Arylpiperidines as a new class of oxidosqualene cyclase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.12.025

The cyclization of oxidosqualene to lanosterol, catalyzed by the enzyme oxidosqualene cyclase (OSC), goes through a number of carbocationic high energy intermediates (HEI), and mimicking these intermediates is a promising approach for the development of O

Full text of DOI:10.1016/j.ejmech.2015.12.025

European Journal of Medicinal Chemistry 109 (2016) 13e22
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Arylpiperidines as a new class of oxidosqualene cyclase inhibitors
Marco Keller a, Annette Wolfgardt a, Christoph Müller a, Rainer Wilcken b,
b
c
c
c
€
Frank M. Bockler , Simonetta Oliaro-Bosso , Terenzio Ferrante , Gianni Balliano ,
,
*
Franz Bracher a
a Department of Pharmacy e Center for Drug Research, Ludwig-Maximilians University, Butenandtstr. 5-13, 81377 Munich, Germany
b Department of Pharmacy, Eberhard-Karls University, Auf der Morgenstelle 8, 72076 Tübingen, Germany
c Department of Drug Science and Technology, University of Torino, Via Pietro Giuria, 9, 10125 Torino, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The cyclization of oxidosqualene to lanosterol, catalyzed by the enzyme oxidosqualene cyclase (OSC),
goes through a number of carbocationic high energy intermediates (HEI), and mimicking these in-
termediates is a promising approach for the development of OSC inhibitors. 3-Arylpiperidines (or tet-
rahydropyridines) were designed as steroidomimetic rings A þ C equivalents containing two protonable
amino groups for mimicking both the pro-C4 HEI and the pro-C20 HEI of the OSC-mediated cyclization
cascade. Inhibitory activity is strongly dependent on the nature of the lipophilic substituent representing
an equivalent of the sterol side chain. Here aromatic residues (substituted benzyl, cinnamyl, naph-
thylmethyl) were found to be most suitable. Docking experiments on a first optimized 3-arylpiperidine
compound led to an isomeric 4-arylpiperidine with submicromolar activity on human OSC. This inhibitor
Received 12 July 2014
Received in revised form
10 December 2015
Accepted 12 December 2015
Available online 17 December 2015
Keywords:
Oxidosqualene cyclase
Enzyme inhibitor
High energy intermediate
Arylpiperidine
reduced total cholesterol biosynthesis in a cellular assay with an IC50 value of 0.26
mM.
© 2015 Elsevier Masson SAS. All rights reserved.
Docking experiments
1. Introduction
this fascinating cyclization.
The resulting sterols exhibit important physiological roles.
Cholesterol in man is a precursor of sexual hormones, mineralo-
corticoids, and glucocorticoids, as well as a risk factor in cardio-
Sterol biosynthesis is a complex biosynthetic pathway in which
the pre-squalene part is devoted to building the open triterpene
2,3-oxidosqualene starting from the common precursor acetyl-CoA,
and in the “tailoring section” the sterol intermediates are remod-
elled to generate the species-specific stereols (cholesterol, ergos-
terol or phytosterols, in animal, fungal or plant cells, respectively).
The two sections are connected by one of the most outstanding
mono-enzymatic reactions in nature, the cyclization of 2,3-
oxidosqualene to the tetracyclic steroids, catalyzed by the enzyme
oxidosqualene cyclases (OSC; EC 5.4.99.7). This reaction produces
lanosterol in non-photosynthetic organisms and, mostly, cyclo-
artenol in photosynthetic organisms. This reaction has been studied
mechanistically in detail over several decades, and is now under-
stood quite well [1,2]. Two crystal structures of human OSC, one
with lanosterol, the product of the enzymatic reaction, and one
with the inhibitor Ro 48e8071, have been published [3]. They
provide unique insights into the three-dimensional structure of the
enzyme and the contribution of the amino acids in its active site to
vascular diseases. Ergosterol is
a crucial component of cell
membranes in pathogenic fungi and protozoa. Therefore, oxi-
dosqualene cyclases are attractive targets for designing cholesterol
lowering [4], antifungal [5,6], and antiparasitic agents [7e9]. Very
recently, anticancer effects of OSC inhibitors against glioblastoma
[10] and breast cancer cells [11,12] have been demonstrated. Thus,
novel potent, selective, and non-toxic OSC inhibitors are highly
desirable.
OSC-catalyzed cyclization of 2,3-oxidosqualene is triggered by
protonation of the epoxide ring of the substrate and proceeds
through a number of carbocationic high energy intermediates
(HEI). This occurs during both the formation of the new carbon-
ecarbon bonds for the construction of the tetracyclic steroid scaf-
fold (in the sequence pro-C4, C10, C8, C13 and C20; Fig. 1A) and the
subsequent rearrangement of the protosteryl carbocation pro-C20
(in the sequence pro-C17, C13, C14, and C8 or C9 depending on
the product) until the removal of a proton and formation of lano-
sterol or cycloartenol [1]. Since HEI generally interact very tightly
with the enzyme, compounds mimicking the HEI have a much
* Corresponding author.
E-mail address: Franz.Bracher@cup.uni-muenchen.de (F. Bracher).
http://dx.doi.org/10.1016/j.ejmech.2015.12.025
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

14
M. Keller et al. / European Journal of Medicinal Chemistry 109 (2016) 13e22
Fig. 1. Oxidosqualene cyclase (OSC)-mediated cyclization cascade starting with protonation of the epoxide, going through five carbocationic high energy intermediates (positions
marked with an *) (A); Selected OSC inhibitors: acyclic azasqualene analogue of the pro-C4 HEI (B), monocyclic OSC inhibitor mimicking the pro-C20 HEI (C), bicyclic inhibitors
mimicking the pro-C8 (D) and pro-C4 (or pro-C10) HEI (E), steroidal pro-C20 HEI analogue (F), and the non-terpenoic benzophenone-type OSC inhibitor Ro 48e8071 (G).
higher affinity (up to three orders of magnitude) for the enzyme
than analogues of the substrate, and thus have the potential to be
highly effective enzyme inhibitors [13]. Based on this rationale, a
considerable number of OSC inhibitors mimicking individual
cationic HEI of the cascade described above have been developed in
the past (Fig. 1). These inhibitors contain aliphatic amino groups
which are protonated under physiological conditions, and thus can
mimic the cationic HEI.
Among already established inhibitors of OSC from various or-
ganisms are azasqualenes (B) [14,15], and various monocyclic (C)
[5,16], bicyclic (D, E) [14,17], and tricyclic [18] steroidomimetic
analogues of carbocationic HEI. Surprisingly, aminosteroids (e.g. F),
which are structurally closely related to the protosteryl cation (pro-
C20 HEI), were found to be poor OSC inhibitors [18,19], whereas
flexible mimics of the pro-C20 HEI (C [5] and 19-azasqualenes [15])
are potent inhibitors. Furthermore, aminoalkyl benzophenones (G)
[4,20,21] and related compounds [9,22e24], fulfilling concise
structural requirements [25], are OSC inhibitors. The mode of
binding of Ro 48e8071 (G) to human OSC has been elucidated by
co-crystallization [3].
