Biphenylquinuclidines as inhibitors of squalene synthase and growth of parasitic protozoa

Article abstract of DOI:10.1016/j.bmc.2005.02.060

In this paper we describe the preparation of some biphenylquinuclidine derivatives and their evaluation as inhibitors of squalene synthase in order to explore their potential in the treatment of the parasitic diseases leishmaniasis and Chagas disease. The compounds were screened against recombinant Leishmania major squalene synthase and against Leishmania mexicana promastigotes, Leishmania donovani intracellular amastigotes and Trypanosoma cruzi intracellular amastigotes. Compounds that inhibited the enzyme, also reduced the levels of steroids and caused growth inhibition of L. mexicana promastigotes. However there was a lower correlation between inhibition of the enzyme and growth inhibition of the intracellular parasites, possibly due to delivery problems. Some compounds also showed growth inhibition of T. brucei rhodesiense trypomastigotes, although in this case alternative modes of action other than inhibition of SQS are probably involved.

Full text of DOI:10.1016/j.bmc.2005.02.060

Bioorganic & Medicinal Chemistry 13 (2005) 3519–3529
Biphenylquinuclidines as inhibitors of squalene synthase and
growth of parasitic protozoa
a
Silvia Orenes Lorente, Rosario Gomez, Carmen Jimenez, Simon Cammerer,
b
b
a
´
´
Vanessa Yardley,c Kate de Luca-Fradley,c Simon L. Croft,c Luis M. Ruiz Perez,b
Julio Urbina,d Dolores Gonzalez Pacanowskab and Ian H. Gilberta,*
aWelsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3XF, UK
b
´ ´
Instituto de Parasitologıa y Biomedicina ‘Lopez-Neyra’, Avda. del Conocimiento s/n, Parque Tecnologico de Ciencias de la Salud,
´
18100 Armilla, Granada, Spain
cDepartment of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street,
London WC1E 7HT, UK
d
´ ´ ´ ´ ´
Laboratorio de Quımica Biologica, Centro de Bioquımica y Biofısica, Instituto Venezolano de Investigaciones Cientıficas (IVIC),
Altos de Pipe, Km. 11, Carretera Panamericana, Caracas 1020, Venezuela
Received 5 November 2004; accepted 22 February 2005
Available online 6 April 2005
Abstract—In this paper we describe the preparation of some biphenylquinuclidine derivatives and their evaluation as inhibitors of
squalene synthase in order to explore their potential in the treatment of the parasitic diseases leishmaniasis and Chagas disease. The
compounds were screened against recombinant Leishmania major squalene synthase and against Leishmania mexicana promastig-
otes, Leishmania donovani intracellular amastigotes and Trypanosoma cruzi intracellular amastigotes. Compounds that inhibited
the enzyme, also reduced the levels of steroids and caused growth inhibition of L. mexicana promastigotes. However there was a
lower correlation between inhibition of the enzyme and growth inhibition of the intracellular parasites, possibly due to delivery
problems. Some compounds also showed growth inhibition of T. brucei rhodesiense trypomastigotes, although in this case alterna-
tive modes of action other than inhibition of SQS are probably involved.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
the abundant supply of cholesterol present in the mam-
malian hosts. There are differences in the enzymes in the
biosynthetic pathways of ergosterol and cholesterol, and
a number of enzymes in the ergosterol biosynthetic
pathway have been investigated as potential drug targets
for these organisms and have shown to have great prom-
ise. Thus C14a-demethylase,2–14 sterol 24-methyltrans-
ferase,2,7,15–21 HMGCoA reductase,22,23 squalene
epoxidase,4,24 squalene synthase and farnesyl pyrophos-
phate synthase,25,26 have been studied both individually
and in combination, with varying degrees of success.
Inhibitors of different steps of the pathway can be used
synergistically.
The different forms of leishmaniasis and Chagas disease
are caused by the protozoan parasites Leishmania spp.
and Trypanosoma cruzi, respectively. The diseases cause
high rates of mortality and morbidity, especially in tropi-
cal regions of the world. The current drugs available to
treat these conditions suffer from poor clinical efficacy,
toxicity and increasing problems due to resistance,1
hence there is urgent need for new drugs in this area.
The enzymes of the sterol biosynthesis pathway are
attractive targets for the specific treatment of these dis-
eases, because the aetiological agents for these diseases
require endogenous ergosterol and other 24-alkylated
sterols for growth and survival and are unable to use
We are interested in squalene synthase, which catalyzes
the condensation of two molecules of farnesyl pyrophos-
phate to produce squalene, the first committed step of
the sterol pathway (Fig. 1). This enzyme has been of
great interest as a potential drug target for inhibition
of cholesterol biosynthesis in humans.27 A number of
*
Corresponding author. Tel.: +44 29 2087 5800; fax: +44 29 2087
4149; e-mail: gilbertih@cf.ac.uk
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.02.060

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S. Orenes Lorente et al. / Bioorg. Med. Chem. 13 (2005) 3519–3529
2 molecules of
O
P
O
P
O
P
O
P
-O
O
O
-O
O
O
-
-
O
O
-
-
O
O
farnesyl pyrophosphate
presqualene synthase
NADPH
squalene
Figure 1. Condensation of two molecules of farnesyl pyrophosphate to produce squalene.
different classes of compounds have been investigated
against mammalian enzymes, including bisphospho-
nates, benzylamines, squalestatins and quinuclidine
derivatives.
nuclidines and investigate them as inhibitors of the L.
major enzyme and for their anti-parasitic activity with
the aim of carrying out some preliminary structure–
activity relationships.
