Novel cholesterol biosynthesis inhibitors targeting human lanosterol 14α-demethylase (CYP51)

Article abstract of DOI:10.1016/j.bmc.2007.10.001

Novel cholesterol biosynthesis inhibitors, a group of pyridylethanol(phenylethyl)amine derivatives, were synthesized. Sterol profiling assay in the human hepatoma HepG2 cells revealed that compounds target human lanosterol 14α-demethylase (CYP51). Structu

Full text of DOI:10.1016/j.bmc.2007.10.001

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 209–221
Novel cholesterol biosynthesis inhibitors targeting human
lanosterol 14a-demethylase (CYP51)
a,ꢀ
a,b,ꢀ
b
a,
*
Tina Koro sˇ ec, Jure A cˇ imovi cˇ ,
Matej Seli sˇ kar, Darko Kocjan,
b
b
a
Klementina Fon Tacer, Damjana Rozman and Uro sˇ Urleb
a
Lek Pharmaceuticals d. d., Drug Discovery, 1000 Ljubljana, Verov sˇ kova 57, Slovenia
Center for Functional Genomics and Bio-chips, Institute of Biochemistry, Faculty of Medicine,
Zalo sˇ ka 4, University of Ljubljana, Slovenia
b
Received 23 April 2007; revised 15 September 2007; accepted 1 October 2007
Available online 4 October 2007
Abstract—Novel cholesterol biosynthesis inhibitors, a group of pyridylethanol(phenylethyl)amine derivatives, were synthesized. Ste-
rol profiling assay in the human hepatoma HepG2 cells revealed that compounds target human lanosterol 14a-demethylase
(
CYP51). Structure–activity relationship study of the binding with the overexpressed human CYP51 indicates that the pyridine
binds within the heme binding pocket in an analogy with the azoles.
2007 Elsevier Ltd. All rights reserved.
ꢀ
1. Introduction
farnesyl pyrophosphate branchpoint of the mevalonate
pathway remains a challenge.
Cholesterol-lowering therapy with 3-hydroxy-3-methyl-
glutaryl coenzyme A (HMG–CoA) reductase inhibitors
The first enzyme below the farnesyl pyrophosphate
branchpoint is squalene synthase. There are potent
inhibitors of squalene synthase that are also being devel-
oped as cholesterol lowering agents. Takeda has one,
lapaquistat (TAK-475), that is in Phase III clinical
(
statins) has been found as an effective method of reduc-
ing mortality and myocardial infarction among patients
with coronary artery disease. However, a significant
number of patients receiving statin therapy continue to
1
,2
7
have high residual risk. Even if statins are considered
3
as relatively safe drugs, considerable attention has been
development.
paid recently to the statin-based risk of muscular ad-
verse drug reactions, hepatotoxicity, and statin resis-
tance. By blocking HMG–CoA reductase, statins
might deplete ubiquinone (Co-Q-10) in the blood. How-
ever, there seems to be insufficient evidence from human
studies to link statin therapy unequivocally to patholog-
ically significantly decreased tissue Co-Q-10 levels. It
was observed that derangements in mevalonate kinase,
but not more distal enzymes of cholesterologenesis, are
associated with a skeletal myopathy, suggesting a criti-
Oxidosqualene cyclase (lanosterol synthase) is the sec-
ond enzyme bellow the farnesyl pyrophosphate branch-
point that has been identified as a target for novel
anticholesterolemic drugs that could complement
8
statins. A novel series of 4-piperidinopyridines and
4-piperidinopyrimidines showed potent and selective
4
,5
9
inhibition of the rat enzyme.
The subsequent enzyme of the cholesterol synthesis path-
way, the lanosterol 14a-demethylase (CYP51), is another
potential anticholesterolemic drug target. This enzyme,
which is the most evolutionarily conserved member of
the cytochrome P450 gene superfamily, is found in both
cal role for the isoprenoids in the maintenance of mus-
6
cle. Consequently, the development of novel
cholesterol lowering agents that are effective below the
1
0
prokaryotes and eukaryotes. CYP51 resides in endo-
1
1
plasmatic reticulum in eukaryotes. In animals it cata-
lyzes the oxidative removal of the 14a-methyl group
Keywords: Cholesterol biosynthesis inhibitors; Human lanosterol 14a-
demethylase (CYP51) inhibitors; Cholesterol intermediates profile;
Pyridylethanolamines.
(C32) of lanosterol and 24,25-dihydrolanosterol to
*
Corresponding author. Tel.: +386 1 580 3349; fax: +386 1 568
340; e-mail: darko.kocjan@sandoz.com
These authors contributed equally to the content of the work.
produce 4,4-dimethyl-5a-cholesta-8,14,24-triene-3b-ol
(FF-MAS). FF-MAS is the substrate for the consecutive
enzyme in cholesterol synthesis, sterol D14-reductase
2
ꢀ
0
968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.001

210
T. Koro sˇ ec et al. / Bioorg. Med. Chem. 16 (2008) 209–221
(
DHCR14), which produces 4,4-dimethyl-5a-cholesta-
,24-diene-3b-ol (T-MAS). Human lanosterol 14a-
2.3. Procedure C
1
2
8
demethylase is a relatively well-characterized target at
the level of gene,
sue-specificity,
ity.
Amides 9a and 9b were prepared by coupling commer-
cially available primary amines with the corresponding
carboxylic acid using EDC and HOBT activation.
Amides 9a and 9b were further reduced by borane-di-
1
3,14
15–18
regulation of expression,
and also protein and enzymatic activ-
However, due to the membrane character of the
tis-
1
9–21
34
2
2–24
enzyme, crystallization attempts have not been so far suc-
cessful. There is also no crystal structure available for the
yeast CYP51 enzymes that are approximately 40% identi-
cal to the human counterparts and represent common
methyl sulfide complex to provide secondary amines
3
5
10a and 10b. By the reaction of reductive alkylation
of the secondary amines using sodium triacetoxyborohy-
3
6
dride, tertiary amines 11a and 11b were prepared. Free
bases 11a and 11b were converted to dihydrobromide
salts 12a and 12b, respectively (Scheme 3).
2
5,26
antifungal drug targets.
Only the crystal structure of
the soluble 14a-sterol demethylase from Mycobacterium
2
7
tuberculosis is known. This bacterial enzyme is 28%
identical to the human CYP51 and provides a template
for analysis of eukaryotic orthologs. So far there have
been many studies directed toward fungal CYP51 inhibi-
tion. Most dealt with azole-type inhibitors and character-
2.4. Procedure D
2-Naphthaleneacetonitrile was reduced by borane-di-
3
5
methyl sulfide complex to provide primary amine 13.
Final compound 16 was prepared with same individual
reaction steps as under procedure B (Scheme 4).
2
8,29
istics of azole binding site.
