmosphere for
72 h. 10 mL Alamar Blue (Trinova, Giessen, Germany)
was then added to each well and incubation continued for
a further 2–4 h.30 The plates are read in a microplate
fluorescence scanner (Spectramax Gemini XS by Mole-
cular Devices) using an excitation wavelength of 536 nm
and an emission wavelength of 588 nm. From the sigmoidal
inhibition curve IC50 values were calculated.
5.22.1. 5-Ethyl-hexadecyl-4-hydroxy-3-methyl-2,5-dihydro-
2-thiophene (12b)
As described in procedure B, starting from 11a (200 mg,
1.26 mmol), sodium hydride (60 mg, 1.51 mmol), butyl-
lithium (0.70 mL of a 2.0 M solution in hexanes, 1.38
mmol) and 1-bromohexadecane (0.38 mL, 0.38 g, 1.26
mmol), 12b was obtained as a yellow oil (57 mg, 12%);
d
H 0.98–1.06 (6H, m, 2ꢃCH3), 1.37 (26H, m, 13ꢃCH2),
Acknowledgements
1.85 (3H, s, CH3), 1,98–2.04 (4H, m, 2ꢃCH2) and 6.80
(1H, br s, OH). Anal. calcd for C23H42O2S: C, 72.2; H,
11.1. Found: C, 72.0; H, 11.4%.
We would like to acknowledge the Cardiff Partnership
Fund for financial support and the EPSRC National

692
S. M. Jones et al. / Bioorg. Med. Chem. 12 (2004) 683–692
Mass Spectrometry Service Centre Swansea for accurate
mass spectrometry.
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