Oxidosqualene cyclase from Saccharomyces cerevisiae, Trypanosoma cruzi, Pneumocystis carinii and Arabidopsis thaliana expressed in yeast: A model for the development of novel antiparasitic agents

Article abstract of DOI:10.1016/j.bmcl.2008.12.040

A series of 25 compounds, some of which previously were described as inhibitors of human liver microsomal oxidosqualene cyclase (OSC), were tested as inhibitors of Saccharomyces cerevisiae, Trypanosoma cruzi, Pneumocystis carinii and Arabidopsis thaliana

Full text of DOI:10.1016/j.bmcl.2008.12.040

Bioorganic & Medicinal Chemistry Letters 19 (2009) 718–723
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Oxidosqualene cyclase from Saccharomyces cerevisiae, Trypanosoma cruzi,
Pneumocystis carinii and Arabidopsis thaliana expressed in yeast: A model
for the development of novel antiparasitic agents
b,
a
a
a
Gianni Balliano a, , Henrietta Dehmlow , Simonetta Oliaro-Bosso , Matilde Scaldaferri , Silvia Taramino ,
*
*
Franca Viola a, Giulia Caron a, Johannes Aebi b, Jean Ackermann b
a Dipartimento di Scienza e Tecnologia del Farmaco, Università di Torino, via P. Giuria 9, 10125 Torino, Italy
b F. Hoffmann-La Roche Ltd, Pharmaceutical Division, CH-4070 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 25 compounds, some of which previously were described as inhibitors of human liver micro-
somal oxidosqualene cyclase (OSC), were tested as inhibitors of Saccharomyces cerevisiae, Trypanosoma
cruzi, Pneumocystis carinii and Arabidopsis thaliana OSCs expressed in an OSC-defective strain of S. cerevi-
siae. The screening identified three derivatives particularly promising for the development of novel anti-
Trypanosoma agents and eight derivatives for the development of novel anti-Pneumocystis agents.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 17 October 2008
Revised 5 December 2008
Accepted 7 December 2008
Available online 14 December 2008
Keywords:
Oxidosqualene cyclase
Lanosterol synthase
Inhibitors
Antiparasitic agents
Enzymes of the last steps of sterol biosynthesis have been re-
garded for a long time as good targets for treatment of fungal dis-
eases. Squalene epoxidase and 14a-demethylase, for instance, are
ment.5 Therefore, the identification of new derivatives endowed
with a selective action on protozoan OSC is a hot topic in medicinal
chemistry.
the targets of the most effective antifungal drugs currently used,
but oxidosqualene cyclase (OSC), 24-methyltransferase and
D24(28) sterolmethylreductase are also considered as good target
candidates.1
Some of the enzymes listed above have been recently addressed
as good targets for development of novel antiparasitic drugs. Sev-
In this study, we were aiming at identifying new starting points
for the development of antiparasitic agents. Therefore a set of com-
pounds, previously described as effective inhibitors of human
oxidosqualene cyclase,6 were tested for their ability to inhibit
oxidosqualene lanosterol cyclases from T. cruzi and P. carinii ex-
pressed in an OSC-defective Saccharomyces cerevisiae strain.7a As
a comparison, the study was extended to two further OSCs ex-
pressed in the same OSC-defective strain, namely OS lanosterol cy-
clase from S. cerevisiae,7a and OS cycloartenol synthase from
Arabidopsis thaliana.7a A preliminary attempt at evaluating the hu-
man versus parasite selectivity was done by testing compounds
against human OSC expressed in the same OSC-defective yeast
strain.7b The structures of the compounds evaluated are depicted
in Figure 1.