Interestingly, ketoconazole
and fluconazole, well-known antifungal agents, showed
differing affinities for human CYP51 and experimental
evidence was presented for structural difference of azole
binding site in CYP51 between human and Mycobacte-
2.5. Procedure E
3
0
rium tuberculosis. A comparison of human CYP51
homology model with that of fungal analog from Candida
3-(Oxiran-2-ylmethoxy)pyridine 17 was prepared by the
reaction of 2-(chloromethyl)oxirane with pyridin-3-ol in
alkaline solution. Oxirane ring opening with phenyle-
thanamine, K CO , and KI afforded secondary amine
3
1
albicans was based on the crystal structure.
2
3
We have identified structurally diverse derivatives of
pyridylethanol(phenylethyl)amines interacting with hu-
man CYP51. Taking into account the importance of
the modified sterol composition after inhibition, a novel
sterol profiling assay has been developed to monitor the
effect of compounds on de novo synthesis of cholesterol.
This allowed us to predict the targeted enzyme and also
to perform a structure–activity relationship study. Bind-
ing studies on the purified, overexpressed human CYP51
enzyme confirmed predictions from the sterol profiling
ex vivo assay demonstrating its predictive power. Our
studies contribute to understanding of structural
requirements for human CYP51 inhibition. Recently
published pharmacophore model based on the three-
dimensional quantitative structure–activity relationship
18. Reductive alkylation using sodium triacetoxyboro-
hydride gave final product 19 (Scheme 5).
3. Biology
A novel ex vivo screening assay has been developed in
human hepatoma HepG2 cells as described in detail in
3
7
the Experimental section. Figure 1a represents an out-
line of the post-lanosterol portion of the cholesterol bio-
synthesis. It indicates sterol intermediates that are
3
measured in our assay. H-labeled de novo synthesized
lanosterol, FF-MAS (follicular fluid meiosis activating
sterol,
4,4-dimethyl-5a-cholesta-8,14,24-triene-3b-ol),
T-MAS (testis meiosis activating sterol, 4,4-dimethyl-
5a-cholesta-8,24-diene-3b-ol), zymosterol, desmosterol,
7-dehydrocholesterol, and cholesterol have been sepa-
rated on a reverse phase column and quantified by the
radioactivity detector. Even if structurally very similar,
these sterols can be separated and unequivocally identi-
fied using reference standards. Figure 1b shows the
relative quantities of de novo produced sterol intermedi-
ates and cholesterol in normal, non-treated HepG2 cells.
De novo produced cholesterol represents 20% of the
radiolabeled sterol pool. Lanosterol (25%) and lathos-
terol (25%) are two bulky sterol intermediates. A mix-
ture of 7-dehydrocholesterol and zymosterol that was
not separated under our experimental conditions
represents 23% of the pool. Two minor intermediates
are FF-MAS (2%) and desmosterol (5%). T-MAS was
not detected by the radio-HPLC assay in HepG2 cells.
T-MAS is a bulky sterol intermediate in mammalian
3
2
study offers a pharmacophore model consisting of
hydrogen bond acceptor and hydrophobic features.
2. Chemistry
Different synthetic approaches have been used for the
synthesis of target compounds with regard to the start-
ing materials.
2
.1. Procedure A
The synthesis of target compounds is shown in Scheme
1
dures described recently.
. Reactions were carried out according to the proce-
3
3
3
8
2
.2. Procedure B
testis where it is identified easily.
3
The synthesis of target compounds is shown in Scheme
Figure 2 shows H labeled sterols isolated from HepG2
cells treated with 10 lM solutions of compounds, poten-
tial cholesterol biosynthesis inhibitors. Normal medium
2
dures described recently.
. Reactions were carried out according to the proce-
3
3

T. Koro sˇ ec et al. / Bioorg. Med. Chem. 16 (2008) 209–221
211
(HBr)
N
N
N
O
a, b
c
Br
NH
OH R1
N
R2
O
1
OH
R
3a, 3d-l (from 2d)
2a, 2d
d
HBr
N
N
e
1
2
e
i
d
g
f
R = Pr R = H
HBr
N
R2
1
2
R = Me R = 3,4-diCl
N
R2
1
OH
R
1
2
1
R = Pr R = 3,4-diCl
OH
5a, 5d-l
R
1
2
R = Pr R = 3,4-diF
4
a, 4d-l
1
2
R = Pr R = 3,4-diOMe
1
2
j
R = Pr R = 3,4-diOH
1
2
h
k
l
R = Pr R = 4-NO
2
1
2
R = Pr R = 4-CF
3
1
2
R = Pr R = 3-CF3
N
N
Cl
Cl
Cl
Cl
N
N
N
N
OH
OH
OH
5
b
4c
, anhyd EtOH, rt, 2h; (b) R NH
4m
1
2
, anhyd EtOH, reflux, 5h; (c) R -phenylacetic acid, HOBt, EDC,
Scheme 1. Reagents and conditions: (a) NaBH
4
2
NMM, DMF, rt, 12 h; (d) BH
3
ÆMe S, THF, reflux, 10 h; (e) HBr, EtOH, 0 ꢁC, 2 h. (a is 4-pyridyl analog of i).
2
(
HBr)
N
a, b
Br
N
N
c
d
8a, 8b-g
N
N
R2
R2
H
OH R1
OH
a, 6b-g
O
6
7a, 7b-g
1
2
a
b
c
d
f
R = Pr R = 3,4-diCl
d
N
1
2
R = Pr R = 2-Cl
8
e
1
2
R = Pr R = 3-Cl
N
1
2
R = Pr R = 4-Cl
OH
1
2
R = Bu R = 3,4-diCl
1
2
g
R = Pr R = 2,4-diCl
7
e
2
Scheme 2. Reagents and conditions: (a) NaBH
4 3
, anhyd EtOH, rt, 2 h; (b) R -phenethylamine, anhyd EtOH, reflux, 12 h; (c) aldehyde, NaBH(OAc) ,
2 2
CH ClCH Cl, rt, Ar 2 h; (d) HBr, EtOH, 0 ꢁC, 2 h. (a is 4-pyridyl analog of d).
and atorvastatin were used as a control (group C). As
expected, atorvastatin showed a complete blockage of
cholesterol biosynthesis. Due to the blockage of
HMG–CoA reductase, an enzyme in the early part
of cholesterol biosynthesis, no post-lanosterol interme-
diates have been observed. Tested compounds have been
grouped according to the sterol profile. Group S inhibi-
tors show no accumulation of cholesterol, low accumu-
lation of post-lanosterol cholesterol biosynthesis
intermediates, and high accumulation of lanosterol, a
substrate for the CYP51 enzyme. Notable accumulation
of lanosterol is observed for group W albeit at lower
amount compared to the S group inhibitors and de novo
synthesis of cholesterol is not completely blocked. With-

212
T. Koro sˇ ec et al. / Bioorg. Med. Chem. 16 (2008) 209–221
N
N
a
O
O
+
OH
R2
NH
2
N
R2
H
9a, 9b
b, c
N
d
1
2a, 12b
N
R2
1
1
2
R
a R = Pr R = 3-F
11a, 11b
1
2
b R = CH CH CH Ph R = 3,4-diCl
2
2
2
3 3
Scheme 3. Reagents and conditions: (a) HOBt, EDC, NMM, DMF, rt, 12h; (b) BH ÆMeS, THF, reflux, 10 h; (c) aldehyde, NaBH(OAc) ,
CH
2
2
ClCH Cl, rt, Ar 12 h; (d) HBr, EtOH, 0 ꢁC.