In continuation of our recent work on the development of in-
hibitors of oxidosqualene cyclases from various organisms
[9,17,22,23] and other enzymes in ergosterol [26] and cholesterol
biosynthesis [27e30] we describe here a novel chemotype of se-
lective inhibitors of human oxidosqualene cyclase. Inspired by the
fact that numerous cationic HEI occur in the course of the con-
struction of the steroid core in this mono-enzymatic reaction, we
intended to develop inhibitors which are able to mimic both the
first occurring HEI (at pro-C4) and the one appearing furthest away
from ring A, namely the pro-C20 HEI (protosteryl cation) (Fig. 2). As
a central building block we selected a benzene ring resembling ring
C of the sterol, to this we attached a piperidine (or tetrahydropyr-
idine) ring as a ring A mimic containing the protonable nitrogen at a
position fitting with C-4 of the sterol. The second protonable amino
group was introduced in the para position in the form of a benzylic
amino group. A methyl substituent at this amino group would
resemble the C-21 methyl group of the sterols, whereas an addi-
tional substituent (SC) was to mimic the lipophilic side chain of the
steroids. Since the arylpiperidines designed this way have struc-
tural similarity to “steroidomimetic” biphenyl derivatives, in-
hibitors of C17,12-lyase in estrogen biosynthesis [31,32], we
expected them to have affinity for the steroid-producing enzyme
OSC. On the other hand, the target compounds should have suffi-
cient structural flexibility to avoid the affinity problems of fully
steroidal mimics of the pro-C20 HEI described above (Fig. 1F).
The newly designed molecules were assayed in a whole-cell
assay suitable for detection of target enzymes in the post-
squalene part of cholesterol biosynthesis [27]. Compounds which
turned out to be OSC inhibitors in this assay were further submitted
to assays on inhibition of OSC from different organisms (Saccha-
romyces cerevisiae; Homo sapiens and Trypanosoma cruzi, both
expressed in the OSC-defective yeast S. cerevisiae) [33], and for
determination of their inhibition of total cholesterol biosynthesis in
a human cell line [27].
2. Results and discussion
2.1. Chemistry
The target 3-arylpiperidines and 3-aryltetrahydropyridines
Fig. 2. The pro-C4 HEI and the pro-C20 HEI of the OSC-mediated cyclization cascade. 3-Arylpiperidines (or tetrahydropyridines) as steroidomimetic rings A þ C equivalents (right).
The two protonable amino groups are designed to mimic both the pro-C4 HEI and the pro-C20 HEI. (SC ¼ lipophilic side chain equivalent).

M. Keller et al. / European Journal of Medicinal Chemistry 109 (2016) 13e22
15
(Fig. 2) were obtained from the central building block 1 (Scheme 1),
which is readily available through Suzuki cross-coupling [34] of 3-
pyridylboroxine and 4-bromo-N-methylbenzylamine. After pro-
tection of the benzylic amino group as the Boc derivative 2, the
pyridine was converted to the N-methylpyridinium salt 3 by reac-
tion with iodomethane. Subsequent reduction with sodium boro-
hydride [35] gave the 1,2,5,6-tetrahydropyridine 4 in good yield.
After deprotection with trifluoroacetic acid, the lipophilic side
chain equivalents were attached to the resulting secondary amine 5
using two different protocols depending on the commercial avail-
ability of the building blocks. We selected branched alkyl chains,
which mimic the sterol side chain very well, different benzyl resi-
dues of similar size (which can be found in a number of
benzophenone-type OSC inhibitors [4,8,9,20,21]), as well as a cin-
namyl group (inspired by the side chain of the squalene epoxidase
or tetrahydropyridine rings of compounds of type 6 and 9,
mimicking the pro-C4 HEI, contributes to OSC inhibition, we pre-
pared pyridine congeners 10a and 10b bearing two of the most
promising side chain equivalents. For this purpose, building block 1
was reacted in the manner described above with 4-
chlorobenzaldehyde/sodium borohydride and cinnamyl bromide/
triethylamine, respectively (Scheme 2).
The role of the central benzene ring (equivalent to ring C of the
steroids) was investigated by replacing it with a 1,2,3-triazole ring,
easily accessible via a modified Huisgen azide-alkyne cycloaddition
[37]. The N-protected racemic 3-azidopiperidine 11 [38] and the
propargylamine 12 [39] bearing the promising 4-chlorobenzyl
residue were reacted under standard conditions (sodium ascor-
bate, copper sulfate) to give the 1,2,3-triazole 13 in good yield. After
removal of the Boc protecting group the piperidine ring was N-
inhibitor naftifine [36]), its dihydro analogue, and
a
naph-
methylated
(Scheme 3).
with
formaldehyde/sodium
cyanoborohydride
thylmethyl group (inspired by the core element of the squalene
epoxidase inhibitors naftifine and terbinafine). The tertiary amines
6a,b,d,e,g, j,l were obtained by a one-pot protocol consisting of
reaction with an appropriate aldehyde followed by reduction with
sodium borohydride. The amines 6c,f,h,i,k were readily obtained
by direct N-alkylation of 5 with the respective alkyl bromides in
DMF in the presence of triethylamine.
As the 4-chlorobenzyl compound 9g had emerged as a very
potent OSC inhibitor, we intended to investigate a considerably
more steroid-like analogue. For this purpose, we introduced a ring
A series of analogous piperidine derivatives 9a,b,c,g,i,k,l were
prepared in the same manner from the secondary amine 8. This
amine was available from tetrahydropyridine 4 by catalytic hy-
drogenation of the olefinic group to give 7, followed by removal of
the Boc protecting group. In this series we deleted a number of side
chain equivalents, which had shown no promise in a preliminary
screening of tetrahydropyridines 6a-l.
Scheme 2. Arylpyridines prepared from 1: Reagents and conditions: For 10a: 4-
chlorobenzaldehyde, NaBH4, MeOH, AcOH, r.t., 18 h (81%); (f) 3-bromo-1-phenyl-1-
propene, Et3N, DMF, r.t., 18 h (53%).
In order to confirm that a protonable nitrogen in the piperidine
Scheme 1. Reagents and conditions: (a) Boc2O, Et3N, MeOH, r.t., 3 h (74%); (b) CH3I, acetone, r.t., 18 h (65%); (c) NaBH4, MeOH, r.t., 18 h (71%); (d) CF3COOH, CH2Cl2, r.t., 1 h (84e92%);
(e) ReCHO, NaBH4, MeOH, AcOH, r.t., 18 h (45e96%); (f) ReCH2Br, Et3N, DMF, r.t., 18 h (20e65%); (g) H2, Pd/C (cat.), MeOH, r.t., 20 h (77%).