eral inhibitors of sterol 24-methyltransferase or sterol 14a-
demethylase, for instance, are good candidates for the treatment
of Chagas disease, a chronic infection caused by the protozoan par-
asite Trypanosoma cruzi.2
In the last few years, a variety of OSC inhibitors have also been
tested on T. cruzi, Trypanosoma brucei, Pneumocystis carinii and
Leishmania mexicana with the aim of developing new agents
against these microorganisms.1c,1d,3
The diseases caused by protozoan parasites are becoming an ur-
gent research topic, not only in tropical regions, but also in once
safer countries.4 These diseases cause high rates of mortality and
morbidity, and few drugs are currently available for their treat-
The preparations of the compounds 1 (Ro 48-8071), 2–9, 10 (BIB
515) and 13 have been previously described.6,8 The syntheses of
benzo[d]isothiazole OSC inhibitors are outlined in Schemes 1 and
2. Alkylation of benzo[d]isothiazole 266 with an excess of 1,4-
dibromobutane gave the bromide 27 and its amination provided
the final amines 11 and 12, as well as the intermediate 28. Acyla-
tion of 28 under basic conditions thus yielded derivative 18. The
compounds 14–17, containing a carbon based linker, were ob-
tained by converting 26 to triflate 29, which was subjected to a
* Corresponding authors. Tel.: +39 011 6707698; fax: +39 011 6707695 (G.B.);
tel.: +41 61 688 90 40; fax: +41 61 688 97 48 (H.D.).
E-mail addresses: gianni.balliano@unito.it (G. Balliano), henrietta.dehmlow@
roche.com (H. Dehmlow).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.040

G. Balliano et al. / Bioorg. Med. Chem. Lett. 19 (2009) 718–723
719
F
O
S
N
CF3
N
S
N
N
O
Br
O
Br
O
O
N
1 Ro 48-8071
11
21
O
CF3
S
N
OH
O
N
S
Br
N
O
Br
O
O
N
N
HO
2
12
22
CF3
O
N
S
N
S
N
Br
O
O
N
O
N
HO
O
S
Br
O
3
4
5
6
13
23
24
25
CF3
CF3
N
N
N
N
O
S
N
O
O
O
N
N
N
N
N
N
O
Br
Br
14
S
N
S
N
N
S
O
Br
Br
O
N
N
N
HO
15
N
S
S
N
N
Br
Br
Br
O
HO
16
S
O
O
Br
HO
7
17
CF3
S
N
O
N
S
N
N
HO
O
Br
O
O
N
HO
8
18
N
O
N
O
O
Cl
O
HO
N
CF3
9
19
N
O
CF3
O
N
N
S
Cl
O
O
N
10
20
Figure 1. Chemical structures of investigated compounds.
Sonogashira reaction to give the intermediates 30a/b and 31,
respectively. Hydrogenation of 30a, followed by in situ formation
of the triflate and amination, provided amine 14 in three steps. Di-
rect mesylation of the alcohols 30a/b and subsequent amination
yielded the products 15 and 16, respectively. Amine 17 was de-
rived from alkynol 31 via phosphonate 32.
the building block 35 as a TFA salt. Treatment of 35 with the
appropriate chloroformate or sulfonyl chloride, followed by ami-
nation provided the carbamate derivative 19 or the sulfonamides
20–22. Starting from 34, ester hydrolysis, amide formation under
standard conditions, followed by BOC deprotection gave the
intermediate amine 36 as an HCl salt. Sulfonamide formation
The syntheses of aminocyclohexanes with carbon-linked spac-
ers are outlined in Schemes 3 and 4. LAH reduction of 34,8c
mesylation and cleavage of the BOC-protecting group yielded
provided the desired amide 23. For the preparation of the a,a-
branched amines 24 and 25 modified Bouveault reaction condi-
tions were used.9

720
G. Balliano et al. / Bioorg. Med. Chem. Lett. 19 (2009) 718–723
R1
R2
S
N
S
N
S
N
11
12
Me
Allyl
a)
b)
R2
N
-(CH2)2-O-(CH2)2-
28 -(CH2)2OH
18 -(CH2)2OH Ac
H
Br
c)
HO
Br
O
R1
O
Br
Br
26
27
Scheme 1. Synthesis of benzo[d]isothiazole OSC inhibitors 11, 12 and 18. Reagents and conditions: (a) 1,4-dibromobutane, K2CO3, acetone, 50 °C (71%); (b) 11: MeNHallyl,
DMA, rt (quant.), 12 HBr: morpholine, DMF, iPr2NEt, rt (50%), 28: 1—HOCH2CH2NH2, DMA, rt; 2—HCl, MeOH, rt (70%); (c) NaOH, Ac2O, iPrOH, rt (86%).