sponse of the purified human CYP51 to ketoconazole
and 5d. It is evident that 5d produces a similar spectral
response as ketoconazole.
a
CN
NH2
O
13
In Table 1 apparent dissociation constants and maxi-
mum spectral responses are compared to lanosterol
accumulation for a selected set of compounds out of
that presented in Figure 2. Type II spectral response
for compounds listed in Table 1, except 5b, is very sim-
ilar to that of 5d and shows a characteristic saturation
curve (Fig. 3c). This is a strong indication that novel
compounds interact with the human lanosterol 14a-
demethylase in a manner similar to that previously rec-
HBr
N
HBr
N
OH Pr
2
3,40
16
ognized for some azoles.
CYP51 inhibitors that do
not have a nitrogen atom for heme iron atom coordina-
tion have spectra quite different from those of ketocon-
azole or fluconazole.
3 2
Scheme 4. Reagents and conditions: (a) BH ÆMe S, THF, reflux, 5 h.
4
1
in the group D compound 12a shows high FF-MAS
accumulation and is potentially a strong sterol D14-
reductase (DHCR14) inhibitor. Group N compounds
are not inhibitors of cholesterol biosynthesis. Their ste-
rol profile shows no effect on de novo synthesis of cho-
lesterol in HepG2 cells.
Ketoconazole and fluconazole show strong and weak
affinity toward human CYP51, respectively, in accor-
dance with the result published previously.
3
0
4. Discussion
Human histidine-tagged CYP51 enzyme (lanosterol 14a-
demethylase) was overexpressed in Escherichia coli and
Due to the massive increase of hyperlipidemias and
cardiovascular diseases in the developed world and an
increasing number of patients not responding to the
currently available therapies searching for novel
hypolipidemic drugs remains a very important task.
39
purified to homogeneity as described previously. Affin-
ities of novel compounds were evaluated as apparent
dissociation constants determined by the spectrophoto-
4
0
metric method. Figure 3 shows type II spectral re-
N
N
O
a
+
Cl
O
O
OH
17
N
b, c
17 +
H N
O
N
2
OH Pr
19
Scheme 5. Reagents and conditions: (a) NaOH, H
CH ClCH Cl, rt, Ar 12 h.
2
O, 35 ꢁC, 12 h; (b) K
2
CO
3
3 2 3
, KI, anhyd EtOH, 80 ꢁC, 4 h; (c) CH CH CHO, NaBH(OAc) ,
2
2

T. Koro sˇ ec et al. / Bioorg. Med. Chem. 16 (2008) 209–221
213
a
b
1
00%
90%
lanosterol
80%
FF-MAS
T- M a s
70%
60%
lathosterol
50%
40%
7-dehydro
cholesterol
+zymosterol
30%
desmosterol
20%
10%
cholesterol
0
%
Figure 1. (a) An outline of the cholesterol biosynthesis pathway. Sterol intermediates are shown that have been separated and identified in this study.
Enzymes CYP51 and DHCR14, targets of the novel compounds, are indicated as 1 and 2, respectively. (b) Relative amount of radiolabeled de novo
synthesized sterols in non-treated HepG2 cells. T-MAS is bellow the detection limit.
Our aim was to synthesize compounds that would inhi-
bit cholesterol synthesis after the farnesyl pyrophos-
phate branchpoint leaving isoprene pathways
untouched in order to avoid statin side effects such as
skeletal myopathy. Several compounds structurally not
related to pyridylethanol(phenylethyl)amines were
already shown to inhibit the post-squalene cholesterol
in the development of human CYP51 inhibitors lies pri-
marily in development of potential anticholesterolemic
drugs and in characterization of structural differences
3
0,31
between human and fungal orthologues.
scopic titration of the two purified enzymes (human
Spectro-
and C. albicans) for ketoconazole showed that the affin-
3
2
ity difference was less than 10-fold. Interestingly, keto-
conazole has higher affinity for the human enzyme than
4
2
biosynthesis. SKF 104976, an analog of lanosterol,
3
0
inhibits human lanosterol 14a-demethylase (CYP51).
AAY 9944 (trans-1,4-bis(2-chlorobenzaminomethyl)
cyclohexane dihydrochloride) inhibits several human
post-squalene cholesterol biosynthesis enzymes: two ste-
rol reductases, DHCR14 and DHCR7, and the sterol
D8-7-isomerase (EBP). SR 31747 (N-cyclohexyl-N-
ethyl-3-(3-chloro-4-cyclohexylphenyl)propen-2-ylamine
hydrochloride) was shown to be a selective EBP
for that from Mycobacterium tuberculosis.
As demonstrated above in Figure 2 series of pyridyleth-
anol(phenylethyl)amines can be divided into four groups
according to the sterol profile obtained from the ex vivo
assay. We demonstrated that sterol profile is indicative
of compound affinity toward human CYP51. Moreover,
in case of specific structural change some compounds ex-
erted notable change in the sterol profile that implies
binding affinity toward post CYP51 enzyme, most prob-
ably DHCR14.
4
3,44
inhibitor.
Research and development of CYP51 inhibitors for the
treatment of fungals infections is now directed toward
efficient and more selective drugs. It was suggested that
increased azole doses to combat resistant candidemias
may well inhibit also the endogenous human CYP51
which may lead to severe side effects. Therefore, interest
Measurements on the purified enzyme (Table 1) con-
firmed the classification based on the sterol profile differ-
ences. Group S compounds showed strong CYP51
inhibition and have Kd values between 0.34 and

214
T. Koro sˇ ec et al. / Bioorg. Med. Chem. 16 (2008) 209–221
1
0
9
8
7
6
5
4
3
2
1
0
e
sterols
C
S
a
W
D
N
compound
a
b
c
d
e
f
Figure 2. The average amount of radiolabeled sterols (a) cholesterol, (b) desmosterol, (c) zymosterol + 7-dehydrocholesterol, (d) lathosterol, (e) FF-
MAS, and (f) lanosterol in cell extracts compared to the normal medium, after treatment with 10 lM solution of novel compounds. (Numerical data
are available in the supplementary data).
Figure 3. Type II cytochrome P450 spectral response of the overexpressed purified human CYP51 (2 lM) to ketoconazole (a) and 5d (b) at
concentrations of 0.5 lM, 1 lM, 2 lM, and 10 lM. (c) Spectrophotometric titration of human CYP51 (0.5 lM) with 5d. DA(A432 À A412) is
absorbance difference between 432 and 412 nm.