16
M. Keller et al. / European Journal of Medicinal Chemistry 109 (2016) 13e22
coupling, followed by reductive amination with methylamine
[45], was protected with the Boc group and then hydrogenated in
the same manner as described for 20 to give the 4-arylpiperidine
26. N-Methylation of the piperidine ring, N-deprotection at the
benzylic position, and N-benzylation with 4-chlorobenzaldehyde
and sodium borohydride gave the target compound 29 (Scheme 5).
2.2. Screenings
2.2.1. Identification of inhibitors of enzymes in cholesterol
biosynthesis
In a first step the compounds were subjected to an in-house
qualitative screening system for the detection of target enzymes
in the post-squalene part of cholesterol biosynthesis [27]. In this
whole-cell assay human HL-60 cells were incubated with the test
compounds. After cell lysis and extraction, the sterol pattern was
analyzed by GCeMS. Inhibition of particular enzymes leads to
typical changes in the sterol pattern. The results are presented in
Table 1. In the tetrahydropyridine series containing 12 different side
chain equivalents (see Scheme 1), compounds 6a-c bear aliphatic
chains, however, despite having the highest similarity to the native
cholesterol side chain, these did not interfere with cholesterol
biosynthesis. In contrast, most of the compounds bearing aromatic
Scheme 3. Reagents and conditions: (a) sodium ascorbate (cat.), CuSO4 x 5H2O (cat.),
n-butanol/water, r.t., 14 h (58%); (b) CF3COOH, CH2Cl2, r.t., 1 h (78%); (c) aqueous HCHO,
NaCNBH3, AcOH (cat.), MeOH, r.t., 3 h (78%).
D equivalent by replacing the central benzyl moiety of 9a with an
indane. This modification was inspired by reports on estrogenic
[40] as well as antiestrogenic [41] compounds bearing aryl-
perhydroindane scaffolds, and on inhibitors of the enzymes 17
hydroxysteroid dehydrogenase [42] and 17 -hydroxylase-17,20-
lyase [31] bearing arylindanes as rings A þ CD equivalents.
b-
a
substituents lead to
a
significant accumulation of 2,3-
oxidosqualene, clearly indicating an inhibition of the enzyme
5-Bromoindan-1-one (16) was coupled with 3-pyridylboroxine
under Suzuki conditions [34], accelerated by microwave irradia-
tion [43], to give pyridylindanone 17 in high yield. Reductive ami-
nation with methylamine and sodium borohydride gave the
methylaminoindane 18. In order to avoid the presence of two
competing secondary amino groups in the forthcoming reactions,
the secondary amino group of 18 was protected as the Boc deriv-
ative (19). High pressure catalytic hydrogenation over palladium in
methanol/acetic acid gave a moderate yield of the piperidine 20 as a
racemic mixture of inseparable diastereomers. N-Methylation of
the piperidine under standard conditions, followed by deprotection
of the amino group at the indane moiety gave the secondary amine
22, which was converted to the N-(4-chlorobenzyl) derivative 23 by
reductive benzylation with 4-chlorobenzaldehyde and sodium
cyanoborohydride (Scheme 4).
Docking experiments (see section below) suggested that the
piperidin-4-yl regioisomer of the until then most attractive inhib-
itor 9g might have even higher affinity for the active site of the
human OSC. Known phenylpyridine 24, readily available from 4-
pyridineboronic acid and 4-bromobenzaldehyde via Suzuki cross-
Scheme 5. Reagents and conditions: (a) Boc2O, Et3N, MeOH, r.t., 3 h (88%); (b) H2
(40 bar), Pd/C (cat.), MeOH-AcOH, r.t., 24 h (29%); (c) aqueous HCHO, NaCNBH3, AcOH
(cat.), MeOH, r.t.,
3 h (70%); (d) CF3COOH, CH2Cl2, r.t., 1 h (75%); (e) 4-
chlorobenzaldehyde, NaBH4, MeOH, AcOH, r.t., 18 h (74%).
Scheme 4. Reagents and conditions: (a) 3-pyridylboroxine, Pd(Ph3P)4 (cat.), Na2CO3, dioxane-water, microwave irradiation, 95 ꢀC, 15 min (83%); (b) CH3NH2eHCl, Et3N, MgClO4
(cat.), 1,2-dichloroethane, r.t., 18 h (69%); (c) Boc2O, Et3N, MeOH, r.t., 3 h (87%); (d) H2 (40 bar), Pd/C (cat.), MeOH-AcOH, r.t., 24 h (29%); (e) aqueous HCHO, NaCNBH3, AcOH (cat.),
MeOH, r.t., 3 h (78%); (f) CF3COOH, CH2Cl2, r.t., 1 h (97%); (g) 4-chlorobenzaldehyde, NaBH4, MeOH, AcOH, r.t., 18 h (71%).

M. Keller et al. / European Journal of Medicinal Chemistry 109 (2016) 13e22
17
Table 1
Target enzymes of the inhibitors in cholesterol biosynthesis and effect on total cholesterol biosynthesis of the OSC inhibitors in a whole-cell assay; general cytotoxicity
determined in a MTT assay on a human cell line (HL-60).
Compound
Target enzyme in cholesterol biosynthesis
Inhibition of total cholesterol biosynthesis IC50
(
m
M)a
Cytotoxicity (MTT assay) IC50 (m
M)a
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
9a
9b
9c
9g
9i
9k
9l
10a
10b
15
23
29
eb
n.d.c
n.d.
n.d.
n.d.
n.d.
2.29
2.14
n.d.
0.68
1.28
n.d.
0.37
n.d.
n.d.
n.d.
1.29
1.10
n.d.
1.32
n.d.
n.d.
n.d.
0.14
0.26
0.31
0.092
n.d.
>50
>50
>50
>50
>50
28
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
40
e
e
e
e
OSCd
OSC
e
OSC
OSC
(OSC)e
OSC
e
e
e
OSC
OSC
(OSC)e
OSC
e
21
44
e
7-DHCRf
OSC
OSC
OSC
OSC
n.d.
>50
>50
>50
>50
8
BIBX 79
Ro 48e8071
Cisplatin
5
a
Experiments carried out in triplicate.
No changes in the sterol pattern downstream squalene detectable.
Not determined.
Inhibition of oxidosqualene cyclase, detected by the accumulation of 2,3-oxidosqualene.