One class of compounds of particular interest are the
arylquinuclidines. These compounds have been shown
to be inhibitors of mammalian SQS and are good start-
ing points for drug discovery. The arylquinuclidines are
protonated at physiological pH and are thought to
mimic a high energy intermediate carbocation interme-
diate in the reaction pathway. Recently the investigation
of 3-(biphenyl-4-yl)-3-hydroxyquinuclidine (BPQ-OH)
as an anti-parasitic has been reported.28 This compound
caused potent non-competitive inhibition of Leishmania
mexicana and T. cruzi SQS (Ki 12–62 nM), inhibition of
growth of L. mexicana promastigotes and T. cruzi epi-
mastigotes, and blockade of sterol biosynthesis at the
level of SQS. We decided to prepare some biphenylqui-
2. Chemistry
A series of arylquinuclidines was prepared according to
the method of Brown et al. (Scheme 1).29 The
biphenylquinuclidines were prepared by condensation
of quinuclidin-3-one with the corresponding lithium
biphenyl. Two biphenyl moieties were used: an
unsubstituted biphenyl moiety and a biphenyl moiety
substituted with a TBDMS protected hydroxy group.
The lithiated biphenyl reagents were obtained by halo-
gen–metal exchange using sec-BuLi. The resulting alco-
hols 4a and 4b were then dehydrated under acidic
conditions. Under these conditions, the TBDMS pro-
OH
(a)
O
R
N
N
Br
R
4a R = H
3
4b R = OTBDMS
2a R = H
2b R = OTBDMS
R
(b)
N
5a R = H
5b R = OH
(c)
Br
OH
Br
OTBDMS
1a
2b
Scheme 1. Reagents and conditions: (a) 2a,b, THF, sec-BuLi, ꢀ78 ꢁC, 5 min, then 1, ꢀ78 ꢁC, 30 min, then rt, 12 h; (b) p-TsOH, toluene, Dean–Stark
trap, reflux, 3 h; (c) TBDMSCl, imidazole, DMF, rt, 48 h.

S. Orenes Lorente et al. / Bioorg. Med. Chem. 13 (2005) 3519–3529
3521
Table 1. Inhibition of L. major SQS, and growth of L. donovani intracellular amastigotes, T. cruzi intracellular amastigotes and T. brucei rhodesiense
trypomastigotes by biphenylquinuclidines
Compound
Inhibition of L. major
SQS IC50 (lM)
L. donovani
T. cruzi
T. brucei rhod.
ED50 (lM)
KB cells
ED50 (lM)
ED50 (lM)
ED50 (lM)
4a
4b
5a
5b
0.013
>1
29.0
n.d.
74.3
9.7
0.36
9.7
20.8
0.098
1.8
76.7
3.1
0.243
0.096
34.8
22.7
>108
14.4
5.4
tecting group was removed from 4b to give the corre-
sponding hydroxy compound 5b.
3.2. Enzyme assays
The E. coli cells were lysed and the cell extract, enriched
in L. major SQS, was used for the assays. The assay was
conducted using radiolabelled FPP as substrate. Follow-
ing extraction of the mixture, the product (newly formed
squalene) was separated from unreacted substrate using
TLC. The band corresponding to squalene was assessed
for radioactivity, allowing an assessment of the amount
of conversion of FPP to squalene.
3. Biology
3.1. Cloning of the Leishmania major SQS
The L. major SQS was cloned and over-expressed in
Escherichia coli for enzyme assays. Full details of the
cloning and characterization of the enzyme will be
reported elsewhere. A double-truncated protein was
produced, lacking 16 residues at the N-terminus
and 40 at the C-terminus. The DNA from the LmSQS
gene was cloned into the pET28a vector and expressed
in E. coli BL21 (DE3) RP cells as a His-tagged fusion
protein.
Compound 4a (BPQ-OH) showed the most potent inhi-
bition of the enzyme with a IC50 of 13 nM (Table 1).
Dehydrating this compound to give 5a reduced the
activity by almost 20-fold, suggesting that either the hy-
droxyl group undergoes important interactions in the
active site, or that in this more rigid conformation, the
Table 2. Effects of 4a on the sterol composition of L. mexicana promastigotes
Sterol
Structure
Control
14.2
0.3 lM
1.0 lM
3.0 lM
Cholesterol
11.5
57.9
>99
HO
HO
HO
HO
HO
HO
Ergosta-8,24(241)-14-methyl-dien-3b-ol
Ergosta-5,7,24(241)-trien-3b-ol (5-dehydroepisterol)
Ergosta-7,24(241)-dien-3b-ol (episterol)
Cholesta-8,24-dien-3b-ol (zymosterol)
Cholesta-7,24-dien-3b-ol
9.4
56.8
15.6
1.9
4.1
70.3
14.1
n.d.
n.d.
n.d.
17.4
24.7
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
2.1
Sterols were extracted from cells exposed to the indicated compound concentration for 96 h; they were separated by silicic acid column chroma-
tography and analyzed by quantitative capillary gas–liquid chromatography and mass spectrometry. Composition is expressed as mass percentages.
n.d. is not detected.

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S. Orenes Lorente et al. / Bioorg. Med. Chem. 13 (2005) 3519–3529
Table 3. Effects of 4b on the sterol composition of L. mexicana promastigotes
Compound
Cholesterol
Structure
Control
14.2
1.0 lM
3.0 lM
11.9
14.9
HO
HO
Ergosta-8,24(241)-14-methyl-dien-3b-ol
Ergosta-5,7,24(241)-trien-3b-ol (5-dehydroepisterol)
Ergosta-7,24(241)-dien-3b-ol (episterol)
Cholesta-5,7,24-trien-3b-ol
9.4
56.8
15.6
n.d.
