Design and evaluation of a novel series of 2,3-oxidosqualene cyclase inhibitors with low systemic exposure, relationship between pharmacokinetic properties and ocular toxicity

Article abstract of DOI:10.1016/j.bmc.2008.04.034

We describe the discovery of novel potent inhibitors of 2,3-oxidosqualene:lanosterol cyclase inhibitors (OSCi) from a focused pharmacophore-based screen. Optimization of the most tractable hits gave a series of compounds showing inhibition of cholesterol biosynthesis at 2 mg/kg in the rat with distinct pharmacokinetic profiles. Two compounds were selected for toxicological study in the rat for 21 days in order to test the hypothesis that low systemic exposure could be used as a strategy to avoid the ocular side effects previously described with OSCi. We demonstrate that for this series of inhibitors, a reduction of systemic exposure is not sufficient to circumvent cataract liabilities.

Full text of DOI:10.1016/j.bmc.2008.04.034

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 6218–6232
Design and evaluation of a novel series of 2,3-oxidosqualene
cyclase inhibitors with low systemic exposure, relationship
between pharmacokinetic properties and ocular toxicity
a,
a
a
a
*
`
´
´ ´
Marie-Helene Fouchet, Frederic Donche, Christelle Martin, Anne Bouillot,
Christophe Junot,b,ꢀ Anne-Benedicte Boullay, Florent Potvain, Sylvie Demaria Magny,
c
c
c
´ ´
c
d
c
a
´
´
Herve Coste, Max Walker, Marc Issandou and Nerina Dodic
aDepartment of Medicinal chemistry, Laboratoire GlaxoSmithKline, 25-27 Avenue du Que´bec, 91951 Les Ulis, France
bDepartment of Drug Metabolism and Pharmacokinetics, Laboratoire GlaxoSmithKline,
25-27 Avenue du Que´bec, 91951 Les Ulis, France
cDepartment of Biology, Laboratoire GlaxoSmithKline, 25-27 Avenue du Que´bec, 91951 Les Ulis, France
dCVU Strategy and Operations, Laboratoire GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA, USA
Received 21 November 2007; revised 9 April 2008; accepted 16 April 2008
Available online 18 April 2008
Abstract—We describe the discovery of novel potent inhibitors of 2,3-oxidosqualene:lanosterol cyclase inhibitors (OSCi) from a
focused pharmacophore-based screen. Optimization of the most tractable hits gave a series of compounds showing inhibition of
cholesterol biosynthesis at 2 mg/kg in the rat with distinct pharmacokinetic profiles. Two compounds were selected for toxicological
study in the rat for 21 days in order to test the hypothesis that low systemic exposure could be used as a strategy to avoid the ocular
side effects previously described with OSCi. We demonstrate that for this series of inhibitors, a reduction of systemic exposure is not
sufficient to circumvent cataract liabilities.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
is a key enzyme in the cholesterol biosynthesis pathway
and controls the conversion of 2,3-mono-epoxysqualene
to lanosterol. OSC is located downstream from HMG-
CoA and its inhibition should be free of isopreno¨ıd
depletion issues.
Hypercholesterolemia constitutes a major-risk factor for
coronary artery disease which is the leading cause of
death and a major source of morbidity in developed
countries.1 The benefits of lowering cholesterol level
via inhibition of cholesterol biosynthesis have been
clearly demonstrated in humans by the Statin class of
drug.2,3 Statins are inhibitors of HMGCoA reductase
and inhibit at an early rate-limiting step the cholesterol
biosynthetic cascade. This results in decreased levels of
cholesterol and depletion of mevalonate, which may be
the cause of Statin side effects such as myopathy at high
doses.4 In our search for lipid-lowering drugs, we tar-
geted an alternative point of inhibition of cholesterol
production. 2,3-oxidosqualene:lanosterol cyclase (OSC)
Several OSC inhibitors (OSCi) have been reported in the
literature, showing in vitro and in vivo potency,5–9 dem-
onstrating that OSC inhibition can deliver drugs with
profound lipid-lowering effects. Moreover, treatment
with OSCi was not associated with the development of
a range of side effects that are reported commonly for
HMGCoA reductase inhibitors, such as gall bladder,
testicular and neurological changes.10 However, other
types of toxicological issues have been reported includ-
ing skin and epididymidal changes in the dog, as well
as severe cataract formation in dogs and rodents.10
Keywords: 2,3-Oxidosqualene:lanosterol cyclase; Hypercholesterol-
emia; Pharmacophore; OSC inhibitor.
U18666A, probably the first reported OSCi,11 is known
to induce cataracts.12 U18666A was shown to intercalate
into a lens lipid model membrane, and it has been
hypothesized that the cataractogenic effect of OSCi
could be related to direct perturbation of lens membrane
*
Corresponding author. Tel.: +33 1 69 29 61 07; e-mail: marie-
helene.fouchet@gsk.com
´ ´
Present address: Laboratoire d’Etude du Metabolisme des Medica-
ꢀ
ments, CEA—Saclay, France.
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.034

M.-H. Fouchet et al. / Bioorg. Med. Chem. 16 (2008) 6218–6232
6219
structure and function.13 If this were the case, then
structural modification might be a way to decrease the
impact of OSCi on cataract induction. Two members
of the aminopyrimidine series of OSCi, with very differ-
ent structure to U18666A, have also been shown to in-
duce equatorial cataracts in the mouse and dog.10 It
has been suggested that for this series of inhibitors, the
mechanism of cataract formation may be rather due to
inhibition of cholesterol in the lens than to lens-mem-
brane perturbation. The critical role of lens sterol
metabolism in the maintenance of lenticular transpar-
ency has already been demonstrated.14,15 Large amounts
of synthesized cholesterol are required for the elonga-
tion of cell fibers and therefore, the inhibition of the
cholesterol pathway leads to decreased membrane pro-
duction and disturbed fiber cell growth. Recently, the
histopathologic findings in hamster and dog associated
with the administration of three different OSCi were
published.16 The treatment (less than four weeks) pro-
duced a similar spectrum of eye lesions to those previ-
ously described for other compounds of this
pharmacological class. However, in this study, dermal
and ocular side effects were less pronounced when the
dogs were given a cholesterol-rich diet, suggesting that
observed changes were related to the inhibitory effect
on cholesterol biosynthesis and not to intrinsic toxicity.
The authors considered that the adverse effects described
were due to supra-pharmacological effects on OSC at
high dose levels, and that a decrease in systemic expo-
sure could reduce these adverse effects. Such a trend
was previously observed with various HMGCoA reduc-
tase inhibitors in the dog for which a strong relationship
was demonstrated between plasma drug level and the
cataractogenic potential.17,18 Unfortunately, no data
concerning the exposure levels compared with those seen
at pharmacological doses were published with the two
previously described toxicological studies of OSCi.10,16
amount of SAR data available in the literature at that
time encouraged us to favor a pharmacophore-based
screening approach rather than high throughput screen-
ing. The pharmacophore was generated from three well-
characterized and structurally diverse published OSCi
(Scheme 1) presenting the chemical features assumed
to be necessary for potency9: a physiologically proton-
ated amine at one extremity of the molecule, a small
lipophilic electron-withdrawing substituent on an aryl
ring at the opposite extremity, and an electrophilic
group incorporated close to this terminal aryl. The dis-
tance between the amine and the aryl is certainly impor-
tant and was held somewhat constant, but the nature of
the spacer was permitted to vary. Finally we assumed an
extended conformation of the inhibitor as suggested by
the chair-boat-chair conformation of both intermediate
and reaction product of the OSC enzymatic reaction
and by the binding mode of RO48-8071 in squa-
lene:hopene cyclase, first hypothesized by modeling19a
and further confirmed by X-ray co-crystallization stud-
ies.19b A five-point pharmacophore that fulfilled these
criteria was selected. As shown in Figure 1, a hydro-
gen-bond acceptor feature was preferred to a protonated
amine, and generic hydrophobic features replaced the
aromatic ring and the electron-withdrawing group in or-
der to enable a more diverse selection of compounds. A
search in the GSK corporate compound collection iden-
tified 4300 virtual hits. Standard ‘lead-like’ filters were
applied to give a preferred set of 3000 compounds for
screening. One hundred and ten compounds showed
some inhibition of OSC, and the 21 most potent, with
an IC50 less than 1.5 lM, were considered for further
profiling. We focused a medicinal chemistry program
on the most tractable and novel series represented by
compound 1, displayed in Scheme 2.
Recently the crystal structure of human OSC co-crystal-
lized with RO48-8071 was published.20 The main fea-
tures defining our pharmacophore are coherent with
the binding mode of RO48-8071 as observed in the crys-
tal structure: the basic nitrogen and the carbonyl are
hydrogen-bonded with the protein and the terminal phe-
nyl group situated in an electron-rich pocket makes an
electron-deficient aromatic ring an optimal group. The
overall shape of the inhibitor is extended but not to
the extent used in our model. As a consequence the dis-
tance between the hydrogen-bond donor and acceptor
features may have been slightly over-estimated in our
3D searches.
Based on the above hypothesis, our goal was to evaluate
the possibility of developing an OSC inhibitor free of
cataract liability as a result of reduced peripheral expo-
sure. A program was initiated to identify a set of small
molecules inhibiting OSC both in vitro and in vivo in or-
der to select compounds with different peripheral expo-
sure parameters and to take them forward into
toxicological studies.
2. Results and discussion
2.1. Pharmacophore-based focused screen
2.2. Chemistry
At the outset of this work no crystal structure of human
oxidosqualene cyclase had been published, but the large
4-Bromophenethylamine 2 was first coupled with the
appropriate benzene sulfonyl chloride and then submit-
F
O
N
O
O
O
O
S
N
N
N
N
N
N
Br
O
Br
Cl
O
ZD-9720
RO48-8071
BIBB 515
Scheme 1. Known OSCi used as starting points for the pharmacophore model generation.