Only very weak accumulation of 2,3-oxidosqualene.
b
c
d
e
f
Weak inhibition of 7-dehydrocholesterol reductase, detected by accumulation of 7-dehydrocholesterol.
oxidosqualene cyclase. In the series of N-benzyl compounds the
nature of the substituents had a strong influence on the biological
activity. Compounds with unsubstituted (6d) or electron-rich (6e)
benzyl residues were inactive, whereas those bearing small
electron-releasing substituents (NO2, Cl in 6f,g) at the benzyl group
showed significant activity. An ester substituent (6h), however, led
to complete loss of activity. Larger aromatic substituents like styryl
(6i,j) and naphthylmethyl (6l) were tolerated well. Virtually iden-
tical results were obtained in the piperidine series (9g,i,l). Replac-
ing the piperidine and tetrahydropyridine rings with a pyridine
ring (10a,b) led to a complete loss of activity. Compound 15, bearing
a 1,2,3-triazole ring instead of the central benzene ring led only to a
very slight accumulation of dehydrocholesterol, indicating an in-
hibition of the enzyme 7-dehydrocholesterol reductase, the very
last enzyme in cholesterol biosynthesis. But in comparison to the
and piperidine-type (9g,i,l) compounds inhibited total cholesterol
biosynthesis with IC50 values in the range of 0.4e2.3 M. The
indane congener 23 and the piperidin-4-yl regioisomer 29 were
even more potent (0.14 and 0.26 M), showing activities compa-
rable to those of the prominent OSC inhibitors BIBX 79 [24] and Ro
48e8071 [4].
m
m
2.2.3. Assay for inhibition of ergosterol biosynthesis in fungi
In order to investigate the antifungal potential [5,6] and the
species selectivity of the new OSC inhibitors, the active compounds
were subjected to our recently developed assay for the identifica-
tion of inhibitors of ergosterol biosynthesis in fungi [44]. This assay
works in a similar mode as the one described above for cholesterol
biosynthesis and was performed on the strains S. cerevisiae, Yar-
rowia lipolytica, and Candida glabrata. Neither of the highly active
inhibitors of human OSC described above led to a recognizable
accumulation of precursor sterols, nor did incubation with these
compounds reduce the ergosterol content of the cells (data not
shown here). This indicates that the inhibitors described here do
not interfere to a mentionable extent with the biosynthesis of
ergosterol in fungi.
phenethyl
tetrahydroisoquinoline-type
7-dehydrocholesterol
reductase inhibitors described by us previously [29], the potency
of 15 is very poor. The piperidin-4-yl regioisomer 29 of the inhibitor
9g was found to be an inhibitor of OSC as well.
2.2.2. Assay for inhibition of total cholesterol biosynthesis in cells
Compounds which led to a significant accumulation of 2,3-
oxidosqualene in the first, qualitative assay were further evalu-
ated for their inhibition of total cholesterol biosynthesis in HL-60
cells [27]. In this assay the cells are incubated with the inhibitors
in the presence of 2-13C-acetate. This leads to an incorporation of
13C atoms into the cholesterol molecules biosynthesized during the
incubation period, and allows to distinguish newly synthesised
cholesterol from unlabelled matrix cholesterol, present in the cells
before incubation, by GCeMS. The IC50 values determined this way
are presented in Table 1. A number of tetrahydropyridine- (6f,g,i,j,l)
2.2.4. Cytotoxic activity
For the detection of undesired cytotoxic properties, the com-
pounds were screened in a MTT assay according to the method of
Mosmann [46] on human leukaemia HL-60 cells with cisplatin
(IC50 ¼ 5
mM) used as reference. The results are shown in Table 1.
Almost all of the compounds were free from cytotoxicity. However,
among the potent OSC inhibitors, the nitrobenzyl compound 6f
showed slight cytotoxicity (IC50 ¼ 28
mM), for this reason this
compound was not investigated further. In contrast, reference OSC

18
M. Keller et al. / European Journal of Medicinal Chemistry 109 (2016) 13e22
inhibitor Ro 48e8071 [10] (Fig.1G) showed noteworthy cytotoxicity
M). BIBX 79 [22], another prominent OSC inhibitor, was
inactive in this assay.
3. Discussion
(IC50 ¼ 8
m
The results presented here demonstrate that the concept of
mimicking two of the cationic high energy intermediates occurring
in the course of the OSC-catalyzed cyclization cascade in one
molecule is feasible. 3-Arylpiperidines and their 1,2,5,6-
tetrahydopyridine congeners are virtually equipotent inhibitors of
human OSC in the case they bear a suitable lipophilic sterol side
chain equivalent. Surprisingly, branched aliphatic residues which
are most similar to the native sterol side chain, proved to be inef-
fective, whereas substituted benzyl, cinnamyl and naphthylmethyl
groups led to potent enzyme inhibitors. This confirms earlier ob-
servations from others [13,16] and our groups [30,47], that the
shape of the group occupying the binding site of the inconspicuous
sterol side chain is of enormous significance for the affinity of sterol
biosynthesis inhibitors.
Docking experiments clearly indicate that the 4-chlorobenzyl
compound 9g occupies the substrate binding site of human OSC
very well and its binding mode is very similar to the one demon-
strated for the established inhibitor Ro 48e8071 in a crystal
structure [3]. Both the piperidine residue designed to mimic the
pro-C4 HEI, and the benzylic amino group designed to mimic the
pro-C20 HEI, contribute significantly to the interaction with the
active site of the enzyme.
2.2.5. Effect of inhibitors on OSCs from different organisms
OSC inhibitors are potential cholesterol-lowering drugs, but also
play a role in treatment of fungal and protozoal infections. In order
to elucidate the respective potential and species-selectivities, we
performed in vitro experiments with human, fungal, and protozoal
OSC. Activity of the compounds as inhibitors of OSC was tested by
incubating homogenates prepared from cell cultures of different
yeast strains with radiolabelled 2,3-oxidosqualene in the presence
of increasing inhibitor concentrations. The results are presented in
Table 2. Only the most active substances from the abovementioned
investigations were analyzed. Tetrahydropyridines 6g,i,l, piperi-
dine congeners 9g,i,l, indane 23, and 29, the 4-piperidyl analogue
of 9g, showed very similar patterns of inhibition. None of these
compounds inhibits OSC from T. cruzi, and S. cerevisiae OSC is only
slightly inhibited by two of the compounds (6g, 29). In contrast,
most of the compounds were identified as moderate to strong in-
hibitors of human OSC. The IC50 values determined for human OSC
were in good concordance with the values determined for inhibi-
tion of total cholesterol biosynthesis in the whole-cell assay.
In contrast, Ro 48-8071 is an unselective inhibitor of the three
OSCs.
In contrast to the unselective reference inhibitor Ro 48e8071
[9], the newly designed inhibitors did not show noteworthy inhi-
bition of yeast and trypanosomal OSC, and were devoid of unde-
sired cytotoxicity.