2.1
6.9
64.7
14.6
n.d.
1.9
4.1
63.5
12.9
2.0
HO
HO
HO
HO
Cholesta-7,24-dien-3b-ol
2.6
Sterols were extracted from cells exposed to the indicated compound concentration for 96 h; they were separated by silicic acid column chroma-
tography and analyzed by quantitative capillary gas–liquid chromatography and mass spectrometry. Composition is expressed as mass percentages.
n.d. is not detected.
biphenyl group is held in a less optimal conformation.
We also investigated the effect of adding a lipophilic
substituent to give 4b. This compound had essentially
no activity suggesting that there is a limitation to the size
of substituent that can be appended to compound 4a.
The analogue of 5a in which there is a hydroxyl group
on the end of the biphenyl substituent, compound 5b,
showed increased enzyme inhibition compared to 5a.
Compound 4b (Table 3) showed no effect on sterol com-
position at 3 lM, suggesting no inhibition of SQS at
these concentrations, whilst compound 5b showed an
activity comparable to that of compound 5a (Table 5).
3.4. Growth inhibition
Compounds were also investigated for growth inhibition
of various parasites (Fig. 2A–D). Against L. mexicana
promastigotes (the same species used for the studies on
sterol composition), compound 4a, had an MIC (mini-
mum inhibitory concentration defined as that required
for complete growth arrest) of 1 lM and caused cell lysis
after 24 h (Fig. 2A). Compound 5a, was more potent as
a growth inhibitor, consistent with a more significant
effect on sterol composition than compound 4a, with
an MIC of <1 lM (Fig. 2C). The TBDMS protected
compound 4b had no effect on L. mexicana cell
growth, which was again consistent with no effect on
the sterol composition (Fig. 2B). Compound 5b had a
similar effect on cell growth as compound 5a (Fig. 2D).
3.3. Studies on lipid composition
The effects of the compounds on the sterol composition
of L. mexicana promastigotes were investigated (Tables
2–5). By monitoring the sterol composition, it should be
possible to establish the effect of the compounds on the
sterol composition in parasites and hence investigate the
mechanism of action of compounds in cellular systems.
For compound 4a (Table 2), there was a dose-dependent
reduction in the relative content of the parasites endoge-
nous sterols (episterol and 5-dehydroepisterol). There
were no significant effects seen on sterol composition
at 0.3 lM concentration, some effect at 1 lM and almost
complete loss of endogenous sterols at 3 lM. This is
consistent with inhibition of ergosterol biosynthesis at
the stage prior to formation of lanosterol (in this case
suggesting inhibition of squalene synthase). A more sig-
nificant effect was seen with compound 5a (Table 4),
where there was complete loss of endogenous sterols at
1 lM.
The compounds were also investigated for their effect on
Leishmania donovani and T. cruzi intracellular amastig-
otes and for toxicity to a mammalian cell line. None
of the compounds showed significant inhibition of L.
donovani intracellular amastigotes. In contrast all the
compounds showed at least moderate growth inhibition
of T. cruzi. In particular, compound 4b showed a potent

S. Orenes Lorente et al. / Bioorg. Med. Chem. 13 (2005) 3519–3529
Table 4. Effects of 5a on the sterol composition of L. mexicana promastigotes
3523
Compound
Structure
Control
0.3 lM
1.0 lM
Cholesterol
14.2
14.3
>99
n.d.
HO
HO
Ergosta-8,24(241)-14-methyl-dien-3b-ol
Ergosta-5,7,24(241)-trien-3b-ol (5-dehydroepisterol)
Ergosta-7,24(241)-dien-3b-ol (episterol)
Cholesta-8,24-dien-3b-ol (zymosterol)
Cholesta-5,7,24-trien-3b-ol
9.4
56.8
15.6
1.9
3.6
51.6
9.5
n.d.
n.d.
n.d.
n.d.
n.d.
HO
HO
HO
HO
HO
n.d.
14.2
6.8
n.d.
2.1
Cholesta-7,24-dien-3b-ol
Sterols were extracted from cells exposed to the indicated compound concentration for 96 h; they were separated by silicic acid column chroma-
tography and analyzed by quantitative capillary gas–liquid chromatography and mass spectrometry. Composition is expressed as mass percentages.
n.d. is not detected.
inhibition of T. cruzi amastigote proliferation
(ED50 = 0.36 lM).
on the growth of L. mexicana promastigotes, whilst
compound 4a showed a slightly smaller effect on both
sterol composition and promastigote growth. Interest-
ingly compound 4a gave slightly more potent inhibition
of L. major SQS than 5a or 5b. Compound 4b had no
effect on sterol composition, promastigote growth or
showed no inhibition of the recombinant enzyme.
As a part of routine screening, compounds were also as-
sayed for their effect on the growth of T. brucei rhodes-
iense blood stream form trypomastigotes. These
compounds showed growth inhibition of this parasite;
in particular 4b was a very potent inhibitor of growth
(ED50 = 0.098 lM).
Despite the potent inhibition of SQS and of strong
inhibition of the growth of L. mexicana promastigotes,
none of the experimental compounds was able to inhi-
bit the growth of intracellular L. donovani amastigotes.