6220
M.-H. Fouchet et al. / Bioorg. Med. Chem. 16 (2008) 6218–6232
Hbond
acceptor
Hydrophobic
Hbond acceptor
Hydrophobic
Aliphatic
Figure 1. Mapping of RO48-8071 on the selected five points pharmacophore model of OSCi.
OH
Reductive amination of intermediate 6 with 4-chloro-
benzaldehyde led to compound 16 (Scheme 4).
O
O
S
The pyrazole derivative 24 was obtained by the treat-
ment of the acetyl intermediate 9a with DMF dimethyl-
acetal, followed by cyclization with hydrazine hydrate
(Scheme 5).
N
N
H
CN
1 EC50 = 100nM
(racemic mixture)
Scheme 2. Compound 1.
The oxadiazole derivative 26 was obtained by the treat-
ment of the cyano intermediate 9b with hydroxylamine
hydrochloride, followed by cyclization of the resulting
amidoxime with acetic anhydride (Scheme 5).
ted to Suzuki coupling. Reductive amination of the
resulting biphenyl carboxaldehyde 4 with N,N-dimethyl-
amine or pyrolidine afforded compounds 10–12
(Scheme 3).
2.3. Lead optimization
A lead optimization process was conducted starting
from compound 1. All compounds meeting an in vitro
potency threshold (IC50 less than 50 nM) were evaluated
in vivo in the rat at a single dose of 2 mg/kg, monitoring
inhibition of cholesterol synthesis in both liver and lens.
In parallel, the appearance of MonoEpoxySqualene
(MES) as a marker of OSC inhibition was evaluated in
Starting from the substituted benzaldehydes 5,21–23
reductive amination with N-Boc-piperazine followed
by the acidic cleavage of the t-Boc group led to the inter-
mediate 6, which was submitted to coupling reaction
with the appropriate benzene sulfonyl chloride or ben-
zene carbonyl chloride to afford the desired compounds.
H
R1
N
S
H
R1
N
NH2
O
O
a
b
S
H
O
O
Br
Br
O
2
4a: meta-CHO, R1= 4-CN-phenyl
4b: para-CHO, R1= 4-CN-phenyl
4c: para-CHO, R1= 4-Cl-phenyl
3a: R1= 4-CN-phenyl
3b: R1= 4-Cl-phenyl
H
N
R1
R2
N
S
c
O
O
R3
10: meta-(N,N-dimethylaminomethyl), R1= 4-CN-phenyl
11: para-(N,N-dimethylaminomethyl), R1= 4-CN-phenyl
12: para-((pyrrolidin-1-yl)-methyl),
R1= 4-Cl-phenyl
Scheme 3. Reagents and conditions: (a) R1SO2Cl, Et3N, CH2Cl2, rt; (b) 3- or 4-formyl-phenyl boronic acid, Pd(PPh3)4, LiCl, Na2CO3, toluene,
reflux; (c) R2R3NH, NaHB(OAc)3, CH2Cl2, rt.

M.-H. Fouchet et al. / Bioorg. Med. Chem. 16 (2008) 6218–6232
6221
O
O
S
NH
N
a, b
f
H
N
N
Cl
N
N
O
N
5a
6a
13
O
R1
R1
R1
R2
NH
a, b
c
N
H
N
N
15 : R1= pyridin-4-yl, R2= 4-chlorophenyl
17 : R1= pyridin-4-yl, R2= 4-methoxyphenyl
O
6b, 6c, 6d, 6e, 6f
5b, 5c, 5d, 5e, 5f
18 : R1=N,N-dimethylaminomethyl, R2= 4-chlorophenyl
R1= b: pyridin-4-yl
c: imidazol-1-yl
20 : R1=N,N-dimethylaminomethyl, R2= 4-OCF3-phenyl
22 : R1=imidazol-1-yl, R2= 4-chlorophenyl
25 : R1=1-methylpyrazol-4-yl, R2= 4-chlorophenyl
27 : R1 =imidazol-1-yl, R2= trifluoroacetyl
d: N,N-dimethylaminomethyl
e: 4-methylpiperazin-1-yl
f: 1-methylpyrazol-4-yl
O
O
R1
d
S
R2
N
N
14 : R1= pyridin-4-yl, R2= 4-chlorophenyl
19 : R1= N,N-dimethylaminomethyl, R2= 4-chlorophenyl
21 : R1= 4-methylpiperazin-1-yl, R2= 4-chlorophenyl
R1
e
R2
N
N
16 : R1= pyridin-4-yl, R2= 4-chlorophenyl
Scheme 4. Reagents and conditions: (a) N-Boc-piperazine, NaHB(OAc)3, CH2Cl2, rt; (b) CF3COOH, CH2Cl2, rt; (c) R2–COCl, NEt3, CH2Cl2, rt; or
R2–CO–O–CO–R2, NEt3, CH2Cl2, rt, or R2-COOH, HOBT, EDCI, NEt3, DMF, rt; (d) R2-SO2Cl, NEt3, CH2Cl2, rt; (e) R2-CHO, NaHB(OAc)3,
CH2Cl2, rt; (f) 4-chlorophenyl-SO2Cl, NEt3, CH2Cl2, rt.
the same organs (inhibition of OSC leads to the inhibi-
tion of the production of cholesterol and to the accumu-
lation of 2,3 mono-epoxysqualene).
The hydroxymethylene spacer was then removed and
the ethylamino spacer was rigidified (Table 2). In order
to maintain an adequate distance between extremity fea-
tures required by the pharmacophore model, compound
14 was prepared and was highly potent in vitro. Once
again, the meta analogue (compound 13) showed a drop
in potency.
Compound 1 was selected as a starting point for medic-
inal chemistry, because of its high chemical tractability
and its good in vitro activity, and even if it was devoid
of any in vivo activity at 2 mg/kg (data not shown).
Chemical modifications depicted in Scheme 6 were
undertaken in order to obtain compounds, with
in vitro potency and in vivo activity comparable to
inhibitors reported earlier in the literature.
On the right side of the molecule, the sulfonamide func-
tion can be replaced by an amide function (compound
15), but the corresponding aminomethyl compound
(compound 16) was 100-fold less potent, showing the
importance of an H-bond acceptor in this part of the
molecule. An aromatic ring is crucial for in vitro activity
(compound 27), and an electron-withdrawing substitu-
ent in the para position of this aromatic ring is impor-
tant for potency (compound 17).
Replacement of the pyridin-3-yl-hydroxymethylene
group by a para-dialkylaminophenyl gave potent OSCi
(compounds 11 and 12) (Table 1). Comparison with
the inactive analogue 10, containing the meta-dialkylam-
inophenyl, showed the importance of an extended con-
formation as expected with the para substitution.
Compound 11 was tested in vivo, but was poorly active
probably due to metabolic instability (high rat micro-
somal clearance) (Table 3).
As pyridine analogues (compounds 14 and 15) gave
moderate in vivo activity in the rat (Table 3), further
optimization of the left side of the molecule was
conducted.

6222
M.-H. Fouchet et al. / Bioorg. Med. Chem. 16 (2008) 6218–6232
O
O
R
R
a
+
N
N
H
HN
N
Cl
Cl
O
8
9a: R= -COCH3
9b: R= CN
23: R= pyrazol-1-yl
7a: R= -COCH3
7b: R= CN
7c: R= pyrazol-1-yl
H
N
b
N
O
9a
N
N
Cl
24
O
c
N
O
9b
N
N
N
Cl
26
Scheme 5. Reagents and conditions: (a) NaHB(OAc)3, CH2Cl2, rt; (b) i:DMFÆDMA, reflux; ii:H2N–NH2ÆH2O, EtOH, reflux; (c) i:NH2OHÆHCl,
AcONa, EtOH/H2O, reflux; ii:(CH3CO)2O, NEt3, CH2Cl2, rt.
improvement of in vivo activity (Table 3), comparable
with BIBB 515.
OH
O
O
S
Replacement of the imidazole by a pyrazole or an oxa-
diazole (compounds 23–26) led to a dramatic loss of po-
tency. As suggested by our pharmacophore model, one
of the heteroatoms of these heterocycles is expected to
give a hydrogen-bond, mimicking the basic nitrogen of
RO48-8071 as shown in Figure 1. This interaction was
confirmed by X-ray observations, as the basic nitrogen
of RO48-8071 is shown to be hydrogen-bonded with
the carboxylic acid of Asp455.20 Moreover, X-ray data
showed that this terminal part of RO48-8071 is located
into a quite small and mainly hydrophobic pocket, apart
from Asp455.20 Although our pharmacophore model is
not precise enough to explain the loss of potency of
compounds 23–26, we can assume from X-ray observa-
tions that a sub-optimal orientation of an hydrogen-
bond donor atom of the heterocycle would prevent it
from forming the hydrogen-bond with Asp455. In addi-
tion, an unfavorable interaction of an heterocycle sub-
N
N
H
CN
modification
of the spacer
modification of the
pyridyl-methanol part
modification of
the substituent
Scheme 6. Chemical modifications performed on compound 1.
The replacement of the pyridine by dialkyl amino
groups such as N,N-dimethylaminomethyl (compounds
18–20) or a N-methylpiperazine (compound 21), main-
tained in vitro potency. The replacement of the pyridine
by an imidazole gave the potent analogue 22. In vivo,
compound 19 showed an improved inhibition of choles-
terol synthesis but a moderate apparition of MES syn-
thesis whereas compounds 20–22 led to a great
Table 1. Effect of replacement of the pyridin-3-yl-hydroxymethylene group of compound 1 on OSC inhibition
4
R1
3
O
O
S
N
H
R2
R2
Compound
R1
OSC inhibition IC50 (nM)
10
11
12
3-[(N,N-Dimethylamino) methyl]
4-[(N,N-Dimethylamino) methyl]
4-[(Pyrrolidin-1-yl) methyl]
CN
CN
Cl
ia at 200 nM
28
63
ia at 200 nM, inactive at 200 nM.