2.3. Docking experiments
For most of the compounds the IC50 values obtained on human
OSC correlated very well with the IC50 values for inhibition of total
cholesterol biosynthesis in a whole-cell assay. From this fact we
may draw the conclusion that for these compounds inhibition of
OSC is the dominating, if not exclusive factor triggering the atten-
uation of cholesterol biosynthesis in cells. Surprisingly, the naph-
thylmethyl compounds (6l, 9l; factors 6 to >25) and the rigid
indane derivative 23 (factor 30) showed significantly higher activ-
ity in the cellular assay on cholesterol biosynthesis than in vitro on
the human OSC. This suggests that other factors, probably the in-
hibition of an enzyme in the pre-squalene part of cholesterol
biosynthesis (which cannot be detected by our assay [25]),
contribute significantly to the overall effects of these compounds.
For this reason the 4-chlorobenzyl compound 9g was selected as
the lead structure for further optimization. Replacing the central
phenyl ring with a 1,2,3-triazole ring completely eliminated OSC
inhibitory activity. However, the achiral 4-piperidyl regioisomer 29,
which was considered on the basis of our docking experiments,
turned out to be even more active (factor 4e5 in both assays) than
In order to understand the requirements for binding of the most
active OSC inhibitors, docking experiments were performed based
on the crystal structure of human OSC co-crystallized with Ro
48e8071 [3]. The results shown in Fig. 3 demonstrate that com-
pound 9g (S enantiomer shown) has a binding pose very similar to
the one of Ro 48e8071. Both molecules show edge-to-face stacking
of the haloarene ring with Trp192 and a cation-p interaction with
Trp581. The only difference is that the protonated piperidine ni-
trogen of 9g appears to be too far away from the carboxylate group
of Asp455 for building up the salt bridge seen with the side chain of
Ro 48e801. The benzylic amino group of 9g is sandwiched between
His232 and Phe696, forming cation-
p interactions. For the R
enantiomer the docking experiments predicted comparable in-
teractions (data not shown). This suggests that both enantiomers of
9g contribute to the observed biological activity. The isomeric
achiral piperidine 29 shows a binding pose very similar to the one
of (S)-9g, but due the more distal position of the ring nitrogen this
isomer is more likely to build up the salt bridge with Asp455, what
can explain the observed increase in inhibitory activity.
Table 2
Effect of inhibitors on oxidosqualene cyclase activity of homogenates prepared from yeast recombinant strains SMY8 expressing H. sapiens and T. cruzi OSC, as well as
S. cerevisiae wild-type strain FL100. For comparison, IC50 values determined for inhibition of total cholesterol biosynthesis in a whole-cell assay (see Table 1).
Compound
IC50 (mM)
OSC from H. sapiensa
OSC from T. cruzia
OSC from S. cerevisiaea
Cholesterol biosynthesis inhibition
6g
6i
6l
9g
9i
9l
23
29
2.51
5.28
>10
2.06
2.06
8.51
4.22
0.51
0.17
>10
>10
>10
>10
>10
>10
>10
>10
0.90
6.35
>10
>10
>10
>10
2.14
0.68
0.37
1.29
1.10
1.32
0.14
0.26
0.092c
>10
>10
4.83
Ro 48e8071b
0.90c
a
Values are the means of two separate experiments, each one carried out in duplicate. The maximum deviations from the mean were less than 10%.
From Ref. [9].
Determined on S. cerevisiae OSC expressed in SMY8.
b
c

M. Keller et al. / European Journal of Medicinal Chemistry 109 (2016) 13e22
19
the 3-piperidyl compound 9g, and favourably compares with the
equipotent, but unselective OSC inhibitor Ro 48e8071. Compounds
9g and 29 show acceptable lipophilicity (calculated logP values
about 4.7), and fulfil Lipinski's rule of five [48], so they can be
considered promising candidates for further development.
4. Experimental
4.1. General Procedure A: introduction of the N-Boc-protecting
group in secondary amines
The amine (1 equiv) was dissolved in methanol (4 mL/mmol),
triethylamine (2 equiv) and di-tert-butyl dicarbonate (2 equiv)
were added, and the mixture was stirred for 3 h at room temper-
ature. The solvent was evaporated, the residue suspended in satd.
aqueous Na2CO3 solution and extracted three times with CH2Cl2.
The combined organic layers were dried over Na2SO4 and evapo-
rated. Purification was done by flash column chromatography.
4.2. General Procedure B: reductive N-alkylation of secondary
amines
To a solution of one equivalent of the amine (0.2e1.0 mmol), the
appropriate aldehyde (1.5e2.5 equiv) and glacial acetic acid (0.5
equiv) in methanol (4 mL/mmol amine), sodium borohydride
(1.85e2 equiv) was added in portions over a period of 10 min with
stirring, and the reaction mixture was stirred for 18 h at room
temperature. The solvent was evaporated under reduced pressure,
the residue taken up in CH2Cl2 (30 mL/mmol amine) and washed
with concentrated sodium carbonate solution (30 mL/mmol
amine). The aqueous solution was extracted with CH2Cl2
(3 ꢁ 20 mL/mmol amine) and the combined organic layers were
dried over MgSO4 and evaporated. The residue was purified by flash
column chromatography (CH2Cl2:CH3OH 9:1 and 1% NEt3).
5. 3.General Procedure C: N-alkylation of secondary amines
A solution of secondary amine (0.55 mmol), the appropriate
alkyl bromide (0.36e0.46 mmol) and triethylamine (0.55 mmol) in
N,N-dimethylformamide (3 mL) was stirred for 18 h at room tem-
perature. The solvent was evaporated under reduced pressure and
concentrated sodium carbonate solution (30 mL) was added to the
crude residue. The aqueous solution was extracted with CH2Cl2
(3 ꢁ 30 mL), the combined organic layers were dried over MgSO4
und evaporated. The crude material was purified by flash column
chromatography (CH2Cl2:CH3OH, 9:1 and 1% NEt3).
5.1. General Procedure D: reductive N-methylation of secondary
amines
The N-Boc-amine (1 equiv) and was dissolved in methanol (1%
acetic acid) (3 mL/mmol), formaldehyde (5 equiv, 37% in H2O) and
sodium cyanoborohydride (2.5 equiv) were added, and the mixture
was stirred for 3 h at room temperature. The solvent was evapo-
rated, the residue suspended in satd. Na2CO3 solution and extracted
three times with CH2Cl2. The combined organic layers were dried
over Na2SO4 and evaporated. Purification was done by flash column
chromatography.