There could be several reasons for this. First the intra-
cellular forms of the Leishmania parasites are found
within a parasitophorous vacuole inside the host mac-
rophages. This means that to access the parasite, the
compounds have to cross the cell membrane of the
host cell, the membrane of the parasitophorus vacuole
and then the membrane of the parasites. In addition
the parasitophorous vacuole has a pH of about 5.5.
Possibly the compounds are fully charged at that pH
and cannot cross the final barrier constituted by the
parasiteÕs membrane. A second possibility is that the
parasite could scavenge required sterols from the host;
4. Discussion
In this paper we report the synthesis of several
biphenylquinuclidines, with activity against trypanoso-
matid parasites. Some of the compounds were potent
inhibitors of L. major SQS. The compounds also showed
effects on the sterol composition of L. mexicana para-
sites (consistent with inhibition of SQS in the parasite)
and on the growth of the L. mexicana promastigotes.
In general there was very good correlation between the
effect on sterol composition and growth of the prom-
astigotes. Thus compounds 5a and 5b showed the most
pronounced effects on both the sterol composition and

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S. Orenes Lorente et al. / Bioorg. Med. Chem. 13 (2005) 3519–3529
Table 5. Effects of 5b on the sterol composition of L. mexicana promastigotes
Compound
Cholesterol
Structure
Control
10.1
0.1 lM
0.3 lM
1.0 lM
9.9
14.2
>99
HO
HO
Ergosta-8,24(241)-14-methyl-dien-3b-ol
Ergosta-5,7,24(241)-trien-3b-ol (5-dehydroepisterol)
Ergosta-7,24(241)-dien-3b-ol (episterol)
Cholesta-5,7,24-trien-3b-ol
n.d.
70.4
19.5
n.d.
n.d.
1.8
70.3
13.7
2.3
n.d.
70.8
15.0
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
HO
HO
HO
HO
Cholesta-7,24-dien-3b-ol
2.0
Sterols were extracted from cells exposed to the indicated compound concentration for 96 h; they were separated by silicic acid column chroma-
tography and analyzed by quantitative capillary gas–liquid chromatography and mass spectrometry. Composition is expressed as mass percentages.
n.d. is not detected.
however many sterol biosynthesis inhibitors can pre-
vent growth of intracellular Leishmania amastigotes,
implying that scavenging of sterols from the host is
not a significant process or at least is not capable of
compensating for the inhibition of the synthesis of
endogenous sterols.
of ergosterol and other 24-alkyl sterols, which they pro-
duce themselves. The compounds tested produced a sig-
nificant inhibition in the growth of T. brucei blood
stream forms. In particular compound 4b was very po-
tent as a growth inhibitor, yet showed no activity against
the recombinant enzyme. This observation suggests that
these compounds have some other mode of action
against these parasites, rather than inhibition of SQS.
This merits further investigation, as compound 4b, in
particular, showed very potent growth inhibition of T.
brucei rhodesiense.
All compounds had significant inhibitory activity on
the growth of T. cruzi intracellular amastigotes. These
intracellular amastigotes are more accessible as whilst
they are intracellular parasites, they are not contained
within a vacuole, but develop freely in the host cellÕs
cytoplasm. Compound 4b was the most potent growth
inhibitor against this stage, although this compound
does not inhibit the L. major SQS. There are a number
of possible explanations for this: this specific com-
pound may have alternative mechanisms of action
independent of SQS; or that it can penetrate into the
host cells and be transformed in the intracellular envi-
ronment to the parental compound 4a; or there may be
subtle differences in the structure of the L. major and T.
cruzi SQS.
In conclusion, the biphenylquinuclidines had potent
inhibitory activity of L. major SQS, and block de novo
sterol synthesis in L. mexicana promastigotes associated
with growth inhibition of these cells. The analogues pre-
pared here showed weak or no inhibition of growth of
intracellular L. donovani amastigotes, but they were ac-
tive against the intracellular stages of T. cruzi, probably
due to differences in parasitic strategies of these
organisms.
The compounds were also investigated against T. brucei
rhodesiense bloodstream form trypomastigotes, as part
of routine screening. These forms do not appear to bio-
synthesize their own sterols and are thought to scavenge
cholesterol from the human host,30,31 whilst procyclics
(present in the insect vector) contain significant amounts
5. Experimental
5.1. Chemistry
Melting points were determined using a Gallenkamp
melting point apparatus and are uncorrected. Infrared

S. Orenes Lorente et al. / Bioorg. Med. Chem. 13 (2005) 3519–3529
3525
Figure 2. Growth inhibition of L. mexicana promastigotes (A) inhibition by compound 4a; (B) inhibition by compound 4b; (C) inhibition by
compound 4c; (D) inhibition by compound 5b.
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 chlo-
ride plates. 1H and 13C NMR spectra were recorded on a
Bruker Advance DPX300 spectrometer operating at 300
and 75 MHz, respectively, with tetramethylsilane as
internal standard, using deuterated chloroform pur-
chased from Goss unless stated otherwise. Assignments
for 13C spectra were made with the aid of the ACD Labs
software. Low resolution mass spectra that is electro-
spray, were recorded using a fisons VG Platform II spec-
trometer. High resolution spectra were obtained on a
VG ZAB spectrometer from the EPSRC Mass Spec-
trometry Service at Swansea University, UK. Micro-
analyses 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 re-
agents 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. Col-
umn chromatography was carried out using Fisons ma-
trix silica 60 (35–70 lm).