M.-H. Fouchet et al. / Bioorg. Med. Chem. 16 (2008) 6218–6232
6223
Table 2. Effect of replacement of the pyridin-3-yl-hydroxymethylene
group and rigidification of the ethylamino spacer of compound 1 on
OSC inhibition
stituent (NH, N, and methyl) with a close amino-acid of
the hydrophobic pocket would be responsible for the
observed biological results for these compounds. Dock-
ing experiments in the X-ray crystal structure of OSC
would be necessary to prove these hypotheses.
X
4
N
R1
N
R2
3
Overall in this study, the observed SAR in our series is
coherent with the published SAR of the compounds
used for the pharmacophore generation. Three inhibi-
tors with in vivo potency comparable to BIBB 515 were
identified (compounds 20–22), and their PK profiles and
duration of action were determined in rat (Table 4).
Compound R1
R2
Cl
Cl
Cl
Cl
X
OSC inhibition
IC50 (nM)
N
13
–SO2– 500
–SO2– 13
3
4
4
4
4
Compounds 20 and 21 showed similar profiles in terms
of potency and duration of action (similar activity after
8 h) both in liver and lens. After iv administration, both
these have a lower exposure in plasma and a much high-
er exposure in liver than BIBB 515. Moreover, com-
pound 21 has high hepatic exposure versus plasma or
lenticular exposure, compared to 20. Interestingly com-
pound 22 showed a significantly different profile in that
almost all activity disappeared by 8 h of treatment both
in liver and lens. However, 22 also had a lower exposure
in plasma and a higher heptic exposure than BIBB 515.
N
14
N
15
–CO–
3
N
16
–CH2– 316
N
17
–OMe –CO– 2511
We selected compounds 21 and 22 based on their dis-
tinct PK profiles. While both these showed a higher li-
ver/lens and a lower plasma/liver exposure ratio
compared to BIBB 515, 21 showed a long duration of
action associated with the highest liver/lens exposure ra-
tio whereas 22 displayed a short duration of action.
Both compounds displayed the same efficacy in vivo at
2 mg/kg and neither demonstrated major pharmacoki-
netic issues (data not shown).
N
18
19
20
21
22
23
24
25
26
Cl
Cl
–CO– 40
–SO2– 32
4
N
4
4
N
N
–OCF3 –CO– 20
2.4. Toxicological study
Cl
Cl
Cl
Cl
Cl
Cl
–SO2–
6
N
Rats (3 males and 3 females) were treated with com-
pounds 21 and 22 for 21 days;
4
N
–CO– 32
N
The compounds were administered po at 2, 10, and
30 mg/kg once a day. On the last day of treatment, plas-
matic area under the curves (AUC) was calculated (data
not shown). A therapeutic window could thus be evalu-
ated as the ratio of plasmatic AUC calculated at the 1st
dose of cataract apparition versus plasmatic AUC calcu-
lated at the pharmacological active dose. Histology was
performed on lens, liver, and additional organs. Oph-
thalmology was conducted using the slit-lamp biomicro-
scope technique. In parallel, activity for OSC inhibition,
and drug exposure were monitored.
4
4
N
–CO– ia at 200 nM
–CO– ia at 200 nM
–CO– ia at 200 nM
–CO– ia at 200 nM
N
H
N
N
4
N
N
4
4
O
N
Noticeably, but as expected from the previously pub-
lished toxicological data, no relevant findings were ob-
served in the area of hematology, blood chemistry,
liver enzymes, macroscopic post-mortem examination,
and liver histology for either compound. However, len-
ticular changes were found.
N
N
O
27
N
ia at 200 nM
N
CF3
N
Compound 21 gave a dose-dependent and very severe
cataract phenotype. This adverse effect was detectable
at the lowest dose of 2 mg/kg, which is also a pharmaco-
logically active dose. Histological examination of the eye
ia at 200 nM, inactive at 200 nM.

6224
M.-H. Fouchet et al. / Bioorg. Med. Chem. 16 (2008) 6218–6232
Table 3. Effects of compounds on hepatic OSC inhibition following single-dose treatment
Compound
Rat microsomes
Clint (mL/min/g)
% Inhibition of cholesterol
synthesis in liver
Induction of MES synthesis in liver
(% of BIB B515-treated animals)
O
N
O
N
2.3
85
100
Cl
BIBB 515
N
O
O
S
9.9
47
6
N
H
CN
11
N
N
O
O
S
N
1.3
66
64
74
83
75
85
45
55
N
Cl
14
O
N
O
6
N
Cl
15
O
S
N
N
N
2.5
3.6
5.4
0.2
55
N
Cl
19
O
N
100
111
156
N
OCF3
20
N
O
O
N
S
N
N
Cl
21
N
N
O
N
N
Cl
22
OFA rats were treated for 3 h with compounds at 2 mg/kg po as indicated in Section 4. Inhibition of neosynthetized cholesterol in liver was calculated
in comparison with vehicle-treated animals. Induction of neosynthetized MES was expressed as percentage of BIBB 515-treated animals used as
standard (100%).
revealed a keratosis and degeneration of lenticular fi-
bers. Despite very low exposure in plasma and a high he-
patic exposure, no therapeutic window was achieved
with compound 21 (Table 5). For compound 22, no

M.-H. Fouchet et al. / Bioorg. Med. Chem. 16 (2008) 6218–6232
6225
Table 4. In vivo profile of compounds 20–22
Compound In vivo lipid synthesis after 3 h
In vivo lipid synthesis after 8 h AUC0–6 h, ng H/mL
(1 mg/kg, iv bolus)
Tissular exposure (1 mg/kg, iv)
Liver Chol/MES Lens Chol/MES Liver Chol/MES Lens Chol/MES
Liver (ng/g)
Eyes (ng/g)
BIBB 515 92/100
79/100
40/100
61/100
1027 72a
318; 362b
95; 97b
C2h = 301 53a
C6h = 169 49a
C2h < 10
C6h < 10
20
21
22
85/112
87/94
64/175
69/241
66/51
C2h = 1882; 2416b C2h = 230; 260b
C6h = 968; 1317b C6h = 189; 194b
67/162
76/104
62/232
20/85
67/29
0/51
C2h = 5937; 6605b C2h = 181; 278b
C6h = 2649; 3272b C6h = 83; 123b
82/135
504; 534b
C2h = 1999; 2013b C2h = 56; 65b
C6h = 585; 660b
C6h < 10
OFA rats were treated for 3 or 8 h with compounds at 2 mg/kg po as indicated in Section 4. At the end of the labeling period, neosynthetized lipids
were extracted from liver and lens. Inhibition of neosynthetized cholesterol was calculated in comparison with vehicle-treated animals. Induction of
neosynthetized MES was expressed as percentage of level of BIBB 515-treated animals used as standard (100%). In parallel, a pharmacokinetic study
was performed in rats at 1 mg/kg following intravenous bolus administration, for the determination of plasmatic area under the curves from 0 to 6 h
(AUC0–6h) and tissue exposures.
a Means SD, six independent experiments.
b Values from two independent experiments.
ical dose of 2 mg/kg. Nevertheless, despite an improved
toxicological profile compared to 21, the therapeutic
window appears very limited (<10) for such a severe
finding and confirmation in higher species would be re-
quired before any further development of the
compound.
Table 5. Toxicological study in rats
Compound
Proof of activity Opacification of the anterior
lens scoring from 0 to 6a
MES appearance
in liver
Control
M
F
À
À
0/0/0
0/0/0
—
—
21
2 mg/kg
M
F
+
+
+
+
+
+
0/1/4 Very slight/slight
1/1/2
3. Conclusion
10 mg/kg
30 mg/kg
M
F
1/1/3 Moderate
4/4/4
The aim of this work was to test the hypothesis that the
cataractogenic potential of an OSCi could be managed
by a decrease in systemic exposure. With that goal in
mind, we undertook a pharmacophore-based focused
screen and discovered compound 1 as a novel and trac-
table OSCi. We optimized this series to select two com-
pounds (21 and 22) displaying good in vitro and in vivo
potency and distinct pharmacokinetic profiles with re-
duced systemic exposure as compared to BIBB 515.
We have demonstrated with compound 21 that a very
low systemic exposure is not sufficient to remove the
ocular side effects. Compound 22 which displayed a
low systemic exposure combined with a short duration
of action showed a somewhat improved therapeutic win-
dow. However, it is not clear that this small improve-
ment is sufficient to enable the liabilities of OSCi to be
adequately managed. These results, in addition to previ-
ously published toxicological data, suggest that the
development of an OSCi for the treatment of dyslipide-
mia free of ocular toxicity remains high-risk.
M
F
4/4/6 Marked/very marked
3/4/6
22
2 mg/kg
M
F
À
+
+
+
+
+
0/0/0 Nothing
0/0/0
10 mg/kg
30 mg/kg
M
F
1/2/3 Slight/moderate
3/3/3
M
F
4/4/5 Very marked
4/5/6
Sprague–Dawley rats (male and female n = 3) were treated for 21 days,
orally once a day, with increasing doses of compounds 21 and 22. At
the end of the treatment, lipids were extracted from the liver and MES
appearance was evaluated as proof of drug activity. Opacification of
the anterior lens was determined and scored between 0 (no abnorm-
alities) to 6 (severe findings).
a 0, no abnormalities; 1, very slight; 2, slight; 3, moderate; 4, marked; 5,
very marked; 6, severe.
abnormalities were detected at 2 mg/kg. In males at
2 mg/kg, surprisingly no OSC inhibition was observed,
but in females, this dose was pharmacologically active.
At the highest dose a very marked cataract phenotype
was observed. Interestingly, only slight keratosis and
moderate degeneration of lenticular fibers appeared at
the intermediate dose of 10 mg/kg (Table 5) for which
the plasma exposure of 22 was 7- to 12-fold higher than
that seen following the 2 mg dose (data not shown).
Therefore, the low plasma/liver exposure ratio combined
with the short duration of action of compound 22, led to
no observable abnormalities in lens at the pharmacolog-
4. Experimental
4.1. Sterol synthesis in HepG2 cells
2,3-oxidosqualene cyclase activity was determined in
the human hepatoma cell line, HepG2, by measuring
the inhibition of [14C]cholesterol synthesis concomi-
tantly with the formation of [14C]2,3-mono-epoxy-
squalene (MES) and [14C]2,3;22,23-diepoxysqualene
(DES).