S
N
N
c, d)
O
S
N
N
S
N
Br
Br
a)
b)
14
26
S
Br
HO
Br
TfO
n
e)
29
30a n=3
30b n=1
N
HO
15 n=3
16 n=1
n
f)
S
N
S
N
h)
Br
Br
R
N
HO
31 R = OH
32 R = OP(=O)(OEt)2
g)
17
Scheme 2. Synthesis of benzo[d]isothiazole OSC inhibitors 14–17. Reagents and conditions: (a) (TfO)2O, pyridine, 0 °C to rt (98%); (b) 30a: PdCl2(PPh3)2, PPh3, CuI,
HCC(CH2)3OH, NEt3, rt to 50 °C (72%), 30b: Pd(PPh3)4, CuI, HCCCH2OH, piperidine, rt to 80 °C (60%); (c) 14: H2, PtO2, EtOH, rt (93%); (d) 1—(TfO)2O, iPr2NEt, CH2Cl2, 0 °C to rt;
2—morpholine, CH2Cl2, rt (86%); (e) 15: 1—MeSO2Cl, DMAP, pyridine, CH2Cl2, 0° to rt; 2—NH(Et)(CH2CH2OH), DMA, rt (54%, 2 steps), 16: 1—MeSO2Cl, DMAP, pyridine, CH2Cl2,
rt; 2—NH(Et)(CH2CH2OH), DMA, rt (31%, 2 steps); (f) PdCl2(PPh3)2, PPh3, CuI, HCCC(Me)2OH, NEt3, rt to 50 °C (61%); (g) nBuLi, ClP(@O)(OEt)2, NEt3,THF, À78 °C to 0 °C (33%);
(h) NH(Et)(CH2CH2OH), Pd(PPh3)4, THF, 50 °C (8%).
A preliminary screening of 10 compounds from different chem-
ical classes, carried out in cell free homogenates,10 identified three
lead compounds, 5, 8 and 9 (Table 1). Compound 5 was one of the
most potent inhibitors of OSC from P. carinii, slightly more potent
then 1 (Ro 48-8071). Furthermore, it belongs to a new heterocyclic
series—the phenyl-benzo[d]isothiazoles, not yet explored in this
context. Compound 9, a substituted cyclohexylamine, was the
most potent inhibitor of OSC from T. cruzi, and one of the most ac-
tive against OSC from P. carinii. In addition, this compound is in a
different chemical class from compounds 1 (Ro 48-8071) and 5.
Compound 8, in the same chemical class as 9, was less potent than
5 and 9 for inhibition of OSC from P. carinii and T. cruzi. This not-
withstanding, compound 8 was used as a lead compound because
it had potency not too different from 9 on human OSC, 2-fold less
in human liver,8c 3-fold less when tested on homogenates from
yeast recombinant strain SMY8.
analogs of the cyclohexylamines 8 and 9 (compounds 19–25) were
selected (Table 2).
In the heterocyclic series, the reduction of the chain length from
n-hexyl to n-butyl, as demonstrated for compounds 5 and 11, did
not influence the potency of the inhibitors for P. carinii and T. cruzi
OSCs. For the inhibition of OS lanosterol cyclase from S. cerevisiae, a
10-fold drop in activity was seen, whereas a significant increase in
potency was found for OS cycloartenol cyclase from A. thaliana.
An increase in inhibitory effect on OSC from P. carinii was seen
when the methylallylamine was replaced by a morpholine or N-
hydroxyethylethylamine residue (11 vs 12 and 13). These changes
did not affect the activity of the inhibitors against S. cerevisiae,
whereasa decreaseinactivitywasobservedfor T. cruziand A. thaliana.