Fig. 3. A: Overview of Ro 48e871 (grey carbons) binding into the OSC active site (from
PDB code 1W6J; dockings gave identical results). Key ligand-protein contacts include
edge-to-face stacking of the bromobenzene moiety with Trp192 and
the central fluorobenzene moiety with Phe696. The protonated tertiary amine side-
chain forms a cation- contact with Trp581 as well as a salt bridge with Asp455. B:
pep stacking of
p
Docking pose of compound 9g (pale red carbons; S isomer shown) docked into the OSC
active site. 9g shows a binding mode very similar to the one of Ro4877, and forms
overall similar interactions. The most notable difference is that its central protonated
5.2. General Procedure E: cleavage of the N-Boc-protecting group
The N-Boc-amine was dissolved in CH2Cl2 and trifluoracetic acid
(1:1) (12 mL/mmol) and the mixture stirred for 1 h at room tem-
perature. The mixture was alkalized with satd. Na2CO3 solution and
extracted three times with CH2Cl2. The combined organic layers
amine moiety is sandwiched between His232 and Phe696, forming cation-
teractions, while its central benzene moiety does not engage in face-to-face
p in-
pep
stacking interactions. C: The docking pose of compound 29 (pale green carbons) is very
similar to the one of compound 9g.

20
M. Keller et al. / European Journal of Medicinal Chemistry 109 (2016) 13e22
were dried over Na2SO4 and evaporated.
(53); HR-MS (EI, 70 eV): m/z
¼
318.2289 [M]þ: calcd for
C19H30N2O2: 318.2307.
5.3. 3-[4-(N-tert-butoxycarbonyl-N-methylaminomethyl)-phenyl]-
1-methylpyridinium iodide (3)
5.6. (RS)-(N-Methyl-4-(1-methylpiperidine-3-yl)-benzylamine (8)
To a solution of tert-butyl N-methyl-N-4-(pyridine-3-yl)-ben-
zylcarbamate (2) (175 mg, 0.557 mmol) in acetone (5 mL) iodo-
Prepared following General Procedure E using 7 (200 mg,
0.628 mmol) to yield 115 mg (84%) of 8 as yellow oil (flash column
chromatography CH2Cl2:CH3OH 9:1 and 1% NEt3); 1H NMR
methane (347
mL, 5.57 mmol) was added and the mixture was
stirred for 18 h at room temperature. The precipitate was collected
by filtration, washed with cold acetone (10 mL) and recrystallized
from acetone to afford 3 as white solid (158 mg, 65%); 1H NMR
(500 MHz, CD2Cl2):
d
¼ 7.45e7.43 ppm (m, 2H), 7.25e7.23 (m, 2H),
3.87 (s, 2H), 3.03e2.94 (m, 3H), 2.46 (s, 3H), 2.38 (s, 3H), 2.20e2.11
(m, 2H), 1.90e1.82 (m, 3H), 1.48e1.41 (m, 1H); 13C NMR (100 MHz,
(500 MHz, CD2Cl2):
d
¼ 9.31 ppm (s, 1H), 8.89e8.85 (m, 2H),
CD2Cl2):
d
¼ 144 ppm, 132.8, 130.5, 128.5, 127.5, 62.3, 55.8, 55.3,
46.1, 42.1, 33.4, 30.6, 25.5; IR (NaCl): n ¼ 3396 cmꢂ1, 2933, 2852,
2779, 2359, 1684, 1517, 1465, 1375, 1284, 1242, 1198, 1157, 1130,
1090, 1060, 1024, 985, 912, 822, 798; MS (CI, CHþ5 ): m/z (%) ¼ 219
(100) [MþH]þ, 188 (30); MS (EI, 70 eV): m/z (%) ¼ 218 (12) [M]þ, 58
(100); HR-MS (EI, 70 eV): m/z ¼ 218.1778 [M]þ: calcd for C14H22N2:
218.1783.
e
8.17e8.14 (m, 1H), 7.87e7.86 (m, 2H), 7.51e7.49 (m, 2H), 4.55 (s,
2H), 4.51 (s, 3H), 2.91 (s, 3H), 1.53e1.47 (m, 9H); 13C NMR (125 MHz,
CD2Cl2):
d
¼ 157.6 ppm, 145.1, 144.9, 144.0, 142.3, 133.8, 129.7, 129.3,
ꢂ1
e
129.0, 126.0, 81.7, 53.3, 49.3, 34.9, 28.8; IR (KBr): n ¼ 3447 cm
,
3022, 2974, 2951, 1675, 1496, 1479, 1421, 1390, 1369, 1325, 1307,
1232, 1200, 1158, 949, 885, 835, 796, 772, 674, 616, 570; MS (CI,
CHþ5 ): m/z (%) ¼ 315 (6), 299 (84), 243 (100), 199 (36); MS (EI,
70 eV): m/z (%) ¼ 314 (16), 242 (100), 197 (46), 168 (62), 142 (54);
HR-MS (EI, 70 eV): m/z ¼ 313.1910 [M]þ: calcd for C19H25N2O2:
313.1916.
5.7. N-(4-Chlorobenzyl)-N-methyl-4-(1-methylpiperidine-3-yl)-
benzylamine (9g)
Prepared following General Procedure B using 8 (200 mg,
5.4. tert-Butyl N-methyl-4-(1-methyl-1,2,5,6-tetrahydropyridine-
3-yl)-benzylcarbamate (4)
0.628 mmol) and 4-chlorobenzaldehyde (111
mL, 0.942 mmol) to
yield 220 mg (70%) of 9g as yellow oil; 1H NMR (500 MHz, CD2Cl2):
d
¼ 7.32e7.28 ppm (m, 6H), 7.20e7.18 (m, 2H), 3.47 (s, 2H), 3.46 (s,
Sodium borohydride (260 mg, 6.76 mmol) was added in por-
2H), 3.07e2.97 (m, 3H), 2.39 (s, 3H), 2.18e2.12 (m, 5H) 1.95e1.79
(m, 3H), 1.46 (qd, J ¼ 3.9 and 12.3 Hz, 1H); 13C NMR (100 MHz,
tions to
a solution of 3-[4-(N-tert-butoxycarbonyl-N-methyl-
aminomethyl)-phenyl]-1-methylpyridinium iodide (3) (595 mg,
1.35 mmol) in methanol (20 mL) over a period of 10 min and the
mixture was stirred for 18 h at room temperature. The reaction
mixture was poured into a conc. aqueous Na2CO3 solution (20 mL)
and extracted with CH2Cl2 (3 ꢁ 30 mL). The combined organic
layers were dried over MgSO4 and evaporated. The residue was
purified by flash column chromatography (CH2Cl2:CH3OH 9:1) to
afford 4 as colourless oil (303 mg, 71%): 1H NMR (400 MHz, CD2Cl2):
CD2Cl2):
d
¼ 142.7 ppm, 138.8, 138.1, 132.7, 130.6, 129.4, 128.6, 127.4,
62.5, 61.8, 61.3, 55.7, 45.9, 42.3, 41.9, 30.9, 25.2; IR (NaCl):
n ¼ 3416 cmꢂ1, 2933, 2781, 1597, 1513, 1498, 1463, 1364, 1284, 1241,
e
1130, 1089, 1060, 1014, 985, 804, 670; MS (CI, CHþ5 ): m/z (%) ¼ 343
(100) [MþH]þ, 231 (6), 188 (18), 168 (16), 125 (12); MS (EI, 70 eV):
m/z (%) ¼ 342 (18) [M]þ, 58 (100); HR-MS (EI, 70 eV): m/
z ¼ 342.1863 [M]þ: calcd for C21H27ClN2: 342.1863.