3
.3 lM. Group W has one order of magnitude larger
Analogs of 5d were prepared with the aim to improve
the inhibitory potential against the human CYP51 and
to study structure–activity relationship.
affinity constants (27–35 lM) compared to group S
and therefore contains weak CYP51 inhibitors.
Compound 5d showed to be a strong human lanosterol
14a-demethylase (CYP51) inhibitor in the ex vivo assay
and this was confirmed also by measurements on the
Lead optimization was directed toward the substitution
pattern on the phenyl ring (5e, 5f, 5g, 5h, 5j, 5k, 5l, 8c,
8d, 8e, 8h, 12a), the amino group has been substituted
by different functions (5i, 8f, 12b), pyridine ring has been
E. coli overexpressed purified human CYP51 enzyme.

T. Koro sˇ ec et al. / Bioorg. Med. Chem. 16 (2008) 209–221
215
Table 1. Apparent dissociation constants (K
selected compounds
d
, lM) and maximal spectral response per nanomole of purified human CYP51 (DAmax/nmol) for
Group
Compound
K
d
(lM)
DAmax/nmol
Lanosterol (ex vivo)
S
5d
8d
16
5g
5f
0.4 ± 0.1
3.3 ± 0.5
0.34 ± 0.06
27 ± 3
0.057 ± 0.003
0.062 ± 0.003
0.053 ± 0.001
0.054 ± 0.002
0.057 ± 0.005
0.050 ± 0.003
6 ± 3
4 ± 2
5 ± 2
S
S
W
W
W
D
N
1.9 ± 0.6
1.8 ± 0.6
1.7 ± 0.5
1.2 ± 0.3
1.0 ± 0.1
29.2 ± 0.5
35 ± 4
8b
*
a
5b
4m
ꢀ1000
260 ± 90
0.11 ± 0.01
40 ± 3
0.06 ± 0.01
0.080 ± 0.001
0.053 ± 0.001
Ketoconazole
Fluconazole
3
Lanosterol (ex vivo)—fold increase in lanosterol, that is, relative quantity of H-lanosterol from the ex vivo assay (numerical data from Fig. 2).
a
No type II spectral response up to 300 lM.
*
2-Pyridyl analog of 5d.
substituted on different positions (5a, 5b, 8a). Deshydr-
oxy pyridylethanol analogs have been prepared (12a,
that lipophilic interactions are very important for the
potent binding of pyridylethanol(phenylethyl)amines to
human CYP51. It seems that these features correlate
well with the pharmacophore model presented by Ekins
et al. although our compounds have in general smaller
molecular weight and direct comparison might not be
trivial.
1
and the opposite (4m, 4c), OCH group has been intro-
2b), phenyl ring has been replaced by pyridine ring,
2
3
2
duced between pyridine ring and the alkyl chain (19) and
different length of the alkyl side chain between tertiary
nitrogen and phenyl ring has been introduced (8e).
The replacement of the phenyl by a naphthalene ring
has also been made (16).
Replacement of the 3-pyridine moiety by 4-pyridine (8a)
showed an activity of the same order of magnitude as in
5d. This was also confirmed within pair of compounds 5i
3
,4-DiCl substitution of the phenyl ring is most suitable
1
for CYP51 inhibition. Mono chloro substitution of the
phenyl ring showed a marked decrease in cholesterol
inhibition with regard to 5d: 4-Cl analog (8d) is more
potent than 3-Cl analog (8c), the least potent is 2-Cl
analog (8b). 8d and 8b have weaker affinity for CYP51
and 5a (R = Me). According to the ex vivo test, 2-
pyridinyl substitution (5b) completely changed the sterol
profile (group D), possibly inhibiting DHCR14 and
other enzymes from the later steps of cholesterol biosyn-
thesis. This modification led to loss of affinity for
CYP51 (Table 1). The distance between the two nitrogen
atoms in the molecule seems to be essential for CYP51
affinity. Same conclusion can be drawn from the loss
(
5
Table 1). 2,4-diCl analog (8g) is weaker inhibitor than
d. Removal of both Cl atoms (5e) additionally de-
creased lanosterol accumulation and cholesterol lower-
ing effect. Compounds 5k and 5l with mono CF3
substitution are as potent as their Cl analogs.
of CYP51 inhibition in 19which has an OCH group in-
2
serted in the alkyl chain. Replacement of the 3-pyridine
moiety by the phenyl group (4c) resulted in loss of affin-
ity for CYP51 and showed a different sterol profile. 4c
most likely inhibits DHCR14 and some other enzymes
from the later steps of cholesterol biosynthesis. This
clearly indicates that the pyridine nitrogen is essential
for strong CYP51 inhibition. Sterol profiles of 4c and
5b are similar, although it seems they are weaker than
12a, presumably, a potent DHCR14 inhibitor.
Replacement of 3,4-dichloro phenyl ring substitution by
3
,4-difluoro substitution (5g) showed a marked decrease
in affinity (Table 1) and reduction of lanosterol accumu-
lation. Introduction of more polar groups, either 3,4-
dimethoxy (5f) or 3,4-dihydroxy (5j) in the phenyl ring,
clearly follows this trend. Replacement of 3,4-dichlor-
ophenyl moiety with 3-pyridyl group (4m) resulted in
the complete lack of cholesterol lowering activity and
in the loss of affinity for CYP51 (Table 1). On the other
side, replacement of the 3,4-dichlorophenyl moiety by a
As demonstrated above we have obtained good correla-
tion of results between Hep G2 cell assay and purified
enzyme assay. Most of our compounds are hydropho-
bic, a property that is favorable both for cell membrane
permeation and CYP51 binding. However, it has to be
pointed out that Hep G2 cell assay was discriminatory
between hydrophilic and hydrophobic statins, potent
2-naphthyl group (16) showed a minor increase in affin-
ity and comparable lanosterol accumulation with regard
to 5d.
Comparison of compounds 5e and 8e showed that the
longer alkyl chain connecting the phenyl moiety to the
tertiary amine nitrogen ((CH )n, n = 2 and 4, respec-
2
4
5
HMG CoA inhibitors. Moreover, we also found that
rosuvastatin was less potent inhibitor of cholesterol syn-
4
6
tively) increased CYP51 inhibition. To study N-alkyl
substitution, compounds 5d (R = Pr), 8f (R = Bu), 5i
thesis than atorvastatin in our Hep G2 cell assay.