For OSCs from P. carinii and S. cerevisiae, the replacement of the
amine moiety by an amide residue (as in compound 18) was detri-
mental to the activity of the inhibitors. This is similar to the effects
previously observed for the inhibition of human liver OSC.8c How-
ever, the inhibition of OS lanosterol cyclase from T. cruzi was hardly
affected and that of OS cycloartenol cyclase from A. thaliana re-
mained submicromolar.
For the next round of testing we wanted to investigate the influ-
ence of both spacer unit and basicity of the amine in the two dis-
tinct series. Therefore, eight analogs of compound
5 (i.e.
heterocyclic analogs of Ro 48-8071: compounds 11–18) and seven
NHBOC
a)
b)
c or d)
N
NH
N
R2
BOC
MsO
R1
R1
TFA
HO2C
EtO2C
33
34
35
R2
19 NEt(CH2CH2OH) 4-ClPhCO2-
20 NMeAllyl
21 NEt2
22 N(CH2CH2OH)2
4-CF3PhSO2-
4-CF3PhSO2-
4-CF3PhSO2-
Scheme 3. Synthesis of aminocyclohexane derivatives 19–22. Reagents and conditions: (a) 1—carbonyldiimidazol, MeOH, rt (99%); 2—NaH, MeI, DMF, 0 °C to rt; 3—LiBH4,
THF, reflux; 4—oxalylchloride, DMSO, NEt3, CH2Cl2, À78 °C to rt; 5—triethyl-phosphono acetate, EtOH, NaOMe, rt (86%, 4 steps); 6—Pd/C, H2, MeOH, rt (94%); 7—LiBH4, THF,
reflux, (73%, 2 steps); 8—oxalylchloride, DMSO, NEt3, CH2Cl2, À78 °C to rt; 9—triethyl-phosphono acetate, EtOH, NaOMe, rt (88%, 2 steps); 10—Pd/C, H2, MeOH, rt (94%); (b) 1—
LAH, THF, rt (97%); 2—MeSO2Cl, NEt3, CH2Cl2, rt; 3—TFA, CH2Cl2, rt (73%, 2 steps); (c) 19 HCl: 1—4-Cl-PhOCOCl, iPr2NEt, CH2Cl2, rt (82%); 2—NH(Et)(CH2CH2OH), MeOH, 60 °C;
3—HCl in EtOAc (56%, 2 steps); (d) 1—4-CF3-PhSO2Cl, iPr2NEt, CH2Cl2, rt (74%); 2—20: NHMeAllyl, MeOH, 60 °C (56%); 21: Et2NH, MeOH, 60 °C (73%); 22: NH(CH2CH2OH)2,
MeOH, 60 °C (60%).

G. Balliano et al. / Bioorg. Med. Chem. Lett. 19 (2009) 718–723
721
CF3
CF3
N
CF3
e)
S
S
O
O
N
N
NH
d)
a, b, c)
N
S
O
34
HCl
N
24
25
O
N
O
O
36
23
f)
N
O
O
Scheme 4. Synthesis of aminocyclohexane derivatives 23–25. Reagents and conditions: (a) aq LiOH, THF; KHSO4, EtOAc (quant.); (b) EDCI, HOBt, NHEt2, NMM, CH2Cl2, rt
(86%); (c) HCl, dioxane, rt (quant.); (d) 4-CF3-PhSO2Cl, iPr2NEt, DMAP, CH2Cl2, 0 °C to rt (85%); (e) MeMgBr, ZrCl4, THF, À10 °C to rt (63%); (f) EtMgBr, ZrCl4, THF, À10 °C to rt
(58%).
Table 1
compounds 13 and 15. This effect seemed to be most pronounced
for the inhibition of OSC from P. carinii. Some of the activity could
be recovered by shortening the spacer unit from pentynyl to pro-
pynyl (15 vs 16). This was most apparent for the activity on S. cere-
visiae, but also visible on T. cruzi.