d
¼ 7.33e7.31 ppm (m, 2H), 7.18e7.16 (m, 2H), 6.14e6.12 (m, 1H),
5.8. tert-Butyl N-methyl-N-[4-(pyridin-4-yl)-benzyl]-carbamate
(25)
4.38 (s, 2H), 3.26 (dd, J ¼ 2.6 and 4.5 Hz, 2H), 2.78 (s, 3H), 2.55 (t,
J ¼ 5.8 Hz, 2H), 2.42 (s, 3H), 2.37e2.32 (m, 2H), 1.50e1.40 (m, 9H);
13C NMR (100 MHz, CD2Cl2):
d
¼ 156.0 ppm,138.8,137.2,134.7,127.1,
Prepared following General Procedure A using 24 (200 mg,
1.01 mmol) to yield 264 mg (88%) of 25 as yellow oil; (flash column
chromatography ethyl acetate/hexanes 1:1 and 2% NEt3); 1H NMR
124.9, 122.1, 79.3, 56.2, 52.2, 51.4, 45.7 33.8, 28.2, 26.5; IR (NaCl):
n ¼ 2973 cmꢂ1, 2934, 2839, 2781, 1903, 1694, 1513,1480, 1455, 1425,
e
1391, 1365, 1289, 1247, 1174, 1142, 1047, 1001, 973, 953, 920, 880,
819, 798, 770, 666; MS (CI, CHþ5 ): m/z (%) ¼ 317 (36) [MþH]þ, 261
(100), 186 (16); MS (EI, 70 eV): m/z (%) ¼ 316 (41) [M]þ, 260 (62),
216 (58), 186 (45), 172 (56), 142 (100), 130 (76), 115 (28); HR-MS (EI,
70 eV): m/z ¼ 316.2146 [M]þ: calcd for C19H28N2O2: 316.2151.
(CD2Cl2, 400 MHz):
d
¼ 8.62 ppm (dd, J ¼ 1.6 and 4.5 Hz, 2H),
7.68e7.62 (m, 2H), 7.52 (dd, J ¼ 1.6 and 4.5 Hz, 2H), 7.38e7.32 (m,
2H), 4.47 (s, 2H), 2.83 (s, 3H), 1.53e1.39 (m, 9H); 13C NMR (CD2Cl2,
125 MHz):
d
¼ 156.4 ppm, 150.7 (2C), 148.1, 140.0, 137.3, 128.5 (2C),
127.5 (2C), 121.8 (2C), 79.9, 52.6 (rotamer 1), 52.0 (rotamer 2), 34.3,
28.5 (3C); IR (NaCl): n ¼ 3028 cmꢂ1, 2974, 2928, 2358, 1690, 1597,
e
5.5. (RS)-tert-butyl N-methyl-4-(1-methylpiperidine-3-yl)-
benzylcarbamate (7)
1487, 1391, 1365, 1245, 1171, 1145, 879, 801; MS (CI, CHþ5 ): m/z
(%) ¼ 299 (100) [MþH]þ; MS (EI, 70 eV): m/z (%) ¼ 242 (48); HR-MS
(EI, 70 eV): m/z ¼ 298.1652 [M]þ, calcd for C18H22N2O2: 298.1681.
A solution of 4 (255 mg, 1.18 mmol) and palladium on carbon
(10%, 20 mg) in methanol (5 mL) was hydrogenated for 20 h at room
temperature. The mixture was filtered through celite, evaporated,
purified by flash column chromatography (CH2Cl2:CH3OH 9:1) to
afford 7 as orange oil (290 mg, 77%): 1H NMR (400 MHz, CD2Cl2):
5.8.1. tert-Butyl N-methyl-N-[4-(piperidin-4-yl)-benzyl]-carbamate
(26)
Pyridine 25 (1.96 g, 6.57 mmol) was dissolved in methanol
(5 mL) and acetic acid (50 mL). Palladium on carbon (10%, 50 mg)
was added and the mixture was stirred in a pressure vessel under
an atmosphere of hydrogen (40 bar) for 24 h at room temperature.
The mixture was filtered over celite, brought to pH 10 with NaOH,
and extracted with CH2Cl2 (3 ꢁ 30 mL). The combined organic
layers were dried over Na2SO4 and evaporated. Purification was
done by flash column chromatography (CH2Cl2/NEt3 9:1) to yield
578 mg (29%) of 26 as a brown oil; (flash column chromatography
d
¼ 7.21e7.19 ppm (m, 2H), 7.16e7.14 (m, 2H), 4.36 (s, 2H),
2.91e2.81 (m, 3H), 2.77 (s, 3H), 2.25 (s, 3H), 1.96e1.85 (m, 3H),
1.79e1.64 (m, 2H), 1.45 (s, 9H), 1.42e1.34 (m, 1H); 13C NMR
(125 MHz, CD2Cl2):
d
¼ 155.6 ppm, 144.2, 136.7, 127.8), 79.6, 63.5,
56.2, 52.6, 51.9, 46.7, 42.9, 34.0, 31.5, 28.6, 26.1; IR (NaCl):
n ¼ 2971 cmꢂ1, 2932, 2854, 2778, 1698, 1514, 1479, 1463, 1450, 1419,
e
1391, 1365, 1306, 1285, 1244, 1174, 1144, 1091, 1061, 1047, 1026, 986,
957, 880, 814, 789, 771; MS (CI, CHþ5 ): m/z (%) ¼ 319 (30) [MþH]þ,
263 (100); MS (EI, 70 eV): m/z (%) ¼ 318 (100) [M]þ, 262 (25), 217
CH2Cl2/NEt3 9:1); 1H NMR (CD2Cl2, 500 MHz):
(m, 4H), 4.36 (s, 2H), 3.16e3.10 (m, 2H), 2.78 (s, 3H), 2.73e2.66 (m,
d
¼ 7.20e7.14 ppm

M. Keller et al. / European Journal of Medicinal Chemistry 109 (2016) 13e22
21
2H), 2.63e2.55 (m, 1H), 1.80e1.74 (m, 3H), 1.63e1.52 (m, 2H), 1.46
(s, 9H); 13C NMR (CD2Cl2, 100 MHz):
for supplying S. cerevisiae strains FL100.