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Cells plated in 96-well plates, were incubated for 24 h in
the absence (control) or presence of compounds in BME
medium containing 10% of fetal calf serum and then la-
beled with 7.4 kBq of [14C]mevalonate (Perkin-Elmer).
At the end of the incubation, lipids were extracted with
heptane/isopropanol (99:1, v/v) and separated by thin-
layer chromatography using cyclohexane/ethylacetate
(90:10, v/v). [14C]cholesterol, MES, and DES were iden-
tified using purified standards and the radioactivity
associated with each individual lipid was quantified
using a Phosphoscreen (Strom, Molecular Dynamics).
separations were performed on a supelcosil LC ABZ+
column (150 · 4.6 mm, 5 lm) under two solvent gradi-
ent conditions at 30 ꢁC with a flow rate of 1 mL/min
and UV detection at 290 nm. Mobile phases consisted
of 50 mM ammonium acetate (A) and acetonitrile (B).
The results were expressed as intrinsic clearances (Clint
,
mL/min/g), assuming 45 mg of microsomal proteins
per gram liver.
4.4. Pharmacokinetics and tissue exposure
OSC inhibitors were administered to OFA rats (n = 10)
by intravenous injection via the penis vein at a dose of
1 mg/kg in 10% DMA and 90% PEG200. Blood samples
were collected at T5, T30, T120, T240, and T360 (min). Tis-
sues (liver and the 2 eyes) were collected at selected times
(T120 and T360). Plasma samples (0.5 mL) were diluted
with 1:1 borate buffer (0.1 M, pH 11.5) and then ex-
tracted with ethyl ether (4 mL). The ethyl ether was
evaporated and the residues resuspended in 300 lL of
mobile phase (water/acetonitrile, 40:60, v/v, ammonium
acetate 5 mM). Liver samples (1 g) and eyes were
homogenized in borate buffer 0.1 M, pH 11.5 (3 and
1.5 mL, for liver and eyes, respectively). The homoge-
nates were centrifuged at 4000 rpm for 15 min at 4 ꢁC.
The liver (1 mL) and the eye (0.5 mL) homogenates were
diluted with 1:1 borate buffer (0.1 M, pH 11.5) and then
extracted with ethyl ether (4 mL). The ethyl ether was
evaporated and the residues resuspended in 1 mL and
300 lL of mobile phase (water/acetonitrile, 40:60, v/v,
ammonium acetate 5 mM), for liver and eye extracts,
respectively. Samples were analyzed by high-perfor-
mance liquid chromatography coupled to tandem mass
spectrometry (LC/MS/MS). Chromatographic separa-
tions were performed on a supelcosil LC ABZ+ column
(150 · 2.1 mm, 5 lm) under two solvent gradient condi-
tions at 30 ꢁC with a flow rate of 0.2 mL/min. Mobile
phases consisted of water (A) and acetonitrile (B), both
containing 5 mM ammonium acetate, and 20 lL was in-
jected into the chromatographic system. The mass spec-
trometer was a Quattro Ultima (Waters), operated in
the positive ion mode. The MS/MS detection was car-
ried out by using the multiple monitoring reaction
mode. The areas under the curve (AUC) were obtained
from SIPHAR (Simed).
Data are expressed as percentage of control for choles-
terol synthesis inhibition.
Reference compound, BIBB 515, showed an IC50 of
0.00585 0.0034 lM (33 measurements).
4.2. Sterol synthesis in rat
For single-dose treatment, OSC activity was determined
in rat by measuring the inhibition of [14C]cholesterol
synthesis concomitantly with the formation of [14C]2,3-
mono-epoxysqualene (MES) and [14C]2,3;22,23-diep-
oxysqualene (DES).
Male OFA rats were orally treated with compounds at
the indicated doses for 2 h. Cholesterol and MES syn-
thesis were assessed by intraperitoneal injection of
370 kBq of [14C]mevalonate for an additional hour.
After sacrifice, liver and lens were removed and lipids
were extracted using isopropanol and separated by
thin-layer chromatography as above. [14C]Cholesterol,
MES, and DES were identified using purified standards
and the radioactivity associated with each individual li-
pid was quantified using a Phosphoscreen (Strom,
Molecular Dynamics). Data are expressed as percentage
of control for cholesterol synthesis inhibition.
For toxicological study, OSC activity was determined in
rat by measuring the formation of MES. On day 21, 3 h
after last administration, animals were sacrificed and he-
patic lipids were extracted and analyzed as described
above. MES formation was assessed by staining the
thin-layer chromatography with 5% phosphomolybdic
acid in isopropanol in order to visualize the different lip-
ids. Quantification of MES mass was done using a den-
sitometer (PDSI, Molecular Dynamics).
4.5. Pharmacophore generation
Molecular modeling studies were performed using Cata-
lyst4.5 (Accelrys) installed on a Silicon Graphics Oc-
tane2 workstation. Conformations were calculated
using an energy cutoff of 15 kcal with the best option.
The number of conformers generated for each molecule
was limited to a maximum of 250. Pharmacophore
hypothesis was generated with the common features
algorithm. The database search was conducted with
the best option.
4.3. Metabolic stability of OSC inhibitors in pooled rat-
liver microsomal incubations
Pooled rat-liver microsomes (Xenotech, Lenexa, KA)
were diluted in Tris buffer 0.1 M, pH 7.4, containing
10 mM MgCl2. The incubation mixtures (1.5 mL total
volume), which contained 0.5 mg/mL of microsomal
proteins and 1.25 lg/mL of OSC inhibitors, were prein-
cubated for 5 min at 37 ꢁC. The reactions were initiated
by addition of NADPH (final 0.48 mM), continued for
30 min, and stopped by the addition of 1 volume of cold
acetonitrile. Incubates were then centrifuged for 15 min
at 4000 rpm and 50 lL of the supernatant was injected
into the chromatographic system. Chromatographic
4.6. Chemistry
All starting materials were commercially available and
used without further purification. All reactions were car-
ried out with the use of standard techniques under an

M.-H. Fouchet et al. / Bioorg. Med. Chem. 16 (2008) 6218–6232
6227
inert atmosphere (Ar or N2). Organic extracts were rou-
tinely dried over anhydrous sodium sulfate. Solvent re-
moval refers to rotary evaporation under reduced
pressure at 30–40 ꢁC. The analytical thin-layer chroma-
tography (TLC) was carried out on E. Merck 60-F254
precoated silica gel plates and components were usually
visualized using UV light, iodine vapor, or Dragendorff
preparation. Flash column chromatography was per-
formed using silica gel, Merck grade 60 (230–400 mesh).
Melting points were determined on a hot-stage Kofler
apparatus and are uncorrected. Proton NMR (1H
NMR) spectra were recorded at ambient temperature
on Bruker Avance 300 DPX spectrometers using tetra-
methylsilane as internal standard and proton chemical
shifts (d) are expressed in ppm in the indicated solvent.
The following abbreviations are used for multiplicity
of NMR signals: s = singlet, d = doublet, t = triplet,
q = quadruplet, dd = double doublet, m = multiplet,
ddd = doublet of doublet of doublet, and br = broad.
Analytical HPLC (method A) was conducted on a X-
Terra MS C18 column (2.5 lm 30 · 3 mm id) eluting
with 0.01 M ammonium acetate in water (solvent A)
and 100% acetonitrile (solvent B), using the following
elution gradient 0–4 min 0–100%B, 4–5 min 100%B at
a flow rate of 1.1 mL/min. The mass spectra were re-
corded on a micromass Platform-LC mass spectrometer
using atmospheric pressure chemical positive ionization
[AP+ve to give [M+H]+ molecular ions] or atmospheric
pressure chemical negative ionization [APÀve to give
[MÀH]À molecular ion] modes (MS-APCI).
4.6.2. N-[2-(4-Bromophenyl)ethyl]-4-chlorobenzenesulf-
onamide (3b). Following the procedure used for 3a, 4-
bromophenethyl amine 2 (5 g, 25 mmol), 4-chloro-
benzenesulfonyl chloride (5.54 g, 26.24 mmol) and tri-
ethylamine (4.2 mL, 30 mmol) were used to give 3b as
a cream solid (9.34 g, 99%); mp: 118 ꢁC; 1H NMR
(CDCl3) d: 7.73 (d, 2H, 8.67 Hz), 7.37 (d, 2H,
8.85 Hz), 7.29 (d, 2H, 8.48 Hz), 6.87 (d, 2H, 8.29 Hz),
4.50 (t, 1H), 3.13 (q, 2H), and 2.67 (t, 2H).
4.6.3. N-[2-(4-(3-Formylphenyl)phenyl)ethyl]-4-cyanoben-
zenesulfonamide (4a). Intermediate 3a (1 g, 2.74 mmol)
and tetrakis triphenylphosphine palladium[0] (0.159 g,
0.14 mmol) in toluene (50 mL) were stirred under nitro-
gen at room temperature for 10 min. 3-Formylbenzen-
eboronic acid (Aldrich, 0.53 g, 3.56 mmol), LiCl
(0.35 g, 8.22 mmol), and Na2CO3 (3.5 mL of a solution
2 M, 6.84 mmol) were added and the mixture was heated
under reflux for 3 h, then cooled, and poured in water.
The aqueous phase was extracted twice with CH2Cl2
and the organic phase was dried (Na2SO4) and evapo-
rated under reduced pressure. The resulting residue
was purified by flash chromatography (CH2Cl2/MeOH,
99:1 then 98:2) to provide 4a as a colorless oil (0.52 g,
1
49%); H NMR (CDCl3) d: 10 (s, 1H), 7.99 (s, 1H),
7.84 (d, 2H, 8.48 Hz), 7.77 (m, 2H), 7.69 (d, 2H,
8.48 Hz), 7.55 (t, 1H, 7.54 and 7.72 Hz), 7.46 (d, 2H,
8.10 Hz), 7.12 (d, 2H, 8.29 Hz), 4.70 (t, 1H), 3.25 (q,
2H), and 2.79 (t, 2H).
4.6.4. N-[2-(4-(4-Formylphenyl)phenyl)ethyl]-4-cyanoben-
zenesulfonamide (4b). Following the procedure used for
4a, 3a (2.85 g, 7.81 mmol), tetrakis triphenylphosphine
palladium[0] (0.45 g, 0.4 mmol), 4-formylbenzeneboron-
ic acid (Aldrich, 1.52 g, 10.15 mmol), LiCl (1 g,
23.42 mmol), and Na2CO3 (10 mL of a solution 2 M,
20 mmol) were used to provide after flash chromatogra-
phy (CH2Cl2 then CH2Cl2/MeOH, 98:2), compound 4b
as a white solid (1.8 g, 59%); mp: 174 ꢁC; 1H NMR
(CDCl3) d: 10.01 (s, 1H), 7.9 (d, 2H, 8.48 Hz), 7.86 (d,
2H, 8.67 Hz), 7.72 (d, 2H, 8.67 Hz), 7.66 (d, 2H,
8.29 Hz), 7.50 (d, 2H, 8.29 Hz), 7.15 (d, 2H, 8.10 Hz),
4.55 (t, 1H), 3.29 (q, 2H), and 2.83 (t, 2H).
Analytical HPLC (method B) was conducted on a X-
Bridge C18 column (2.5 lm 30 · 3 mm id) eluting with
0.01 M ammonium acetate in water (solvent A) and
100% acetonitrile (solvent B) using the following elution
gradient: 0–0.5 min 5% B, 0.5–3.5 min 5% B–100% B,
3.5–4 min 100% B, 4–4.5 min 100% B–5% B, 4.5–
5.5 min, 5% B at a flow rate of 1.3 mL/min with a tem-
perature of 40 ꢁC. The mass spectra were recorded on a
micromass LCT mass spectrometer using electrospray-
positive ionization [ES+ve to give [M+H]+ molecular
ion] or electrospray-negative ionization [ESÀve to give
[MÀH]À molecular ion] modes (HRMS-ESI).
The purity of the compounds was determined on two
analytic HPLC systems by UV detection (100% purity
in both methods was obtained for the examples de-
scribed below).
4.6.5.
N-[2-(4-(4-Formylphenyl)phenyl)ethyl]-4-chloro-
benzenesulfonamide (4c). Following the procedure used
for 4a, 3b (5 g, 13.35 mmol), tetrakis triphenylphosphine
palladium[0] (1.54 g, 1.33 mmol), 4-formylbenzenebo-
ronic acid (Aldrich, 2.6 g, 17.36 mmol), LiCl (1.7 g,
40 mmol), and Na2CO3 (17.2 mL of a solution 2 M,
34.4 mmol) were used to provide after flash chromatog-
raphy (CH2Cl2 then CH2Cl2/MeOH, 98:2), compound
4.6.1. N-[2-(4-Bromophenyl)ethyl]-4-cyanobenzenesulf-
onamide (3a). To a solution of 4-bromophenethyl amine
2 (Aldrich, 2.5 g, 12.49 mmol) in CH2Cl2 (50 mL) were
added 4-cyanobenzenesulfonyl chloride (Aldrich,
2.64 g, 13.12 mmol), then triethylamine (2.1 mL,
15 mmol) and the mixture was stirred at room tempera-
ture for 30 min and then diluted with CH2Cl2 (100 mL).
The organic phase was washed with water, dried
(Na2SO4), and evaporated under reduced pressure to
provide 3a as a colorless oil which crystallized (4.55 g,
1
4c as a white solid (3.8 g, 71%); H NMR (CDCl3) d:
9.99 (s, 1H), 7.87 (d, 2H, 8.48 Hz), 7.66 (d, 2H,
8.67 Hz), 7.64 (d, 2H, 8.10 Hz), 7.47 (d, 2H, 8.29 Hz),
7.37 (d, 2H, 8.67 Hz), 7.12 (d, 2H, 8.29 Hz), 4.36 (t,
1H), 3.22 (q, 2H), and 2.78 (t, 2H).
4.6.6. N-[3-(Pyridin-4-yl)phenylmethyl]-piperazine (6a).
To a solution of 3-(pyridin-4-yl)-benzaldehyde21 (0.8 g,
4.37 mmol) in CH2Cl2 (50 mL) were added N-tertbutyl-
oxycarbonylpiperazine (Fluka, 0.896 g, 4.81 mmol) and
sodium triacetoxyborohydride (1.11 g, 5.24 mmol) and
1
99%); mp: 140 ꢁC; H NMR (CDCl3) d: 7.79 (d, 2H,
8.67 Hz), 7.69 (d, 2H, 8.67 Hz), 7.29 (d, 2H, 8.48 Hz),
6.88 (d, 2H, 8.29 Hz), 4.63 (t, 1H), 3.18 (q, 2H), and
2.69 (t, 2H).