Effect of inhibitors (first series) on oxidosqualene cyclase activity of homogenates
prepared from yeast recombinant strains SMY8 expressing S. cerevisiae, T. cruzi, P.
carinii, A. thaliana and H. sapiens OSCs7
a
Compound
IC50
(
lM)
OSCb
OSCb
OSCb
OSCc
OSCb
The activity of the compounds was not changed when introduc-
ing substituents as branching points alpha to the amine (16 vs 17)
for OSC from P. carinii, whereas it dropped 2- to 7-fold for the other
OSCs.
S. cerevisiae
T. cruzi
P. carinii
A. thaliana
H. sapiens
1 fumarate
2 HCl
3 HCl
4 fumarate
5 fumarate
6 fumarate
7 fumarate
8 fumarate
9 citrate
10
0.21
0.32
0.18
0.29
0.07
0.28
0.21
0.27
0.05
0.67
0.90
0.60
1.50
3.00–5.00
0.34
0.34
0.05
0.20
ꢀ5
1.75
0.25
0.26
2.60
0.95
10.00
3.00
3.00
1.00
2.5
0.17
0.25
0.27
2.30
0.16
20.34
90.00
0.45
0.14
1.15
In the series of cyclohexylamines, changes both in the spacer re-
gion and at the amine part of the molecules were also investigated.
In the both subseries, the carbamates (9 vs 19) and the sulfona-
mides (8 vs 20), the allylmethyl amine residue was the most prom-
ising group. In the sulfonamide subseries, modifications at the
amine did not significantly influence the activity against OS cyclo-
artenol cyclase (compounds 8, 20–22). The amide 23 was not an
inhibitor of the lanosterol cyclases, but it is still effective against
the cycloartenol cyclase. Disubstitution by methyl residues alpha
to the amino group led to a 2-fold drop in activity against lanos-
terol cyclase from P. carinii and S. cerevisiae and a detrimental drop
in the activity against T. cruzi enzyme (24 vs 21). An improved po-
tency was observed for the cyclopropyl derivative 25 for all OSCs
with the exception of lanosterol cyclase of P. carinii.
Altogether, while most of the inhibitors showed identical or
comparable activity against both T. cruzi and P. carinii OSCs, five
compounds (2, 12, 13, 14 and 24) proved to be specific for P. carinii
enzyme showing at least a 10-fold selectivity, but even more than
100-fold for the best compounds (13 and 24). On the other hand
inhibitor 9, the most effective on T. cruzi enzyme (IC50 0.07
lM),
was only 5-fold more active than on the P. carinii enzyme. Com-
pound 11 was the only inhibitor of A. thaliana OSC with an
IC50 < 0.1 lM, but it showed only a weak selectivity. Surprisingly
enough, the amides 18 and 23 inhibited A. thaliana OSC, whereas
they were inactive or only slightly active against the other OSCs.
All the compounds were also tested as inhibitors of human OSC
expressed in the same defective strain of S. cerevisiae, in order to
gain a preliminary evaluation of the human versus parasite selec-
tivity (Table 1). The compounds showed a poor human versus T.
cruzi selectivity. The IC50 values on human enzyme were compara-
ble or even lower than those on the parasite OSC. Similar results
were obtained with P. carinii enzyme, with the exception of com-
pounds 2 and 13, which proved 5 and 10 times as effective against
parasite enzyme, respectively. A higher selectivity (40–80 times)
was seen for the less active compounds 6 and 7.
0.23
0.55
1.20
3.00
0.34
ꢀ5
ꢀ5
3.00–5.00
1.60
0.07
ꢀ5
a
Values are the means of two separate experiments with duplicate incubations,
each. The maximum deviations from the mean were less than 10%.
b
Lanosterol cyclase.