5.1.1. 4-Bromo-40[(tert-butyldimethylsilyl)oxy]biphenyl
(2b). A mixture of 4-bromo-40-hydroxybiphenyl (2 g,
8.03 mmol), tert-butyldimethylsilyl chloride (1.88 g,
12.04 mmol) and imidazole (0.82 g, 12.4 mmol) in
DMF (20 mL) was stirred at room temperature for
48 h. The reaction mixture was diluted with chloroform,
washed with water (3 · 50 mL), dried over sodium sul-
fate and reduced in vacuo to yield a white solid

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S. Orenes Lorente et al. / Bioorg. Med. Chem. 13 (2005) 3519–3529
(2.23 g, 76%); 1H NMR (300 MHz, CDCl3) d = 0.20
(6H, s, SiCH3), 0.99 (9H, s, C(CH3)3), 6.85 (2H, d
J = 9 Hz, 7-CH), 7.34–7.50 (9H, m, ar–CH); 13C NMR
(75 MHz, CDCl3) d = ꢀ3.9 (SiCH3), 18.7 (10-C), 26.1
(11-CH3), 120.9 (7-CH), 121.2 (70-CH), 128.4 (CH),
128.8 (CH), 132.2 (CH), 133.4 (CH), 140.2 (4-C), 156.0
(8-C).
CH), 127.9 (ar–CH), 128.2 (ar–CH), 129.4 (ar–CH),
135.2 (ar-C), 142.1 (ar-C), 143.5 (ar-C), 157.3 (13-C).
5.1.4. 3-(Biphenyl-4-yl)-2,3-dehydroquinuclidine (5a). 4-
Toluenesulfonic acid (0.492 g, 2.6 mmol) and 2 (0.24 g,
0.86 mmol) were heated under reflux in toluene
(50 mL) for 3 h. using a Dean–Stark water separator.
The toluene was evaporated and the residue dissolved
in 1 M NaOH (50 mL). The aqueous mixture was ex-
tracted with chloroform (2 · 50 mL) and the organic
layer was dried and concentrated to yield a white solid
(0.083 g, 37%); Rf 0.52 (20% MeOH/CHCl3); m/z
(ES+) 261 (M+, 100%); HRMS calculated for C19H20N
([M+H]+) 262.1595, found 262.1596; 1H NMR
(300 MHz, CDCl3) d 1.65 (2H, m, 8-CH2), 1.85 (2H,
m, 5-CH2), 2.72 (2H, m, 6-CH2), 3.01 (2H, m, 7-CH2),
3.27 (1H, s, 4-CH), 6.94 (1H, s, 2-CH), 7.31–7.67 (9H,
m, ar–CH); 13C NMR (75 MHz, CDCl3) d 28.7 (CH2),
29.6 (4-CH), 49.6 (CH2), 126.6 (2-CH), 127.4 (ar–CH),
127.8 (ar–CH), 129.2 (ar–CH), 136.2 (9-C), 141.1 (10-
C), 146.7 (11-C).
s
5.1.2. 3-(Biphenyl-4-yl)-3-hydroxyquinuclidine (4a). Bu-
Li in cyclohexane (5 mL, 6.4 mmol) was added to a stir-
red solution of 4-bromobiphenyl (1.27 g, 5.4 mmol) in
THF (20 mL) at ꢀ78 ꢁC. The mixture was stirred for
5 min, and a solution of quinuclidin-3-one (0.62 g,
4.9 mmol) in THF (10 mL) was added during 20 min.
Stirring was continued at ꢀ78 ꢁC for 30 min and the
mixture allowed to reach room temperature overnight;
2 N HCl (30 mL) was added below 10 ꢁC and the aque-
ous layer washed with Et2O (2 · 50 mL) before the addi-
tion of excess 10 M NaOH to pH 14. The mixture was
extracted with ethyl acetate, which had been heated to
50 ꢁC and the extract allowed to cool, dried and evapo-
rated. TLC of the ethyl acetate residue showed mainly
presence of the starting material. Quinuclidine 2 had re-
mained in the diethyl ether layer. Chromatography over
silica gel with MeOH/CHCl3 (0%!25%) as the eluent
afforded 2 as a white solid (0.343 g, 25%); Rf 0.32
(20% MeOH/CHCl3); mp 165–166 ꢁC; m/z (ES+) 280.1
(M+H+, 100%); HRMS calculated for C19H22NO
([M+H]+) 280.1701, found: 280.1699; 1H NMR
(300 MHz, CDCl3) d = 1.47 (3H, m, 4-CH, 8-CH2),
2.18 (2H, m, 5-CH2), 2.72 (2H, t, J = 8 Hz, 6-CH2),
3.01 (2H, m, 7-CH2), 3.16 (1H, s), 3.45 (1H, d,
J = 14 Hz, –OH), 7.24–7.52 (9H, m, ar–CH); 13C
NMR (75 MHz, CDCl3) 21.4 (CH2), 22.9 (CH),
32.7 (4-CH), 46.2 (CH2), 47.1 (CH2), 61.8 (2-CH2),
72.5 (3-C), 126.8 (ar–CH), 127.4 (ar–CH), 127.8 (ar–
CH), 129.2 (ar–CH), 140.5 (10-C), 140.8 (11-C), 144.9
(9-C).