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M.-H. Fouchet et al. / Bioorg. Med. Chem. 16 (2008) 6218–6232
the mixture was stirred at room temperature overnight.
The reaction was diluted with CH2Cl2 (100 mL), washed
with water, and the organic phase was dried (Na2SO4),
and evaporated under reduced pressure. The residue
was purified by flash chromatography on silica gel
(CH2Cl2/MeOH, 99:1 then 98:2) to afford 1-(tertbutyl-
oxycarbonyl)-4-[3-(pyridin-4-yl)phenylmethyl]-pipera-
zine as a colorless oil (1.17 g, 76%). 1H NMR (CDCl3) d:
8.64 (d, 2H, 5.84 Hz), 7.60 (s, 1H), 7.52 (m, 3H), 7.44 (t,
1H, 7.35 and 7.72 Hz), 7.39 (d, 1H, and 7.35 Hz), 3.58 (s,
2H), 3.44 (m, 4H), 2.42 (m, 4H), and 1.45 (s, 9H). To a
solution of 1-(tertbutyloxycarbonyl)-4-[3-(pyridin-4-
yl)phenylmethyl]-piperazine (1.15 g, 3.26 mmol) in
CH2Cl2 (80 mL) was added dropwise trifluoroacetic acid
(5 mL), and the mixture was stirred at room temperature
for 3 h, diluted with CH2Cl2 (100 mL), and washed with
a NaOH solution 1 N, and then with water. The organic
phase was dried over Na2SO4 and evaporated under re-
duced pressure to provide 6a as a colorless oil; (0.8 g,
4.6.9. N-[4-(Dimethylaminomethyl)phenylmethyl]-pipera-
zine (6d). Following the procedure used for 6a, 4-(N-
dimethylaminomethyl)-benzaldehyde23 (4.8 g, 29.45 mmol),
N-tertbutyloxycarbonylpiperazine (Fluka, 6 g, 32.26
mmol), and sodium triacetoxyborohydride (7.5 g,
35.34 mmol) were used to provide 1-(tertbutyloxycar-
bonyl)-4-[4-(N-dimethylaminomethyl)phenylmethyl]-
1
piperazine as a colorless oil; H NMR (CDCl3) d: 7.18
(m, 4H), 3.42 (s, 2H), 3.35 (m, 4H), 3.33 (s, 2H), 2.30
(t, 4H), 2.16 (s, 6H), and 1.38 (s, 9H). Following the pro-
cedure used for compound 6a, 1-(tertbutyloxycarbonyl)-
4-[4-(N-dimethylaminomethyl)phenylmethyl]-piperazine
was hydrolyzed with trifluoroacetic acid (50 mL) to pro-
vide 6d as a pale yellow oil (5 g, 73%); 1H NMR (CDCl3)
d: 7.02 (m, 4H), 3.24 (s, 2H), 3.16 (s, 2H), 2.64 (t, 4H),
2.17 (m, 4H), and 2 (s, 6H).
4.6.10. N-[4-(4-Methyl-piperazin-1-yl)phenylmethyl]-pipera-
zine (6e). Following the procedure used for 6a, 4-(4-
methyl-piperazin-1-yl)-benzaldehyde22 (3 g, 14.71 mmol),
1
97%); H NMR (CDCl3) d: 8.58 (d, 2H, 6.22 Hz), 7.54
(s, 1H), 7.45 (m, 3H), 7.36 (t, 1H, 7.72 and 6.78 Hz),
7.33 (d, 1H, 7.35 Hz), 3.50 (s, 2H), and 2.83 (m, 4H),
2.38 (m, 4H).
N-tertbutyloxycarbonylpiperazine
(Fluka,
3.01 g,
16.17 mmol), and sodium triacetoxyborohydride
(3.74 g, 17.65 mmol) were used to provide after flash
chromatography (CH2Cl2/MeOH, 95:5 then 90:10), 1-
(tertbutyloxycarbonyl)-4-[4-(4-methyl-piperazin-1-yl)phen-
ylmethyl]-piperazine as a yellow oil (5.3 g, 96%); 1H
NMR (CDCl3) d: 7.31 (d, 2H, 8.67 Hz), 7.00 (d, 2H,
8.67 Hz), 3.56 (s, 2H), 3.55 (m, 4H), 3.34 (m, 4H), 2.95
(m, 2H), 2.71 (m, 4H), 2.49 (s+m, 5H), 1.59 (s, 9H). Fol-
lowing the procedure used for compound 6a, 1-(tertbu-
tyloxycarbonyl)-4-[4-(4-methyl-piperazin-1-yl)phenyl-
methyl]-piperazine (1 g, 2.67 mmol) was hydrolysed with
trifluoroacetic acid (5 mL) to provide 6e as a colorless
oil (0.63 g, 86%); 1H NMR (CDCl3) d: 7.11 (d, 2H,
8.67 Hz), 6.79 (d, 2H, 8.67 Hz), 3.34 (s, 2H), 3.13 (t,
4H), 2.81 (t, 4H), 2.50 (t, 4H), 2.33 (m, 4H), and 2.18
(br s, 3H).
4.6.7. N-[4-(Pyridin-4-yl)phenylmethyl]-piperazine (6b).
Following the procedure used for 6a, 4-(pyridin-4-yl)-
benzaldehyde21 (1.5 g, 8.2 mmol), N-tertbutyloxycar-
bonylpiperazine (Fluka, 1.6 g, 8.6 mmol) and sodium
triacetoxyborohydride (2.08 g, 9.84 mmol) were used to
provide
1-(tertbutyloxycarbonyl)-4-[4-(pyridin-4-yl)
phenylmethyl]-piperazine as a colorless oil which crys-
tallized on standing (2.86 g, 99%); mp 76–78 ꢁC; 1H
NMR (CDCl3) d: 8.56 (d, 2H, 6.22 Hz), 7.51 (d, 2H,
8.29 Hz), 7.43 (d, 2H, 6.03 Hz), 7.35 (d, 2H, 8.10 Hz),
3.49 (s, 2H), 3.37 (t, 4H), 2.35 (t, 4H), and 1.38 (s,
9H). Following the procedure used for compound 6a,
1-(tertbutyloxycarbonyl)-4-[4-(pyridin-4-yl)phenyl-
methyl]-piperazine (2.86 g, 8.1 mmol) was hydrolyzed
with trifluoroacetic acid (10 mL) to provide 6b as a
4.6.11. N-[4-(1-Methyl-pyrazol-4-yl)phenylmethyl]-piper-
azine (6f). Following the procedure used for 6a, 4-(1-
methyl-pyrazol-4-yl)-benzaldehyde22 (2 g, 10.75 mmol),
1
white amorphous solid (1.8 g, 88%); H NMR (CDCl3)
d: 8.61 (d, 2H, 6.22 Hz), 7.56 (d, 2H, 8.29 Hz), 7.47 (d,
2H, 6.22 Hz), 7.41 (d, 2H, 8.10 Hz), 3.53 (s, 2H), 2.91
(t, 4H), 2.45 (m, 4H), and 2.35 (s, 1H).
N-tertbutyloxycarbonylpiperazine
(Fluka,
2.20 g,
11.83 mmol) and sodium triacetoxyborohydride
(3.42 g, 16.13 mmol) were used to provide 1-(tertbutyl-
oxycarbonyl)-4-[4-(1-methyl-pyrazol-4-yl)phenyl-
4.6.8. N-[4-(Imidazol-1-yl)phenylmethyl]-piperazine (6c).
Following the procedure used for 6a, 4-(imidazol-1-yl)-
benzaldehyde22 (2.1 g, 12.2 mmol), N-tertbutyloxycar-
bonylpiperazine (Fluka, 2.5 g, 13.42 mmol) and sodium
triacetoxyborohydride (2.84 g, 13.42 mmol) were used to
provide after flash chromatography (CH2Cl2/MeOH,
95:5), 1-(tertbutyloxycarbonyl)-4-[4-(imidazol-1-yl)phenyl-
methyl]-piperazine as a colorless oil (4 g, 96%); 1H
NMR (CDCl3) d: 7.83 (s, 1H), 7.36 (d, 2H, 8.48 Hz),
7.26 (d, 2H, 8.48 Hz), 7.2 (br s, 1H), 7.14 (br s, 1H),
3.49 (s, 2H), 3.37 (t, 4H), 2.35 (t, 4H), and 1.38 (s,
9H). Following the procedure used for compound 6a,
1-(tertbutyloxycarbonyl)-4-[4-(imidazol-1-yl)phenyl-
methyl]-piperazine (4 g, 11.7 mmol) was hydrolysed with
trifluoroacetic acid (15 mL) to provide 6c as a colorless
oil (2.73 g, 96%); 1H NMR (CDCl3) d: 7.85 (s, 1H), 7.43
(d, 2H, 8.48 Hz), 7.33 (d, 2H, 8.48 Hz), 7.28 (br s, 1H),
7.2 (br s, 1H), 3.54 (s, 2H), 2.95 (t, 4H), and 2.47 (m,
5H).
methyl]-piperazine as a white solid (3.8 g, 99%); 1H
NMR (CDCl3) d: 7.54 (s, 1H), 7.39 (s, 1H), 7.21 (d,
2H, 8.10 Hz), 7.09 (d, 2H, 8.10 Hz), 3.73 (s, 3H), 3.29
(s, 2H), 3.22 (t, 4H), 2.19 (t, 4H), and 1.24 (s, 9H). Fol-
lowing the procedure used for 6a, 1-(tertbutyloxycar-
bonyl)-4-[4-(1-methyl-pyrazol-4-yl)phenylmethyl]-piper-
azine (3.8 g, 10.67 mmol) was hydrolyzed with
trifluoroacetic acid (10 mL) to provide 6f as a white so-
1
lid (2.2 g, 80%); H NMR (CDCl3) d: 7.67 (s, 1H), 7.52
(s, 1H), 7.34 (d, 2H, 8.10 Hz), 7.23 (d, 2H, 8.10 Hz), 3.87
(s, 3H), 3.41 (s, 2H), 2.82 (t, 4H), and 2.35 (m, 4H).
4.6.12. 1-[4-Chlorobenzoyl]-4-[4-acetylphenylmethyl]-piperazine
(9a). To a solution of N-(4-chlorobenzoyl)-piperazine 8
(Enamine BB, 1.59 g, 7.08 mmol) in CH2Cl2 (50 mL)
were0 added 4-acetyl-benzaldehyde 7a (Aldrich, 1 g,
6.76 mmol) and then portionwise sodium triacetoxy-
borohydride (2.15 g, 10.13 mmol). The mixture was stir-