Appendix A. Supplementary data
d
¼ 156.5 ppm, 146.4, 136.4,
127.9 (2C), 127.3 (2C), 79.6, 52.5 (rotamer 1), 51.9 (rotamer 2), 47.5
(2C), 43.2, 35.0 (2C), 34.0, 28.6 (3C); IR (NaCl): n ¼ 2974 cmꢂ1, 2930,
e
2851, 2807, 2735,1694,1480,1449,1391,1365,1246,1173,1143, 880;
MS (CI, CHþ5 ): m/z (%) ¼ 305 (22) [MþH]þ; MS (EI, 70 eV): m/z
(%) ¼ 304 (2) [M]þ; HR-MS (EI, 70 eV): m/z ¼ 304.2148 [M]þ, calcd
for C18H28N2O2: 304.2151.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.12.025.
References
5.8.2. tert-Butyl N-methyl-N-[4-(1-methylpiperidin-4-yl)-benzyl]-
carbamate (27)
Prepared following General Procedure D using 26 (185 mg,
0.608 mmol) to yield 136 mg (70%) of 27 as colourless oil (flash
column chromatography ethyl acetate/hexanes 1:1 and 2% NEt3);
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1H NMR (CD2Cl2, 500 MHz):
d
¼ 7.21e7.18 ppm (m, 2H), 7.16e7.13
(m, 2H), 4.36 (s, 2H), 2.94e2.89 (m, 2H), 2.77 (s, 3H), 2.49e2.41 (m,
1H), 2.25 (s, 3H), 2.04e1.97 (m, 2H), 1.81e1.68 (m, 4H), 1.45 (s, 9H);
13C NMR (CD2Cl2, 100 MHz):
d
¼ 156.3 ppm, 146.0, 136.4, 127.8 (2C),
127.3 (2C), 79.6, 56.7 (2C), 52.5 (rotamer 1), 51.9 (rotamer 2), 46.7,
42.1, 34.0 (3C), 28.6 (3C); IR (NaCl): n ¼ 2973 cmꢂ1, 2934, 2846,
e
2780, 2735, 2678, 2361, 2342, 1697, 1448, 1420, 1391, 1365, 1278,
1246, 1174, 1142, 880; MS (CI, CHþ5 ): m/z (%) ¼ 319 (74) [MþH]þ; MS
(EI, 70 eV): m/z (%) ¼ 318 (18) [M]þ; HR-MS (EI, 70 eV): m/
z ¼ 318.2294 [M]þ, calcd for C19H30N2O2: 318.2307.
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5.8.3. N-Methyl-1-[4-(1-methylpiperidin-4-yl)-phenyl]-
methanamine (28)
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G. Balliano, ChemMedChem 2 (2007) 226e233.
Prepared following General Procedure E using 27 (118 mg,
0.371 mmol) to yield 62 mg (75%) of 28 as colourless wax; 1H NMR
(CD2Cl2, 500 MHz):
d
¼ 7.25e7.22 ppm (m, 2H), 7.20e7.17 (m, 2H),
3.68 (s, 2H), 2.96e2.91 (m, 2H), 2.62 (s, 1H), 2.49e2.41 (m, 1H), 2.40
(s, 3H), 2.27 (s, 3H), 2.07e1.99 (m, 2H), 1.81e1.70 (m, 4H); 13C NMR
(CD2Cl2, 100 MHz):
d
¼ 145.7 ppm, 138.2, 128.7 (2C), 127.2 (2C), 56.7
(2C), 55.8, 46.5, 42.0, 36.0, 33.8 (2C); IR (NaCl): n ¼ 3012 cmꢂ1, 2934,
2844, 2780, 2735, 2680, 2361, 2342, 1688, 1514, 1464, 1445, 1378,
1200, 1133, 994, 824, 764; MS (CI, CHþ5 ): m/z (%) ¼ 219 (100)
[MþH]þ; MS (EI, 70 eV): m/z (%) ¼ 218 (40) [M]þ; HR-MS (EI, 70 eV):
m/z ¼ 218.1832 [M]þ, calcd for C14H22N2: 218.1783.
e
5.8.4. N-(4-Chlorobenzyl)-N-methyl-1-[4-(1-methylpiperidin-4-
yl)-phenyl]-methanamine (29)
Prepared following General Procedure B using 28 (50 mg,
0.23 mmol) and 4-chlorobenzaldehyde (80 mg, 0.57 mmol) to yield
58 mg (74%) of 29 as colourless oil (flash column chromatography
ethyl acetate/hexanes 9:1 and 2% NEt3); 1H NMR (CD2Cl2,
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(2009) 8123e8137.
400 MHz):
d
¼ 7.34e7.25 ppm (m, 6H), 7.20e7.16 (m, 2H), 3.46 (s,
€
[27] M. Giera, F. Plossl, F. Bracher, Steroids 72 (2007) 633e642.
2H), 3.45 (s, 2H), 2.94e2.87 (m, 2H), 2.48e2.39 (m, 1H), 2.24 (s, 3H),
€
[28] M. Giera, D. Renard, F. Plossl, F. Bracher, Steroids 73 (2008) 299e308.
2.12 (s, 3H), 2.03e1.94 (m, 2H), 1.80e1.67 (m, 4H); 13C NMR (CD2Cl2,
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7614e7622.
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108e122.
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T. Lauterbach, R.W. Hartmann, J. Med. Chem. 51 (2008) 5009e5018.
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M. Yamaoka, M. Kusaka, A. Tasaka, Bioorg. Med. Chem. 12 (2004) 2251e2273.
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S.P.T. Matsuda, G. Balliano, Lipids 37 (2002) 1171e1176.
100 MHz):
d
¼ 145.8 ppm, 138.9, 137.4, 132.6, 130.6 (2C), 129.2 (2C),
128.6 (2C), 127.1 (2C), 61.9, 61.3, 56.8 (2C), 46.7, 42.3, 42.2, 34.1 (2C);
IR (NaCl): n ¼ 3022 cmꢂ1, 2934, 2883, 2842, 2780, 2735, 2679, 1513,
e
1489, 1461, 1445, 1378, 1364, 1279, 1134, 1087, 1068, 1031, 1015, 994,
837, 803; MS (CI, CHþ5 ): m/z (%) ¼ 345 (40) [MþH]þ, 343 (100)
[MþH]þ; MS (EI, 70 eV): m/z (%) ¼ 344 (12) [M]þ, 342 (34) [M]þ;
HR-MS (EI, 70 eV): m/z ¼ 342.1865 [M]þ, calcd for C21H27ClN2:
342.1863.
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V.V. Fokin, J. Am. Chem. Soc. 127 (2005) 210e216.
Acknowledgements
Technical support by M. Klimt and M. Stadler is greatly
acknowledged. We thank the University of Torino (Progetti di
Ricerca finanziati ex 60%-2013) for financial support of this research
to S. O. B. Thanks are due to Professor S. Matsuda (Rice University,
Houston, TX, USA) for supplying S. cerevisiae strains SMY8[pBJ1.21],
expressing the T. cruzi OSC and to Professor F. Karst (Colmar, France)
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