5.1.5. 3-(Biphenyl-4-yl-40-hydroxy)-2,3-dehydroquinucl-
idine (5b). 4-Toluenesulfonic acid (0.400 g, 2.1 mmol)
and 3 (0.286 g, 0.70 mmol) were heated under reflux in
toluene (30 mL) for 10 h. The toluene was evaporated
and the residue dissolved in 1 M NaOH (50 mL). The
aqueous mixture was extracted with chloroform
(5 · 50 mL) and the organic layer was dried and concen-
trated to yield a white solid (0.093 g, 48%); Rf 0.30 (40%
MeOH/CHCl3); m/z (ES+) 278 (M+H+, 100%); HRMS
calculated for C19H20NO ([M+H]+) 278.1539, found
1
278.1538; H NMR (300 MHz, CDCl3) d 1.54 (2H, m,
8-CH2), 1.75 (2H, m, 5-CH2), 2.59 (2H, m, 6-CH2), 2.95
(2H, m, 7-CH2), 3.17 (1H, s, 4-CH), 3.74 (1H, s, –OH),
6.69 (1H, s, 2-CH), 6.83 (2H, d, J = 9 Hz, 12-CH), 7.26–
7.47 (6H, m, ar–CH); 13C NMR (75 MHz, CDCl3) d 27.9
(CH2), 29.4 (4-CH), 49.3 (CH2), 116.1 (2-CH), 125.6
(ar–CH), 127.1 (ar–CH), 128.4 (ar–CH), 132.4 (11-C),
134.6 (ar–CH), 134.8 (ar–CH), 140.9 (10-C), 147.0 (9-
C), 157.0 (13-C).
5.1.3. 3-(Biphenyl-4-yl-40[(tert-butyldimethylsilyl)oxy)]-3-
hydroxyquinuclidine (4b). BuLi in cyclohexane (3.5 mL,
s
4.1 mmol) was added to a stirred solution of 4-bromo-
40[(tert-butyldimethylsilyl)oxy]biphenyl 1 (0.99 g, 2.7
mmol) in THF (20 mL) at ꢀ78 ꢁC. The mixture was stir-
red for 5 min, and a solution of quinuclidin-3-one
(0.26 g, 2.0 mmol) in THF (10 mL) was added during
20 min. Stirring was continued at ꢀ78 ꢁC for 30 min
and the mixture allowed to reach room temperature
overnight. The reaction mixture was reduced to vacuum.
Flash chromatography with MeOH/CHCl3 (0%!20%)
as the eluent afforded 3 as a white solid (0.43 g, 50%);
Rf 0.52 (40% MeOH/CHCl3); m/z (ES+) 279 (M+-
OTBDMS, 100%), 410 (M+, 58%); HRMS calculated
for C25H36NO2Si ([M+H]+) 410.2510, found: 410.2504;
1H NMR (300 MHz, CD3OD) d 0.19 (6H, s, 13-CH3),
0.98 (9H, s, 14-CH3), 1.78 (1H, m, 4-CH), 1.94 (2H,
m, 8-CH2), 2.50 (2H, m, 5-CH2), 3.29 (2H, m, 6-CH2),
3.40 (2H, m, 7-CH2), 3.49 (2H, m, –OH), 3.99 (1H, m,
–OH), 6.88 (2H, d, J = 9 Hz, 12-CH), 7.49 (2H, d,
J = 9 Hz, ar–CH), 7.60 (4H, m, ar–CH); 13C NMR
(75 MHz, CD3OD) d ꢀ3.8 (14-CH3), 19.5 (15-C), 20.2
(CH2), 21.4 (CH2), 26.6 (15-CH3), 33.2 (4-CH), 47.2
(CH2), 48.1 (CH2), 60.9 (2-CH2), 72.6 (3-C), 122.0 (12-
6. Enzyme assays
6.1. Cloning of enzyme
To test the potential for inhibition of L. major SQS by
the different compounds, protein extracts of E. coli cells
containing the plasmid pET28a-LmSQS2 were used as
enzyme source. A double truncated L. major SQS pro-
tein, that lacks 16 residues at the N-terminus and 40 at
the C-terminus, was expressed in E. coli BL21 (DE3)
RP cells. Briefly, the DNA fragment was amplified from
the LmSQS gene and cloned in the pET28a vector
(Novagen). The resulting plasmid was introduced in
bacterial cells; the recombinant truncated L. major
SQS is expressed as a His-tagged fusion protein when
cells are induced with 1 mM IPTG during 2 h at
25 ꢁC. After induction, cells were disrupted by sonica-
tion in a buffer containing 20 mM phosphate buffer
(pH 7.4), 2 mM MgCl2, 500 mM NaCl, 10 mM CHAPS,
10% glycerol, 10 mM b-mercaptoethanol and protease

S. Orenes Lorente et al. / Bioorg. Med. Chem. 13 (2005) 3519–3529
3527
inhibitors (0.02 mg/mL leupeptine, 0.05 mg/mL aproti-
nine, 10 mM phenanthroline, 0.05 mg/mL trypsine
inhibitor, 1 mM benzamidine and 50 lM PMSF). Solu-
ble extracts were used as enzyme source (3 lg).
in a capillary high resolution column (25 m · 0.20 mm
i.d. Ultra-2 column, 5% phenyl-methyl-siloxane,
0.33 lm film thickness) in a Hewlett–Packard 6890 Plus
gas chromatograph equipped with a HP5973A mass sen-
sitive detector. The lipids were injected in choloroform
and the column was kept a 50 ꢁC for 1 min, then the
temperature was increased to 270 ꢁC at a rate of
25 ꢁC/min and finally to 300 ꢁC at a rate of 1 ꢁC/min.
The carrier gas (He) flow was kept constant at 0.5 mL/
min. Injector temperature was 250 ꢁC and the detector
was kept at 280 ꢁC.