M.-H. Fouchet et al. / Bioorg. Med. Chem. 16 (2008) 6218–6232
6229
red at room temperature overnight. The reaction was di-
luted with CH2Cl2 (150 mL), washed with water, and the
organic phase was dried (Na2SO4), and evaporated un-
der reduced pressure. The oily residue was crystallized
by trituration with i-Pr2O to provide 9a as white crystals
(1.6 g, 66%); mp: 122 ꢁC; H NMR (CDCl3) d: 7.93 (d,
2H, 8.29 Hz), 7.43 (d, 2H, 8.29 Hz), 7.38 and 7.34 (2d,
4H, 8.85 Hz), 3.78 (m, 2H), 3.60 (s, 2H), 3.46 (m, 2H),
2.61 (s, 3H), and 2.47 (br m, 4H).
procedure used for 10, 4c (0.25 g, 0.63 mmol), pyrroli-
dine (Aldrich, 0.13 mL, 1.56 mmol) and sodium triacet-
oxy borohydride (0.16 g, 0.75 mmol) were used to
provide, after flash chromatography (CH2Cl2/MeOH,
98:2 then 93:7) and trituration of the resulting oily resi-
due with i-Pr2O, 12 as white crystals (0.165 g, 58%); mp:
140 ꢁC; 1H NMR (CDCl3) d: 7.68 (d, 2H, 8.67 Hz), 7.47
(m, 4H), 7.42 (d, 2H, 8.10 Hz), 7.39 (d, 2H, 8.67 Hz),
7.09 (d, 2H, 8.10 Hz), 4.52 (br s, 1H), 3.80 (s, 2H),
3.20 (m, 2H), 2.76 (m, 6H), and 1.86 (m, 4H); MS-APCI
1
4.6.13.
1-[4-Chlorobenzoyl]-4-[4-cyanophenylmethyl]-
455.10
[M+H]+;
HRMS-ESI:
calculated
for
piperazine (9b). Following the procedure used for com-
pound 9a, N-(4-chlorobenzoyl)-piperazine 8 (Enamine
BB, 3 g, 13.36 mmol), 4-cyano-benzaldehyde 7b (Al-
drich, 1.75 g, 13.36 mmol) and sodium triacetoxyboro-
hydride (4.25 g, 20 mmol) were used to provide, after
trituration with i-Pr2O, 9b as white crystals (3.92 g,
C25H27Cl1N2O2S1 [M+H]+ 455.1560, found 455.1565;
retention time = 2.94 min.
4.6.17.
1-[4-Chlorophenylsulfonyl]-4-[3-(pyridin-4-yl)
phenylmethyl]-piperazine (13). To a solution of 6a
(0.25 g, 0.99 mmol) in CH2Cl2 (30 mL) were added 4-
chlorobenzenesulfonyl chloride (Aldrich, 0.219 g,
1.04 mmol) and then triethylamine (0.16 mL,
1.18 mmol). The mixture was stirred at room tempera-
ture for 30 min and then diluted with CH2Cl2 (50 mL).
The organic solution was washed with water, dried
(Na2SO4), and evaporated under reduced pressure.
The residue was purified by flash chromatography
(CH2Cl2/MeOH, 99:1, then 95:5). The resulting oil
was crystallized by trituration with i-Pr2O to provide
13 as cream crystals (0.183 g, 43%); mp: 128–130 ꢁC;
1H NMR (CDCl3) d: 8.58 (d, 2H, 6.03 Hz), 7.6 (d,
2H, 8.48 Hz), 7.50 (d, 2H, 5.65 Hz), 7.46 to 7.42
(m, 2H), 7.43 (d, 2H, 8.29 Hz), 7.35 (t, 1H,
7.54 Hz), 7.28 (dd, 1H, 7.54 2.45 Hz), 3.7 (s, 2H),
3.1 (m, 4H), and 2.65 (m, 4H); MS-APCI 428.10
[M+H]+; HRMS-ESI: calculated for C22H22Cl1
N3O2S1 [M+H]+ 428.1199, found 428.1194; retention
time = 3.21 min.
1
86%); mp: 157 ꢁC; H NMR (CDCl3) d: 7.62 (d, 2H,
8.29 Hz), 7.46 (d, 2H, 8.29 Hz), 7.39 (d, 2H, 8.67 Hz),
7.34 (d, 2H, 8.85 Hz), 3.79 (m, 2H), 3.60 (s, 2H), 3.45
(m, 2H), and 2.47 (m, 4H).
4.6.14. N-[2-(4-(3-Dimethylaminomethylphenyl)phenyl)
ethyl]-4-cyanobenzenesulfonamide (10). To a solution of
4a (0.2 g, 0.51 mmol) in CH2Cl2 (30 mL) were added
dimethylamine (Aldrich, solution 2 M/THF, 0.51 mL,
1 mmol) and then portionwise sodium triacetoxyboro-
hydride (0.13 g, 0.61 mmol). The mixture was stirred at
room temperature overnight. The organic solution was
diluted with CH2Cl2, washed with water, then dried
(Na2SO4), and evaporated under reduced pressure.
The oily residue was purified by flash chromatography
(CH2Cl2/MeOH, 98:2 then 9:1). The resulting oil was
crystallized by trituration with i-Pr2O to provide 10 as
white crystals (0.14 g, 65%); mp: 97 ꢁC; 1H NMR
(CDCl3) d: 7.9 (d, 2H, 8.48 Hz), 7.75 (d, 2H, 8.48 Hz),
7.59 (s, 1H), 7.53 (d, 2H, 8.10 Hz), 7.5 (d, 1H,
7.72 Hz), 7.42 (t, 1H, 7.54 Hz), 7.32 (d, 1H, 7.35 Hz),,
7.15 (d, 2H, 8.10 Hz), 4.82 (br s, 1H), 3.59 (s, 2H),
3.32 (t, 2H), 2.85 (t, 2H), and 2.35 (s, 6H); MS-APCI
4.6.18. 1-[4-Chlorophenylsulfonyl]-4-[4-(pyridin-4-yl)phenyl-
methyl]-piperazine (14). Following the procedure used
for 13, 6b (0.3 g, 1.18 mmol), 4-chlorobenzene sulfonyl
chloride (Aldrich, 0.262 g, 1.24 mmol) and triethylamine
(0.2 mL, 1.42 mmol) were used to provide after flash
chromatography (CH2Cl2/MeOH, 99:1 then 97:3) and
trituration of the resulting oil with i-Pr2O, 14 as white
420.10
[M+H]+;
HRMS-ESI:
calculated
for
C24H25N3O2S1 [M+H]+ 420.1746, found 420.1783;
retention time = 2.64 min.
1
crystals (0.345 g, 68%); mp: 136 ꢁC; H NMR (CDCl3)
4.6.15. N-[2-(4-(4-Dimethylaminomethylphenyl)phenyl)
ethyl]-4-cyanobenzenesulfonamide (11). Following the
procedure used for 10, 4b (0.3 g, 0.77 mmol), dimethyl-
amine (Aldrich, 0.77 mL of a solution 2 M in THF,
1.54 mmol) and sodium triacetoxy borohydride (0.2 g,
0.92 mmol) were used to provide, after flash chromatog-
raphy (CH2Cl2/MeOH, 98:2 then 9:1) and trituration of
the resulting oily residue with i-Pr2O, 11 as white crys-
d: 8.65 (d, 2H, 6.22 Hz), 7.7 (d, 2H, 8.67 Hz), 7.57 (d,
2H, 8.29 Hz), 7.52 (d, 2H, 8.67 Hz), 7.48 (d, 2H,
6.03 Hz), 7.37 (d, 2H, 8.29 Hz), 3.57 (s, 2H), 3.07 (m,
4H), 2.58 (m, 4H); MS-APCI 428.10 [M+H]+; HRMS-
ESI: calculated for C22H22Cl1N3O2S1 [M+H]+
428.1199, found 428.1201; retention time = 3.28 min.
4.6.19. 1-[4-Chlorophenylsulfonyl]-4-[4-(dimethylamino-
methyl)phenylmethyl]-piperazine (19). Following the
procedure used for 13, 6d (0.5 g, 2.14 mmol), 4-chloro-
benzene sulfonyl chloride (Aldrich, 0.455 g, 2.14 mmol)
and triethylamine (0.3 mL, 2.14 mmol) were used to pro-
vide 19 as a colorless oil (0.87 g, quantitative); 1H NMR
(CDCl3) d: 7.6 (d, 2H, 8.67 Hz), 7.41 (d, 2H, 8.48 Hz),
7.15 (d, 2H, 8.10 Hz), 7.10 (d, 2H, 8.29 Hz), 3.4 (s,
2H), 3.31 (s, 2H), 2.95 (t, 4H), 2.44 (t, 4H), and 2.15
(s, 6H); MS-APCI 408.10 [M+H]+; HRMS-ESI: calcu-
lated for C20H26Cl1N3O2S1 [M+H]+ 408.1512, found
408.1523; retention time = 2.51 min.
1
tals (0.215 g, 67%); mp: 174 ꢁC; H NMR (DMSO d6)
d: 8.05 (d, 2H, 8.67 Hz), 7.94 (d, 2H, 8.67 Hz), 7.61 (d,
2H, 8.10 Hz), 7.56 (d, 2H, 8.10 Hz), 7.39 (d, 2H,
8.29 Hz), 7.25 (d, 2H, 8.10 Hz), 3.46 (s, 2H), 3.25 (br
s, 1H), 3.09 (m, 2H), 2.75 (t, 2H), and 2.21 (s, 6H);
MS-APCI 420.1 [M+H]+; HRMS-ESI: calculated for
C24H25N3O2S1 [M+H]+ 420.1746, found 420.1768;
retention time = 2.62 min.
4.6.16. N-[2-(4-(4-((Pyrolidin-1-yl)methyl)phenyl)pheny-
l)ethyl]-4-chlorobenzenesulfonamide (12). Following the