1
1
1
R = Me), and 12b (R = CH CH CH Ph) were synthe-
1
(
In conclusion, novel pyridylethanol(phenylethyl)amines
show a post-lanosterol cholesterol biosynthesis inhibi-
tion effect. For majority of compounds biological data
2
2
2
sized. They all showed the CYP51 inhibition ability of
the same order of magnitude. These findings confirmed

216
T. Koro sˇ ec et al. / Bioorg. Med. Chem. 16 (2008) 209–221
suggest CYP51 as a main target of inhibition. Studies on
the purified human CYP51 enzyme confirmed predic-
tions from the sterol profiling ex vivo assay demonstrat-
ing its predictive power. Replacement of the pyridine
ring with a phenyl ring resulted in a loss of CYP51 affin-
ity indicating that the pyridine nitrogen is important for
CYP51 inhibition. Moreover, spectroscopy titration
provided evidence that the pyridine nitrogen corre-
sponds to that in azoles. We demonstrated that lipo-
philic properties of the structure moiety apparently
play a crucial role in the strength of CYP51 inhibition
in accordance with the previous experimental and theo-
retical work.
5.1.1.4. 2-((3,4-(Dichlorophenethyl(propyl)amino)-1-
(pyridin-3-yl)ethanol dihydrobromide (5d). Yield: 9%.
H NMR d 094 (3H, t, J = 7.5 Hz), 1.78 (2H, m),
3.10–3.54 (6H, m), 5.48 (1H, bs), 7.34–7.74 (3H, m),
1
8.06 (1H, dd, J = 8.1 Hz, J = 5.7 Hz), 8.63 (1H, d,
2
1
1
3
J = 8.1 Hz), 8.91 (1H, d, J = 5.1 Hz), 9.04 (1H, s);
C
NMR d 10.84, 16.41, 27.93, 52.51, 53.73 or 53.90,
55.29, 57.20, 64.32, 126.31, 129.51 or 129.61, 130.67,
131.05, 138.20, 140.25, 141.72, 142.22, 142.92; mp 170-
172 ꢁC; IR (KBr) 3411, 3168, 2954, 2685, 1626, 1598,
À1
1536, 1473, 1348, 1210, 1129, 1028, 805, 680 cm ; MS
+
(FAB): M + H : 353; Anal. Calcd for C H Cl N
1
8
24
2
2
OBr + 4/5 H O: C, 40.83, H, 4.87, N, 5.29. Found C,
2
2
4
0.86, H, 4.74, N, 5.21.
5
. Experimental
5.1.1.5. 2-(Phenethyl(propyl)amino)-1-(pyridin-3-
yl) ethanol dihydrobromide (5e). Yield: 13%. H NMR
d 0.92 (3H, t), 1.76 (2H, m), 2.48 (2H, m), 3.05–3.51
1
5
.1. General chemical methods
(6H, m), 5.42 (1H, bs), 7.35 (1H, m), 7.23–7.37 (5H,
m), 8.03 (1H, dd, J = 8.0 Hz, J = 5.5 Hz), 8.58 (1H,
Starting materials, reagents, and solvents were pur-
chased from commercial suppliers and used without fur-
ther purification. Analytical TLC was performed on
silica gel (60 F 254) plates (0.25 mm) and components
visualized with ultraviolet light. Column chromatogra-
phy was carried out on silica gel 60 (particle size 240–
1
2
d, J = 8.1 Hz), 8.88 (1H, d, J = 4.6 Hz), 9.00 (1H, s,
1H); mp 127–130 ꢁC; IR (KBr) 3267, 2955, 1589, 1455,
À1
+
1075, 806, 704 cm ; MS (FAB): M + H : 285; Anal.
Calcd for C H N OBr : C, 48.45, H, 5.87, N, 6.28.
Found C, 48.38, H, 6.09, N, 6.08.
1
8
26
2
2
4
stage microscope and are uncorrected. H NMR and
C NMR experiments were carried out at 300 MHz
or 600 MHz in CDCl3 or DMSO-d6 solution with
TMS as an internal standard.
00 mesh). Melting points were determined on a hot
1
5.1.1.6. 2-((3,4-Dimethoxyphenethyl)(propyl)amino)-1-
(pyridin-3-yl)ethanol dihydrobromide (5f). Yield: 25%. H
NMR d 0.93 (3H, t, J = 7.2 Hz), 1.75 (2H, m), 3.00 (2H,
m), 3.22–3.42 (6H, m, 6H), 3.73 (6H, m), 5.27 (1H, bs),
1
3
1
6
.78–6.97 (3H, m), 7.66 (1H, dd, J = 7.9 Hz in
1
3
3
5
.1.1. General synthesis procedure A
.1.1.1. 2-((3,4-(Dichlorophenethyl)(methyl)amino)-1-
pyridin-4-yl)ethanol dihydrobromide (5a). Yield: 14%.
J = 5.2 Hz), 8.16 (1H, d, J = 8, 1 Hz), 8.67 (1H, dd,
2
J = 5.2 Hz, J = 1.5 Hz), 8.79 (1H, s); C NMR d
1
3
5
1 2
(
11.27, 16.79, 28.95, 53.51 or 53.72, 55.19, 55.52, 55.92,
58.09, 65.17, 112.37, 113.08 or 113.19, 121.18, 124.98,
129.58, 137.62, 138.54, 146.03, 147.16, 148.10, 149.16;
mp 147–149 ꢁC; IR (KBr) 3452, 3176, 2959, 1626,
1537, 1473, 1345, 1208, 1074, 1208, 1074, 1028, 895,
811. 681, 620 cm ; MS (EI): M + H : 345; Anal. Calcd
for C H N O Br : C, 47.45, H, 5.97, N, 5.53. Found
C, 47.37, H, 6.06, N, 5.51.
1
H NMR d 2.49 (3H, s), 2.97–3.57 (6H, m, 6H), 5.44
1H, bs), 7.35 (1H, m), 7.62–7.70 (2H, m), 8.10 (2H, d,
(
J = 6.1 Hz), 8.97 (2H, d, J = 6.3 Hz); mp 185–190 ꢁC;
IR (KBr): 3210, 2964, 2706, 1632, 1595, 1498, 1395,
À1
+
264, 1135, 1079, 1029, 809 cm ; MS (FAB): M + H :
À1
+
1
3
4
25; Anal. Calcd for C H Cl N OBr : C, 39.46, H,
2
.14, N, 5.75. Found C, 39.46, H, 4.22, N, 5.59.
1
6
20
2
2
20 29
2
3
2
5.1.1.2. 2-((3,4-Dichlorophenethyl)(propyl)amino)-1-
4-methylpyridin-2-yl)ethanol dihydrobromide (5b). Yield:
5.1.1.7. 2-((3,4-Difluorophenethyl)(propyl)amino)-1-
(pyridin-3-yl)ethanol dihydrobromide (5g). Yield: 17%.