Cycloartenol cyclase.
c
Table 2
Effect of the second series of inhibitors on oxidosqualene cyclase activity of
homogenates prepared from yeast recombinant strains SMY8 expressing S. cerevisiae,
T. cruzi, P. carinii, A. thaliana and H. sapiens OSCs7
a
Compound
IC50
(lM)
OSCb
S. cerevisiae
OSCb
T. cruzi
OSCb
P. carinii
OSCc
A. thaliana
OSCb
H. sapiens
11
0.70
0.60
0.70
0.40
5.00
0.30
2.20
100
0.10
0.30
3.60
1.50
>100
7.00
0.50
0.25
1.10
6.50
1.10
>10
2.30
4.20
5.50
2.00
0.30
2.40
2.70
>100
>100
0.40
0.20
0.07
0.05
0.05
2.25
0.80
1.00
100
2.50
0.12
0.60
0.40
>100
1.00
1.30
0.07
0.48
2.30
0.65
1.70
0.55
1.25
0.80
0.70
0.44
1.00
1.50
2.00
4.10
0.32
0.26
0.12
0.52
0.10
0.23
4.60
11.5
>1
12 HBr
13 HCl
14
15
16
17
18
19 HCl
20
21
4.12
0.1
2.44
0.72
>10
4.93
0.44
22
23
24
25
a
Values are the means of two separate experiments with duplicate incubations,
each. The maximum deviations from the mean were less than 10%.
b
Lanosterol cyclase.
Cycloartenol cyclase.
c
The effect of some derivatives on oxidosqualene cyclases from
T. cruzi and P. carinii was also tested on spheroplasts11 prepared
from recombinant yeast cells expressing OSCs from these patho-
gens (Tables 3 and 4). As recently observed,11 spheroplasts from
these recombinant yeast strains can make sterols, although the
post-squalene section of the biosynthetic pathway appears rather
depressed as indicated by the higher squalene/sterols ratios
Modification of the linker of the side chain from oxygen to car-
bon (12 vs 14) did not influence the potency of the inhibitors for all
OSCs tested.
Introducing rigidity in the spacer unit caused a drop in the
activity against the lanosterol cyclases, when comparing

722
G. Balliano et al. / Bioorg. Med. Chem. Lett. 19 (2009) 718–723
Table 3
Table 4
Effect of the most representative inhibitors on sterol biosynthesis in spheroplasts of S.
cerevisiae expressing T. cruzi OSC11: % radioactivity incorporated into non-saponifiable
lipids separated on TLC
Effect of the most representative inhibitors on sterol biosynthesis in spheroplasts of S.
cerevisiae expressing P. carinii OSC11: % radioactivity incorporated into non-saponi-
fiable lipids separated on TLC
% Radioactivity incorporated into non-saponifiable lipidsa
Squalene Oxido Lanosterol Mono Ergosterol
methylsterols
2.82
Compound
% Radioactivity incorporated into non-saponifiable lipidsa
Compound
Squalene
Oxido
Lanosterol
Mono
Ergosterol
squalene
methylsterols
squalene
1.72
Control
50.22
6.10
18.01
3.37
22.30
Control
70.33
10.42
14.71
1 fumarate
1 fumarate
0.1
lM
52.35
42.59
6.59
38.31
26.20
11.09
1.75
1.94
13.11
6.07
0.1
l
M
71.45
60.54
11.06
30.96
9.46
3.36
1.06
1.54
6.97
3.60
1
lM
1 lM
2 HCl
0.1
2 HCl
0.1
l
M
44.89
47.22
9.88
34.59
22.12
6.77
2.47
2.66
20.64
8.76
l
M
72.47
62.92
1.80
27.80
13.01
5.16
2.20
1.12
10.52
3.00
1
lM
1
lM
3 HCl
0.1
3 HCl
0.1
1 lM
l
M
48.34
40.90
8.69
33.39
22.99
12.38
2.72
2.29
17.26
11.04
l
M
62.92
64.27
2.50
16.7
17.43
10.21
3.25
1.99
13.90
6.83
1
lM
5 fumarate
0.1
1 lM
5 fumarate
0.1
l
M
44.47
52.38
33.26
33.37
10.29
5.08
1.32
1.62
10.66
7.55
l
M
66.59
63.37
8.23
24.96
10.78
5.20
2.91
1.39
11.49
5.08
1
lM
9 citrate
0.1
6 fumarate
0.1
1 lM
l
M
52.31
54.58
37.42
38.65
3.33
1.71
1.43
1.29
5.51
3.77
l
M
65.98
67.66
1.17
1.44
14.49