6230
M.-H. Fouchet et al. / Bioorg. Med. Chem. 16 (2008) 6218–6232
4.6.20. 1-[4-Chlorophenylsulfonyl]-4-[4-(4-methylpipera-
zin-1-yl)phenylmethyl]-piperazine (21). Following the
procedure used for 13, 6e (8.8 g, 32.12 mmol), 4-chloro-
benzene sulfonyl chloride (Aldrich, 7.12 g, 33.72 mmol)
and triethylamine (5.36 mL, 38.54 mmol) were used to
provide, after flash chromatography (CH2Cl2/MeOH,
99:1 then 95:5) and trituration of the resulting oil with
i-Pr2O, 21 as white crystals (11.92 g, 82%); mp: 155–
157 ꢁC; 1H NMR (CDCl3) d: 7.69 (d, 2H, 8.48 Hz),
7.51 (d, 2H, 8.67 Hz), 7.12 (d, 2H, 8.67 Hz), 6.85 (d,
2H, 8.67 Hz), 3.42 (s, 2H), 3.21 (t, 4H), 3.03 (m, 4H),
2.59 (t, 4H), 2.51 (t, 4H), and 2.37 (s, 3H); MS-APCI
procedure used for 13, 6f (0.3 g, 1.17 mmol), 4-chloro-
benzoyl chloride (Aldrich, 0.156 mL, 1.23 mmol) and
triethylamine (0.2 mL, 1.41 mmol) were used to provide,
after flash chromatography (CH2Cl2/MeOH, 99:1 then
95:5) and trituration of the resulting oil with i-Pr2O,
25 as white crystals (0.42 g, 91%); mp: 241 ꢁC; 1H
NMR (CDCl3) d: 7.97 (s, 1H), 7.82 (s, 1H), 7.65 (d,
2H, 8.29 Hz), 7.6 (d, 2H, 8.10 Hz), 7.57 (d, 2H,
8.85 Hz), 7.52 (d, 2H, 8.10 Hz), 4.17 (s, 3H), 3.99 (m,
2H), 3.76 (s, 2H), 3.66 (m, 2H), 2.74, and 2.65 (2m,
4H); MS-APCI 395.1 [M+H]+; HRMS-ESI: calculated
for C22H23Cl1N4O1 [M+H]+ 395.1638, found 395.1655;
retention time = 2.63 min.
449.1
[M+H]+;
HRMS-ESI:
calculated
for
C22H29Cl1N4O2S1 [M+H]+ 449.1778, found 449.1819;
retention time = 2.74 min.
4.6.25.
1-[4-Methoxyphenylcarbonyl]-4-[4-(pyridin-4-
yl)phenylmethyl]-piperazine (17). To a solution of 6b
(0.25 g, 0.99 mmol) in DMF (30 mL) were added 4-
methoxybenzoic acid (Aldrich, 0.18 g, 1.18 mmol),
HOBT (0.16 g, 1.18 mmol), EDCI (0.227 g, 1.18 mmol),
and triethylamine (0.16 mL, 1.18 mmol) and the mixture
was stirred at room temperature overnight and then di-
luted with CH2Cl2. The organic solution was washed
with a NaOH solution 1 N, then water, dried (Na2SO4),
and evaporated under reduced pressure. The residue was
purified by flash chromatography (CH2Cl2/MeOH,
95:5). The resulting oil was crystallized by trituration
with i-Pr2O to provide 17 as white crystals (0.26 g,
4.6.21. 1-[4-Chlorophenylcarbonyl]-4-[4-(pyridin-4-yl)phen-
ylmethyl]-piperazine (15). Following the procedure used
for 13, 6b (0.3 g, 1.19 mmol), 4-chlorobenzoyl chloride
(Aldrich, 0.16 mL, 1.24 mmol) and triethylamine
(0.2 mL, 1.42 mmol) were used to provide, after flash
chromatography (CH2Cl2/MeOH, 99:1 then 97:3) and
trituration of the resulting oil with i-Pr2O, 15 as white
1
crystals (0.358 g, 77%); mp: 160 ꢁC; H NMR (CDCl3)
d: 8.66 (d, 2H, 6.03 Hz), 7.61 (d, 2H, 8.10 Hz), 7.51 (d,
2H, 6.22 Hz), 7.45 (d, 2H, 8.10 Hz), 7.38–7.39 (2d, 4H,
8.85 Hz), 3.80 (m, 2H), 3.61 (s, 2H), 3.46 (m, 2H), and
2.5 (m, 4H); MS-APCI 392.20 [M+H]+; HRMS-ESI:
calculated for C23H22Cl1N3O1 [M+H]+ 392.1530, found
392.1510; retention time = 2.86 min.
1
68%); mp: 162 ꢁC; H NMR (CDCl3) d: 8.57 (d, 2H,
6.03 Hz), 7.52 (d, 2H, 8.29 Hz), 7.42 (d, 2H, 6.03 Hz),
7.36 (d, 2H, 8.29 Hz), 7.30 (d, 2H, 8.85 Hz), 6.82 (d,
2H, 8.85 Hz), 3.76 (s, 3H), 3.52 (s, 2H), 2.42 (m, 4H),
and 1.61 (m, 4H); MS-APCI 388.2 [M+H]+; HRMS-
ESI: calculated for C24H25N3O2 [M+H]+ 388.2025,
found 388.2049; retention time = 2.58 min.
4.6.22.
1-[4-Chlorophenylcarbonyl]-4-[4-(imidazol-1-
yl)phenylmethyl]-piperazine (22). Following the proce-
dure used for 13, 6c (13.4 g, 55.37 mmol), 4-chloroben-
zoyl chloride (Aldrich, 7.39 mL, 58.14 mmol) and
triethylamine (9.24 mL, 66.45 mmol) were used to pro-
vide, after flash chromatography (CH2Cl2/MeOH, 99:1
then 95:5) and trituration of the resulting oil with i-
Pr2O, 22 as cream crystals (11.55, 55%); mp: 148 ꢁC;
1H NMR (CDCl3) d: 8.03 (s, 1H), 7.62 (d, 2H,
8.29 Hz), 7.55 (d, 2H, 8.85 Hz), 7.53 (d, 2H, 9.04 Hz),
7.51 (d, 2H, 8.48 Hz), 7.45 (br s, 1H), 7.38 (br s, 1H),
3.96 (m, 2H), 3.77 (s, 2H), 3.65 (m, 2H), and 2.67 (m,
4H); MS-APCI 381.10 [M+H]+; HRMS-ESI: calculated
for C21H21Cl1N4O1 [M+H]+ 381.1482, found 381.1506;
retention time = 2.50 min.
4.6.26. 1-[4-(Trifluoromethoxy)phenylcarbonyl]-4-[4-(dimeth-
ylaminomethyl)phenylmeth yl]-piperazine (20). Following
the procedure used for 17, 6d (0.23 g, 1 mmol), 4-triflu-
oromethoxybenzoic acid (Aldrich, 0.226 g, 1.1 mmol),
HOBT (0.148 g, 1.1 mmol), EDCI (0.21 g, 1.1 mmol),
and triethylamine (0.15 mL, 1.1 mmol) were used to pro-
vide, after flash chromatography (CH2Cl2/MeOH, 9:1),
1
20 as a colorless oil (0.16 g, 38%); H NMR (CDCl3)
d: 7.68 (d, 2H, 8.48 Hz), 7.51 (br s, 4H), 7.49 (d, 2H),
4.02 (m, 2H), 3.77 (s, 2H), 3.67 (m, 4H), 2.76 (m, 2H),
2.64 (m, 2H), and 2.49 (s, 6H); MS-APCI 422.2
[M+H]+; HRMS-ESI: calculated for C22H26F3N3O2
[M+H]+ 422.2055, found 422.2074; retention
time = 2.53 min.
4.6.23. 1-[4-Chlorophenylcarbonyl]-4-[4-(dimethylamino-
methyl)phenylmethyl]-piperazine (18). Following the pro-
cedure used for 13, 6d (0.23 g, 1 mmol), 4-chlorobenzoyl
chloride (Aldrich, 0.13 mL, 1 mmol) and triethylamine
(0.16 mL, 1.2 mmol) were used to provide, after flash
chromatography (CH2Cl2/MeOH, 90:10), 18 as colorless
oil (0.29 g, 79%); 1H NMR (CDCl3) d: 7.39 (d, 2H,
8.67 Hz), 7.35 (d, 2H, 8.29 Hz), 7.28 (br s, 4H), 3.77
(m, 2H), 3.54 (s, 2H), 3.49 (s, 2H), 3.44 (m, 2H), 2.52
(m, 2H), 2.41 (m, 2H), and 2.27 (s, 6H); MS-APCI
4.6.27. 1-[4-Trifluoroacetyl]-4-[4-(imidazol-1-yl)phenyl-
methyl]-piperazine (27). To a solution of 6c (0.2 g,
0.83 mmol) in CH2Cl2 (30 mL) were added trifluoroace-
tic anhydride (Aldrich, 0.17 mL, 1.24 mmol) and then
triethylamine (0.14 mL, 1 mmol) and the mixture was
stirred at room temperature overnight and then diluted
with CH2Cl2. The organic solution was washed with a
solution of NaOH 1 N, then water, dried (Na2SO4),
and concentrated under reduced pressure. The residue
was purified by flash chromatography (CH2Cl2/MeOH,
98:2, then 95:5) to provide 27 as a cream oil (0.19 g,
372.2
[M+H]+;
HRMS-ESI:
calculated
for
C21H26Cl1N3O1 [M+H]+ 372.1843, found 372.1866;
retention time = 2.35 min.
1
4.6.24. 1-[4-Chlorophenylcarbonyl]-4-[4-(1-methyl-pyra-
zol-4-yl)phenylmethyl]-piperazine (25). Following the
68%); H NMR (CDCl3) d: 7.85 (s, 1H), 7.37 (d, 2H,
8.67 Hz), 7.29 (d, 2H, 8.48 Hz), 7.22 (br s, 1H), 7.16