6.2. Enzyme assay
A standard SQS activity assay contained 50 mM phos-
phate buffer (pH 7.4), 20 mM MgCl2, 5 mM CHAPS,
1% Tween 80, 10 mM DTT, 0.025 mg/mL BSA,
0.25 mM NADPH, 2.1 mM 6-phosphate glucose,
0.125 mg/mL 6-phosphate glucose dehydrogenase and
0.5 lM FPP (10080 dpm/pmol) as substrate. The reac-
tion was started with the protein extract and the final
volume of the reaction was 200 lL. After incubation
at 37 ꢁC for 10 min, 40 lL of 10 M NaOH were added,
followed by 10 lL of a (50:1) mixture of 70% EtOH
and squalene. Resulting mixtures were mixed vigor-
ously by vortexing, then 20 lL aliquots were applied
to 2.5 · 10 cm channels of a silica gel thin layer chroma-
togram, and newly formed squalene was separated
from unreacted substrate by chromatography in
toluene–EtOAc (9:1). The region of the squalene band
was removed, immersed in Hydrofluoro liquid scintilla-
tion fluid, and assessed for radioactivity using a Phar-
macia LKB liquid scintillation counter. Negative
controls were reactions containing soluble extracts of
E. coli BL21 (DE3) RP cells transformed with pET28a
(not overexpressing L. major SQS). No activity was
observed using this extract as an enzyme source. IC50
values were calculated from the hyperbolic plot of
percentage of inhibition versus concentration of
inhibitor.
8. Growth inhibition studies
8.1. L. mexicana promastigotes
L. mexicana promastigotes were cultivated in LIT
medium supplemented with lactabulmin and 10% foetal
calf-serum (Gibco)28 at 26 ꢁC, without agitation. The
cultures were initiated with a cell density of 2.106 cells/
mL and the drug was added at a cell density of 0.5–
1.107 cell/mL. Cell densities were measured with an elec-
tronic particle counter (model ZBI; Coulter Electronics
Inc., Hielah, Fla.) and by direct counting with an haemo-
cytometer. Cell viability was followed by Trypan blue
exclusion.
8.2. L. donovani amastigotes
Peritoneal exudate macrophages were harvested from
CD1 mice, 24 h after starch induction. After washing
the macrophages were dispensed into Lab-tekTM 16-well
tissue culture slides and maintained in RPMI1640 +
10% heat-inactivated foetal calf serum (HIFCS) at
37 ꢁC, 5% CO2/air mixture for 24 h. L. donovani
(MHOM/ET/67/L82) amastigotes were harvested from
an infected Golden hamster spleen and were used to in-
fect the macrophages at a ratio of 5 parasites:1 macro-
phage. Infected cells were left for a further 24 h and
then exposed to drug32 for a total of 5 days, with the
overlay being replaced on day 3.33 The top concentra-
tion for the test compounds was 30 lg/mL and all con-
centrations were carried out in quadruplicate. On day
5 the overlay is removed, the slides fixed (100% metha-
nol) and stained (10% Giemsa, 10 min) before being
evaluated microscopically. ED50 values were calculated
using Msxlfit. The ED50 value for the positive control
drug, Pentostamꢀ, is usually 3–8 lg SbV/mL.
7. Effect on steroid composition
L. mexicana amazonensis promastigotes were cultivated
in LIT medium supplemented with lactoalbumin and
10% foetal calf-serum (Gibco) (3) at 26 ꢁC, without agi-
tation. The cultures were initiated with a cell density of
2.106 cells/mL and the drug was added at a cell density
of 0.5–1.107 cells/mL. Cell densities were measured with
an electronic particle counter (model ZBI; Coulter Elec-
tronics Inc., Hielah, Fla.) and by direct counting with a
haemocytometer. Cell viability was followed by Trypan
blue exclusion using light microscopy.
For the analysis of the effects of drugs on the lipid com-
position of promastigotes, total lipids from control and
drug-treated cells were extracted and fractionated into
neutral and polar lipid fractions by silicic acid column
chromatography and gas–liquid chromatography.6,7,12,13
The neutral lipid fractions were first analyzed by thin
layer chromatography (on Merck 5721 silica gel plates
with heptane–isopropyl ether–glacial acetic acid
[60:40:4] as developing solvent) and conventional gas–
liquid chromatography (isothermic separation in a 4-m
glass column packed with 3%OV-1 on Chromosorb
100/200 mesh, with nitrogen as carrier gas at 24 mL/
min and flame ionization detection in a Varian 3700
gas chromatograph). For quantitative analysis and
structural assignments the neutral lipids were separated
8.3. T. cruzi amastigotes
Murine (CD1) peritoneal macrophages were harvested
24 h after starch induction. One hundred microlitre
was dispensed into 96-well plates at a concentration of
4 · 105/mL. After 24 h the cells were infected with T.
cruzi Tulahuan LAC-Z trypomastigotes, harvested from
L6 feeder layer cultures. Twenty-four hour later the in-
fected cells were exposed to the drug20 for 3 days. Fifty
microlitres of 500 lM CPRG:1% nonidet P-40 was
added to each well. The plates were read after 2–5 h,
k570.34 ED50 (ED90) values were calculated using
Msxlfit. L6 fibroblasts are also used as host cells.

3528
S. Orenes Lorente et al. / Bioorg. Med. Chem. 13 (2005) 3519–3529
9. Molina, J.; Martins, O.; Brener, Z.; Romanha, A. J.;
Loebenberg, D.; Urbina, J. A. Antimicrob. Agents Che-
mother. 2000, 44, 150.
10. Urbina, J. A.; Vivas, J.; Ramos, H.; Larralde, G.; Aguilar,
Z.; Avilan, L. Mol. Biochem. Parasitol. 1988, 30,
185.