14.29
2.90
3.44
15.46
13.17
1
lM
11
1
10 lM
9 citrate
0.1
l
M
42.41
46.47
28.74
24.37
7.03
3.20
3.19
8.19
18.63
17.77
l
M
70.38
67.53
9.03
24.95
8.89
2.39
1.81
1.06
9.89
4.07
1
lM
14
1
10
20
0.1
12 HBr
0.1
l
lM
M
45.04
46.17
15.86
24.76
19.33
4.65
2.01
7.52
17.76
16.90
l
M
71.04
69.63
0.68
1.72
9.55
15.78
3.57
2.82
15.16
10.05
1
lM
13 HCl
0.1
lM
54.01
56.42
34.86
37.51
4.15
2.40
1.40
1.01
5.58
2.66
l
M
71.03
69.90
2.39
15.72
9.93
5.81
3.76
2.80
12.89
5.77
1
lM
1
lM
a
Values are the means of two separate experiments with duplicate incubations,
14
0.1
each. The maximum deviations from the mean were less than 10%.
l
M
M
69.01
69.62
1.30
5.44
12.55
9.84
4.02
3.91
13.12
11.19
1
l
a
Values are the means of two separate experiments with duplicate incubations,
compared with intact recombinant cells. Results confirmed data
from experiments with homogenates: all the compounds caused
a dose-dependent increase of the ratio between radioactive oxido-
squalene and sterols extracted from treated cells, thus indicating
that OSC was inhibited. The tested compounds did not substan-
tially affect the fraction of radioactive squalene, suggesting that
squalene epoxidase was not affected.
each. The maximum deviations from the mean were less than 10%.
Furthermore, we sincerely thank Felix Gruber, Barbara Huber, Mar-
ie-Paule Imhoff, Hans-Jakob Krebs, and Ann Petersen, for the prep-
aration of the compounds.
In conclusion, the screening of 25 compounds as inhibitors of P.
carinii and T. cruzi OSCs has identified several promising deriva-
tives as starting points for the development of novel antiparasitic
agents. The compounds 9, 11 and 20, which showed an activity
Thanks are due to Professor Seiichi Matsuda (Rice University,
Houston, TX, USA) for supplying S. cerevisiae strains SMY8[pBJ1.21],
expressing the OSC of T. cruzi; SMY8[pSM61.21], expressing the
wild-type yeast OSC; SMY8[pBJ4.21] expressing the P. carinii OSC;
SMY8[pSM60.21] expressing the A. thaliana OSC.
60.3 lM, are particularly promising as novel anti-Trypanosoma
agents, while the compounds 2, 3, 5, 11, 12, 13, 14 and 20 show
promise as novel anti-Pneumocystis agents. The derivatives 11
and 20 showed similar potency against both parasital OSCs. As a
rule, the P. carinii enzyme was more susceptible than T. cruzi en-
zyme to the action of the inhibitors. While three out of the four
most active compounds against P. carinii OSC were analogs of com-
pound 5, the most active compound against the T. cruzi enzyme
was the cyclohexylamine derivative 9, the only compound more
active against T. cruzi than against P. carinii OSC.
Homology models built taking advantage of the established
structure of human OSC12 could be useful to relate the structure
and activity of compounds to the differences in the active site of tar-
get enzymes. In silico studies are in progress with the aim of finding
novel active molecules and improving the parasite versus human
selectivity of the most active compounds of the present series.
References and notes
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We thank the University of Turin and the regional government
(Regione Piemonte) for the financial support of this research.

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