M.-H. Fouchet et al. / Bioorg. Med. Chem. 16 (2008) 6218–6232
6231
(br s, 1H), 3.64 (t, 2H), 3.56 (t, 2H), 3.52 (s, 2H), and
HRMS-ESI: calculated for C21H21Cl1N4O1 [M+H]+
381.1482, found 381.1493; retention time = 2.49 min.
2.46 (t, 4H); MS-APCI 243.20 [(MÀCOCF3)+H]+.
4.6.28. 1-[4-Chlorobenzyl]-4-[4-(pyridin-4-yl)phenylmethyl]-
piperazine (16). To a solution of 6b (0.25 g, 0.99 mmol)
in CH2Cl2 (30 mL) were added 4-chlorobenzaldehyde
(Aldrich, 0.139 g, 0.99 mmol) and then portionwise so-
dium triacetoxyborohydride (0.31 g, 1.48 mmol). The
mixture was stirred at room temperature overnight.
The organic solution was diluted with CH2Cl2, washed
with water, then dried (Na2SO4), and evaporated under
reduced pressure. The oily residue was purified by flash
chromatography (CH2Cl2/MeOH, 97:3 then 93:7). The
resulting oil was crystallized by trituration with i-Pr2O
to provide 16 as white crystals (0.23 g, 58%); mp:
130 ꢁC; 1H NMR (CDCl3) d: 8.65 (d, 2H, 6.22 Hz),
7.59 (d, 2H, 8.29 Hz), 7.51 (d, 2H, 6.22 Hz), 7.44 (d,
2H, 8.29 Hz), 7.28 (br s, 4H), 3.58 (s, 2H), 3.49 (s,
2H), and 2.51 (m, 8H); MS-APCI 378.20 [M+H]+;
HRMS-ESI: calculated for C23H24Cl1N3 [M+H]+
378.1737, found 378.1758; retention time = 3.52 min.
4.6.31. 1-[4-Chlorophenylcarbonyl]-4-[4-(5-methyl-1,2,4-
oxadiazol-3-yl)phenylmethyl]-piperazine (26). A mixture
of 9b (1 g, 2.94 mmol), hydroxylamine hydrochloride
(Aldrich, 0.33 g, 4.71 mmol) and sodium acetate
(0.725 g, 8.84 mmol) in EtOH (50 mL) and water
(10 mL) was heated under reflux overnight and then
poured in to water. The aqueous phase was extracted
twice with CH2Cl2 and the organic phase was dried
(Na2SO4) and evaporated under reduced pressure to
provide, after trituration with i-Pr2O of the oily residue,
the amidoxime intermediate as a white solid (0.7 g,
64%). The amidoxime intermediate (0.25 g, 0.67 mmol)
was dissolved in CH2Cl2 (20 mL). Acetic anhydride
(0.13 mL, 1.34 mmol) and then triethylamine (0.11 mL,
0.8 mmol) were slowly added and the mixture was stir-
red at room temperature for 1 h and then under reflux
overnight. After cooling, the mixture was poured in to
water and neutralized with a saturated NaHCO3 solu-
tion. The aqueous phase was extracted twice with
CH2Cl2 and the organic phase was dried (Na2SO4)
and evaporated under reduced pressure. The crude
product was purified by flash chromatography
(CH2Cl2/MeOH, 97:3) The resulting oily residue was
triturated with i-Pr2O to provide 26 as white crystals
4.6.29. 1-[4-Chlorophenylcarbonyl]-4-[4-(pyrazol-1-yl)phenyl-
methyl]-piperazine (23). To a solution of 4-(pyrazol-1-
yl)benzaldehyde22 (0.3 g, 1.74 mmol) in CH2Cl2
(30 mL) were added N-(4-chlorobenzoyl)-piperazine 8
(0.43 g, 1.92 mmol) and then portionwise sodium tri-
acetoxyborohydride (0.55 g, 2.62 mmol). The mixture
was stirred at room temperature overnight. The organic
solution was diluted with CH2Cl2, washed with water,
then dried (Na2SO4), and evaporated under reduced
pressure. The oily residue was purified by flash chroma-
tography (CH2Cl2/MeOH, 99:1, then 95:5). The result-
ing oil was crystallized by trituration with i-Pr2O to
provide 23 as white crystals (0.35 g, 53%); mp: 149 ꢁC;
1H NMR (CDCl3) d: 7.91 (d, 1H, 2.26 Hz), 7.72 (d,
1H, 1.51 Hz), 7.64 (d, 2H, 8.48 Hz), 7.40 (d, 2H,
8.48 Hz), 7.38 (d, 2H, 8.85 Hz), 7.33 (d, 2H, 8.85 Hz),
6.46 (t, 1H, 2.07 Hz), 3.78 (m, 2H), 3.56 (s, 2H), 3.43
(m, 2H), 2.52, and 2.41 (2m, 4H); MS-APCI 381.1
[M+H]+; HRMS-ESI: calculated for C21H21Cl1N4O1
[M+H]+ 381.1482, found 381.1526; retention
time = 2.76 min.
1
(95 mg, 36%); mp: 142 ꢁC; H NMR (CDCl3) d: 7.94
(d, 2H, 8.29 Hz), 7.36 (d, 2H, 8.10 Hz), 7.31 (d, 2H,
8.10 Hz), 7.27 (d, 2H, 8.10 Hz), 3.71 (m, 2H), 3.53 (s,
2H), 3.37 (m, 2H), 2.58 (s, 3H), 2.44 and 2.36 (m, 4H);
MS-APCI 397.1 [M+H]+; HRMS-ESI: calculated for
C21H21Cl1N4O2 [M+H]+ 397.1431, found 397.1429;
retention time = 2.80 min.
Acknowledgment
The authors acknowledge Dr. Andrew Brewster for
helpful discussions and comments on the manuscript.
References and notes
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macother. 2006, 7, 170.
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J. Lipid Res. 1997, 38, 564.
6. Mark, M.; Mueller, P.; Maier, R.; Eisele, B. J. Lipid Res.
1996, 37, 148.
7. Morand, O. H.; Aebi, J. D.; Dehmlow, H.; Ji, Y.-H.;
Gains, N.; Lengsfeld, H.; Himber, J. J. Lipid Res. 1997,
38, 373.
8. Brown, G. R.; Hollinshead, D. M.; Stokes, E. S. E.;
Clarke, D. S.; Eakin, M. A.; Foubister, A. J.; Glossop, S.
C.; Griffiths, D.; Johnson, M. C.; McTaggart, F.; Mirrlees,
D. J.; Smith, G. J.; Wood, R. J. Med. Chem. 1999, 42,
1306.
4.6.30. 1-[4-Chlorophenylcarbonyl]-4-[4-(pyrazol-3-yl)phenyl-
methyl]-piperazine (24). A solution of intermediate 9a
(1 g, 2.8 mmol) in DMF dimethylacetal (20 mL) was
heated under reflux overnight then cooled, and evapo-
rated under reduced pressure. The resulting residue
was dissolved in EtOH (30 mL) and then hydrazine hy-
drate (0.2 mL, 4.13 mmol) was slowly added and the
mixture was heated under reflux for 4 h and then poured
in to water. The aqueous phase was extracted twice with
CH2Cl2 and the organic phase was dried (Na2SO4) and
evaporated under reduced pressure. The crude product
was purified by flash chromatography (CH2Cl2/MeOH,
95:5 then 90:10) to provide 24 as a cream solid (0.7 g;
89%); mp: 96 ꢁC; 1H NMR (CDCl3) d: 7.59 (d, 2H,
8.10 Hz), 7.49 (d, 1H, 2.26 Hz), 7.26 (d, 2H, 8.85 Hz),
7.24 (d, 2H, 7.54 Hz), 7.22 (d, 2H, 8.85 Hz), 6.49 (d,
1H, 2.26 Hz), 3.66 (m, 2H), 3.45 (s, 2H), 3.32 (m, 2H),
2.41, and 2.32 (2m, 4H); MS-APCI 381.1 [M+H]+;

6232
M.-H. Fouchet et al. / Bioorg. Med. Chem. 16 (2008) 6218–6232
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