11. Urbina, J. A.; Lazardi, K.; Aguirre, T.; Piras, M. M.;
Piras, R. Antimicrob. Agents Chemother. 1991, 35,
730.
12. Urbina, J. A.; Payares, G.; Molina, J.; Sanoja, C.;
Liendo, A.; Lazardi, K.; Piras, M. M.; Piras, R.; Perez,
N.; Wincker, P.; Ryley, J. F. Science 1996, 273,
969.
13. Urbina, J. A.; Payares, G.; Contreras, L. M.; Liendo, A.;
Sanoja, C.; Molina, J.; Piras, M.; Piras, R.; Perez, N.;
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14. Urbina, J. A.; Lira, R.; Visbal, G.; Bartroli, J. Antimicrob.
Agents Chemother. 2000, 44, 2924.
15. Magaraci, F.; Jimenez Jimenez, C.; Rodrigues, C. O.;
Rodrigues, C. F.; Vianna Braga, M.; Yardley, V.; de
Luca-Fradley, K.; Croft, S. L.; de Souza, W.; Ruiz-Perez,
L. M.; Urbina, J. A.; Gonzalez Pacanowska, D.; Gilbert,
I. H. J. Med. Chem. 2003, 46, 4714.
16. Maldonado, R. A.; Molina, J.; Payares, G.; Urbina, J. A.
Antimicrob. Agents Chemother. 1993, 37, 1353.
17. Rodrigues, J. C. F.; Attias, M.; Rodriguez, C.; Urbina, J.
A.; de Souza, W. Antimicrob. Agents Chemother. 2002, 46,
487.
18. Urbina, J. A.; Vivas, J.; Visbal, G.; Contreras, L. M. Mol.
Biochem. Parasitol. 1995, 73, 199.
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20. Vivas, J.; Urbina, J. A.; deSouza, W. Int. J. Antimicrob.
Agents 1996, 7, 235.
21. Vivas, J.; Urbina, J. A.; deSouza, W. Int. J. Antimicrob.
Agents 1997, 8, 1.
22. Concepcion, J. L.; Gonzalez-Pacanowska, D.; Urbina, J.
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8.4. T. brucei rhodesiense trypomastigotes
T. brucei rhodesiense STIB900 blood stream form (bsf)
trypomastigotes were maintained in HMI-18 medium35
with 15% heat-inactivated foetal calf serum (HIFCS)
[Harlan-SeraLab, UK] at 37 ꢁC, 5%, CO2/air mixture.
Trypomastigotes were washed and resuspended in fresh
medium at a concentration of 2 · 105/mL. The top con-
centration for the test compounds was 30 lg/mL. The
ED50 for pentamidine is usually between 1.0 and
0.1 ng/mL. Plates were incubated for 72 h at 37 ꢁC
in 5% CO2/95% air mixture. At 72 h the plates were as-
sessed microscopically before Alamar Blue was added.36
Plates were read after 5–6 h on a Gemini Fluorescent
plate reader (Softmax Pro. 3.1.1, Molecular Devices,
UK) at EX/EM 530/585 nm with a filter cut-off at
550 nm. ED50 values were calculated with Msxlfit
(IDBS, UK).
8.5. Cytotoxicity against vertebrate cells
Plates were seeded with 100 lL KB cells @ 4 · 104/mL,
RPMI 1640 + 10% HIFCS and incubated at 37 ꢁC, 5%
CO2 for 24 h. The overlay was removed and replaced
by test drugs20 in fresh medium @ 300, 30, 3 and
0.3 lg/mL. The positive control drug was podophyllo-
toxin (Sigma, UK). Dilutions were carried out in tripli-
cate. Plates were incubated for a further 72 h, at 37 ꢁC,
5% CO2. The wells assessed were microscopically for cell
growth. The overlay was removed and wells washed
with PBS (pH 7.0) · 3. Then 100 lL PBS + 10 lL Ala-
marBlueTM were added per well and plates incubated
for 2–4 h (37 ꢁC, 5% CO2) before reading at EX/EM
530/585 nm (cut-off 550 nm) in a Gemini plate reader.
ED50 (ED90) values were calculated compared to blanks
and untreated controls.
Acknowledgements
We would like to acknowledge the EU INCO-DEV pro-
gramme (ICA4-2000-10028), the Spanish Plan Nacional
(SAF2003-01593) and WHO/TDR for funding (S.L.C.
and V.Y.) and the EPSRC National Mass Spectrometry
Service Centre in Swansea for accurate mass spectro-
25. Martin, M. B.; Arnold, W.; Heath, H. T.; Urbina, J. A.;
Oldfield, E. Biochem. Biophys. Res. Commun. 1999, 263,
754.
´
metry. We thank Sofıa Vargas for technical assistance.
26. Martin, M. B.; Grimley, J. S.; Lewis, J. C.; Heath, H. T.;
Bailey, B. N.; Kendrick, H.; Yardley, V.; Caldera,
A.; Lira, R.; Urbina, J. A.; Moreno, S. N. J.; Docampo,
R.; Croft, S. L.; Oldfield, E. J. Med. Chem. 2001, 44,
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27. Biller, S. A.; Neuenschwander, K.; Ponpipom, M. M.;
Poulter, C. D. Curr. Pharm. Des. 1996, 2, 1.
28. Urbina, J. A.; Concepcion, J. L.; Rangel, S.; Visbal, G.;
Lira, R. Mol. Biochem. Parasitol. 2002, 125, 35.
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S. Orenes Lorente et al. / Bioorg. Med. Chem. 13 (2005) 3519–3529
3529
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