H NMR d 0.87 (3H, t, J = 7.2 Hz), 1.66 (2H, m),
2.99 (2H, m), 3.15 (2H, m), 3.35–3.39 (4H, m), 5.20
(
1
1
9
2
7
%. H NMR d 0.92 (3H, t, J = 7.3 Hz), 1.74 (2H, m),
.43 (3H, s), 3.06–3.57 (8H, m), 5.19 (1H, bs), 7.32–
.65 (5H, m), 8.49 (1H, d, J = 5.0 Hz); mp 109–112 ꢁC;
(1H, dd, J = 10.5 Hz in J = 3.0 Hz), 7.09–7.33 (3H,
2
1
À1
IR (KBr) 3426, 1706, 1637, 1474, 820, 780 cm ; MS
(
OCl Br : C, 43.13, H, 4.95, N, 5.29. Found C, 42.65,
m), 7.79 (1H, dd, J = 7.9 Hz in J = 5.3 Hz), 8.38
1 2
(1H, d, J = 8.1 Hz), 8.61 (1H, dd, J = 5.4 Hz in
1
+
FAB): M + H : 367; Anal. Calcd for C H N
19
26
2
J2 = 1.3 Hz), 8.70 (1H, d, J = 1.5 Hz); mp 147–
149 ꢁC; IR (KBr) 3166, 2955, 1612, 1520, 1451,
2
2
H, 5.34, N, 4.79.
À1
1
345, 1289, 1210, 1121, 1072, 943, 809, 680 cm
;
+
5
.1.1.3. 2-(((3,4-Dichlorophenethyl)(propyl)amino)-1-
MS (FAB): M + H : 321; Anal. Calcd for
C H N OF Br : C, 44.83, H, 5.02, N, 5.81. Found
C, 44.71, H, 5.10, N, 5.91.
1
phenylethanol (4c). Yield: 8%. H NMR d 0.97 (3H, t,
J = 7.3 Hz), 1.50–1.60 (2H, m), 2.53–2.91 (8H, m), 4.67
1
8
24
2
2
2
(
J = 8.3 Hz, J = 2.0 Hz), 7.31–7.43 (7H, m));
NMR d 11.72, 20, 28, 32.84, 55.29, 55.79, 62.93, 69.37,
1H, dd, J = 10.3 Hz in J = 3.4 Hz), 7.08 (1H, dd,
1 2
1
3
C
5.1.1.8. 2-((4-Nitrophenethyl(propyl)amino)-1-(pyridin-3-
yl) ethanol dihydrobromide (5h). Yield: 7%. H NMR d
1
2
1
1
1
1
25.74, 127.43, 128.10, 128.31, 130.33, 130.57, 140.28,
42.16; IR (NaCl) 3422, 2956, 2870, 1466, 1395, 1130,
0.93 (3H, t, J = 7.5 Hz), 1.79 (2H, m) 3.26–3.54 (8H,
m), 5.46 (1H, bs), 7.65 (2H, m), 8.05 (2H, dd,
J = 8.1 Hz, J = 5.6 Hz), 8.22 (1H, dd, J = 8.3 Hz in
À1
+
031, 888, 700 cm ; MS (FAB): M + H : 352; HRMS
1
2
1
+
m/z Calcd for C H Cl NO [MH ] 352.123495. Found
1
J2 = 4.6 Hz), 8.61 (1H, d, J = 8.2 Hz), 8.88 (1H, dd,
J = 5.6 Hz, J = 1.1 Hz), 9.02 (1H, d, J = 1.7 Hz); mp
9
23
2
3
52.124200.
1
2

T. Koro sˇ ec et al. / Bioorg. Med. Chem. 16 (2008) 209–221
217
+
2
1
14–218 ꢁC; IR (KBr) 3405, 3178, 2951, 1598, 1524, 1344,
212, 1072, 857, 746, 680. cm ; MS (FAB): M + H : 330;
m/z Calcd for C H N O [MH ] 286.191938. Found
17 23 3
286.192060.
À1
+
Anal. Calcd for C H ClN OBr : C, 44.01, H, 5.13, N,
2
1
8
25
2
3
3
8
.55. Found C, 44.15, H, 5.33, N, 8.25.
5.1.2. General synthesis procedure B
.1.2.1. 2-((3,4-(Dichlorophenethyl(propyl)amino)-1-
(pyridin-4-yl)ethanol dihydrobromide (8a). Yield: 20%.
5
5.1.1.9. 2-((3,4-(Dichlorophenethyl(methyl)amino)-1-
pyridin-3-yl)ethanol dihydrobromide (5i). Yield: 15%.
H NMR d 2.49 (3H, s), 2.98–3.53 (6H, m), 5.40 (1H,
bs), 7.35 (1H, m), 7.60–7.70 (2H, m), 8.02 (1H, dd,
1
(
H NMR d 0.94 (3H, t, J = 7.5 Hz), 1.76 (2H, m),
1
3.07–3.57 (8H, m), 5.47 (1H, bs), 7.32–7.73 (3H, m),
8.07 (2H, d, J = 6.4 Hz), 8.94 (2H, d, J = 6.4 Hz); mp
156–158 ꢁC; IR (KBr) 3433, 3252, 2966, 1631, 1590,
J = 7.9 Hz, J = 5.5 Hz), 8.53 (1H, d, J = 7.9 Hz), 8.87
1
2
À1
(
(
1H, d, J = 4.4 Hz), 8.98 (1H, s); mp 157–161 ꢁC; IR
KBr) 3228, 2932, 2852, 1588, 1470, 1131, 1029,
1498, 1475, 1402, 1228, 1076, 807, 762 cm ; MS
+
(FAB): M + H : 353; Anal. Calcd for C H Cl N
1
8
24
2
2
À1
+
14 cm ; MS (FAB): M + H : 325; Anal. Calcd for
8
C H Cl N OBr : C, 39.46, H, 4.14, N, 5.75. Found
C, 39.52, H, 4.31, N, 5.59.
OBr : C, 41.97, H, 4.70, N, 5.44. Found C, 41.71, H,
2
4.64, N, 5.19.
1
6
20
2
2
2
5.1.2.2. 2-((2-Chlorophenethyl(propyl)amino)-1-(pyri-
din-4-yl)ethanol dihydrobromide (8b). Yield: 20%. H
1
5.1.1.10. 4-(2-((2-Hydroxy-2-(pyridin-3-yl)ethyl)(pro-
pyl)amino)ethyl)benzene-1,2-diol dihydrobromide (5j).
Yield: 4 %. H NMR d 0.91 (3H, t, J = 7.3 Hz), 1.73
NMR d 0.95 (3H, t, J = 7.5 Hz), 1.77 (2H, m) 3.23–
3.51 (8H, m), 5.40 (1H, bs), 7.32–7.53 (4H, m), 7.96
(1H, dd, J = 8.0 Hz in J = 5.3 Hz), 8.49 (1H, d,
1
(2H, m), 2.90 (2H, m), 3.17–3.50 (6H, m), 5.37 (1H,
bs), 6.50–6.70 (3H, m), 8.05 (1H, m), 8.60 (1H, d,
1
2
J = 7.5 Hz), 8.84 (1H, d, J = 5.3 Hz), 8.94 (1H, s); mp
184–187 ꢁC; IR (KBr) 3414, 3172, 2930, 1626, 1537,
1476, 1348, 1209, 1094, 808, 763, 681 cm ; MS
J = 8.4 Hz), 8.87 (1H, d, J = 5.2 Hz), 8.97 (1H, s); IR
(
À1
NaCl) 3374, 1609, 1528, 1458, 1364, 1287, 1119,
À1
+
06 cm ; MS (FAB): M + H : 317; Anal. Calcd for
+
8
C H N O Br + 5/3 H 0: C, 42.54, H, 5.82, N, 5.51.
Found C, 42.69, H, 6.10, N, 5.34.
(FAB): M + H : 319; Anal. Calcd for C H ClN OBr :
1
8
25
2
2
C, 44.98, H, 5.24, N, 5.83. Found C, 44.89, H, 5.47, N,
5.55.
1
8
26
2
3
2
2
5.1.1.11. 2-(Propyl(4-(trifluoromethyl)phenethyl)amino)-
-(pyridin-3-yl)ethanol dihydrobromide (5k). Yield: 1%.
H NMR d 0.95 (3H, t, J = 7.2 Hz), 1.78 (2H, m),
5.1.2.3. 2-((3-Chlorophenethyl(propyl)amino)-1-(pyri-
din-4-yl)ethanol dihydrobromide (8c). Yield: 4%.
1
1
H
1
NMR d 0.91 (3H, t, J = 7.5 Hz), 1.71 (2H, m) 3.04–
3.40 (8H, m), 5.27 (1H, bs), 7.25–7.43 (4H, m), 7.87
(1H, dd, J = 7.9 Hz in J = 5.5 Hz), 8.39 (1H, d,
3
.24–3.53 (8H, m), 5.44 (1H, bs), 7.60-7.76 (4H, m),
.08 (1H, dd, J = 8.0 Hz in J = 5.6 Hz), 8.60 (1H, d,
8
J = 8.2 Hz), 8.90 (1H, d, J = 4.6 Hz), 9.02 (1H, s);
1
2
1
2
1
3
C
J = 8.2 Hz), 8.72 (1H, dd, J = 5.4 Hz, J = 1.2 Hz),
1 2
NMR d 20.81, 26.40, 38.77, 62.60 or 63.69, 64.05 or
8.81 (1H, d, J = 1.5 Hz); mp 133–135 ꢁC; IR (KBr)
3465, 3168, 2963, 1616, 1525, 1448, 1288, 1209, 1071,
6
1
1
1
5.27, 67.18 or 67.28, 74.36, 132.49, 153.44, 136.33,
36.99, 137.40, 137.81, 139.85, 150.27, 151.95, 152.25,
52.91; mp 192–195 ꢁC; IR (KBr) 1636, 1534, 1324,
À1
+
943, 807, 679 cm ; MS (FAB): M + H : 319; Anal.
Calcd for C H ClN OBr : C, 44.98, H, 5.24, N, 5.83.
Found C, 44.53, H, 5.16, N, 5.89.
1
8
25
2
2
À1
+
127 cm ; MS (FAB): M + H : 353; Anal. Calcd for
C H F N OBr : C, 44.38, H, 4.90, N, 5.45. Found
1
9
25
3
2
2
C, 44.15, H, 5.00, N, 5.32.
5.1.2.4. 2-((4-Chlorophenethyl(propyl)amino)-1-(pyri-
din-4-yl)ethanol dihydrobromide (8d). Yield: 8%.
1
H
5.1.1.12. 2-(Propyl(2-(trifluoromethyl)phenethyl)amino)
1-(pyridin-3-yl)ethanol dihydrobromide (5l). Yield: 6%.
H NMR d 0.96 (3H, t, J = 7.2 Hz), 1.78 (2H, m, 2H),
NMR d 0.92 (3H, t, J = 7.5 Hz), 1.75 (2H, m, 2H)
3.04–3.49 (8H, m, 8H), 5.36 (1H, bs), 7.33 (2H, d,
J = 8.5 Hz), 7.43 (2H, d, J = 7.8 Hz), 7.95 (1H, dd,
J = 7.8 Hz in J = 5.6 Hz), 8.47 (1H, d, J = 7.9 Hz),
-
1
3
.28–3.56 (8H, m), 5.44 (1H, bs), 7.49–7.78 (4H, m),
.07 (1H, dd, J = 7.8 Hz in J = 5.9 Hz), 8.61 (1H, d,
1
2
8
J = 7.8 Hz), 8.91 (1H, d, J = 5.4 Hz), 9.00 (1H, s);
8.83 (1H, d, J = 4.4 Hz), 8.94 (1H, s); mp 194–196 ꢁC;
IR (KBr) 3406, 3176, 2956, 1624, 1538, 1489, 1414,
1
2
1
3
C
À1
NMR d 10.81, 16.19, 26.37, 52.79 or 53.57, 54.38 ali
1348, 1210, 1092, 811, 680, 645 cm ; MS (FAB):
M + H : 319; Anal. Calcd for C H ClN OBr : C,
+
5
1
5.00, 57.35, 64.36, 126.03, 126.48, 127.25, 127.77,
32.16, 132.95, 135.09, 140.36, 141.45, 142.56, 142.75;
1
8
25
2
2
44.98, H, 5.24, N, 5.83. Found C, 44.72, H, 5.41, N,
5.47.
mp 180–183 ꢁC; IR (KBr) 3410, 3166, 2984, 1626,
1
6
537, 1452, 1315, 1177, 1126, 1045, 807, 775,
81 cm ; MS (FAB): M + H : 353; Anal. Calcd for
À1
+
5.1.2.5. 2-((4-Phenylbutyl)(propyl)amino)-1-(pyridin-3-
1
C H F N OBr : C, 44.38, H, 4.90, N, 5.45. Found
2
yl)ethanol dihydrobromide (8e). Yield: 8%. H NMR d
0.92 (3H, t, J = 7.3 Hz), 1.58–1.74 (6H, m), 2.55–2.68
1
9
25
3
2
C, 44.30, H, 4.97, N, 5.42.
(2H, m), 3.11–3.47 (6H, m), 5.35 (1H, d, J = 7.8 Hz),
7.17–7.34 (5H, m), 8.06 (1H, dd, J = 8.0 Hz in
5
.1.1.13. 2-(Propyl-3-yl)ethyl)amino)-1-(pyridin-3-
1
1
yl) ethanol (4m). Yield: 6 %. H NMR d 0.92 (3H, t,
J = 7.3 Hz), 1.55–1.63 (2H, m), 3.20–3.30 (2H, m),
3.47–3.73 (6H, m), 5.04 (1H, dd, J1 = 8.1 Hz,
J2 = 2.9 Hz), 7.25–7.36 (2H, m), 7.65–7.81 (2H, m),
.42–8.62 (4H, m); IR (NaCl) 3359, 2966.1635, 1426,
193, 1078, 714 cm ; MS (FAB): M + H : 286; HRMS
J2 = 5.7 Hz), 8.59 (1H, d, J = 7.9 Hz), 8.87 (1H, d,
J = 5.7 Hz), 8.95 (1H, s); mp 129–131 ꢁC; IR (KBr)
3449, 3170, 2363, 1609, 1545, 1472, 1379, 1322, 1258,
À1
+
1076, 908, 807, 753, 702 cm ; MS (FAB): M + H :
313; Anal. Calcd for C H N OBr : C, 50.65, H, 6.38,
N, 5.91. Found C, 50.29, H, 6.64, N, 5.79.
8
1
2
0
30
2
2
À1
+

218
T. Koro sˇ ec et al. / Bioorg. Med. Chem. 16 (2008) 209–221
5
.1.2.6. 2-(Butyl(3,4-dichlorophenethyl)amino)-1-(pyri-
in 1,2-dichloroethane (10 mL) and then treated with
NaBH(OAc) (0.337 g, 1.6 mmol). The mixture was stir-
1
din-3-yl)ethanol dihydrobromide (8f). Yield: 19%.
NMR d 0.91 (3H, t, J = 7.2 Hz), 1.33–1.40 (2H, m),
.73 (2H, m), 3.12 (2H, m), 3.32–3.61 (6H, m), 5.47
1H, dd, J = 10.5 Hz in J = 3.0 Hz), 7.23 (1H, d,
J = 6.8 Hz), 7.48 (2H, d, J = 8.3 Hz), 8.08 (1H, dd,
J = 8.3 Hz in J = 6.0 Hz), 8.61 (1H, d, J = 8, 7 Hz),
H
3
red at rt under an Ar atmosphere overnight. The reac-
tion mixture was quenched by adding aq saturated
NaHCO3 (20 mL) solution and the product was ex-
tracted with EtOAc (20 mL). The EtOAc extract was
dried (Na SO ) and the solvent was evaporated under
1
(
1
2
1
2
2
4
8
1
1
.77 (1H, d, J = 5.6 Hz), 8.88 1H, d, J = 1.7 Hz); mp
34–137 ꢁC; IR (KBr) 3479, 3232, 2932, 1726, 1517,
420, 1264, 1159, 1028, 811, 682 cm ; MS (FAB):
reduced pressure to give the crude free base, which
was chromatographed on silica (MeOH/EtOAc, 2:10)
to give a colorless oil 11b; yield: 0.26 g, 62%. The prod-
uct was converted to the dihydrobromide salt 12b; yield:
0.27 g, 74% (96% pure by area% HPLC analysis).
À1
+
M + H : 367; Anal. Calcd for C H N OCl Br : C,
1
4
5
9
26
2
2
2
3.13, H, 4.95, N, 5.29. Found C, 43.33, H, 5.13, N,
.12.
1
Overall yield: 17%. H NMR d 2.00–2.11 (2H, m), 2.64–
2.69 (2H, m), 3.07–3.13 (2H, m), 3.28–3.59 (6H, m),
5.1.2.7. 2-(Butyl(2,4-dichlorophenethyl)amino)-1-
pyridin-3-yl)ethanol dibenzenesulfonate (8g). Yield: 8%.
H NMR d 0.94 (3H, t, J = 7.5 Hz), 1.36 (2H, m), 1.72
2H, m), 3.48 (8H, m), 5.31 (1H, bs), 7.28–7.34 (10H,
m), 7.46–7.60 (3H, m), 7.88 (1H, dd, J = 8.0 Hz in
(
7.19–7.70 (8H, m), 8.05 (1H, dd, J = 8.0 Hz, J =
1
2
1
5.6 Hz), 8.59 (1H, d, J = 8.1 Hz), 8.87 (1H, d,
J = 5.6 Hz), 9.00 (1H, d, J = 1.5 Hz); mp 154–157 ꢁC;
(
À1
IR (KBr) 3460, 1636, 1557, 1474, 1032, 819, 684 cm
MS (FAB): M + H : 413; Anal. Calcd for C H Cl N
;
1
+
J = 5.3 Hz); mp 133–135 ꢁC; IR (KBr) 3390, 2960,
2
24 28
2
2
1
8
633, 1561, 1476, 1444, 1382, 1235, 1167, 1123, 1015,
20, 728, 611, 563 cm ; MS (FAB): M + H : 367; Anal.
Br : C, 50.11, H, 4.91, N, 4.87. Found C, 50.05, H, 4.43,
2
N, 5.09.
À1
+
Calcd for C H Cl N O S : C, 54.46, H, 5.31, N, 4.10.
3
1
36
2
2
7 2
Found C, 54.70, H, 5.50, N, 4.11.
5.1.3.2. N-(3-Fluorophenethyl)-N-(2-(3-pyridinyl) ethyl)
propan-1-amine dihydrobromide (12a). Yield: 22%. H
1
5
.1.3. Synthesis procedure C
.1.3.1. N-(3,4-Dichlorophenethyl)-3-phenyl-N-(2-
pyridin-3-yl)ethyl)propan-1-amine dihydrobromide (12b).
To a solution of 3,4-dichlorophenethylamine (5.00 g,
6.3 mmol) and 2-(pyridin-3-yl)acetic acid hydrochlo-
NMR d 0.94 (3H, t, J = 7.5 Hz), 1.76 (2H, m) 3.09–
3.53 (10 H, m), 7.07–7.43 (4H, m), 8.05 (1H, dd,
J = 7.9 Hz in J = 5.8 Hz), 8.60 (1H, dd, J = 11.6 Hz
5
(
1
2
1
in J = 1.5 Hz), 8.87 (1H, d, J = 5.5 Hz), 9.01 (1H, s);
2
1
3
2
C NMR d 10.86, 16.66, 25.87, 28.78, 51.55, 52.80,
ride (4.90 g, 28.2 mmol) in DMF (20 mL) was added
HOBT (5.80 g, 28.2 mmol). The pH of the solution
was adjusted to 8 by adding N-methylmorpholine.
EDC (5.63 g, 29.1 mmol) was added. After the reaction
mixture was stirred at rt overnight, the solvent was evap-
orated under reduced pressure and the residue dissolved
in EtOAc (30 mL). The organic layer was washed with
53.67, 113.70, 115.74, 125.13, 126.66, 130.52, 136.64,
139.73, 141.05, 142.81, 146.13, 163.83; mp 142–144 ꢁC;
IR (KBr) 3424, 2938, 1612, 1561, 1473, 1442, 1243,
À1
+
1146, 938, 780, 678 cm ; MS (FAB): M + H : 287;
Anal. Calcd for C H ClN OBr : C, 48.23, H, 5.62,
N, 6.25. Found C, 48.22, H, 5.81, N, 6.08.
1
8
25
2
2
aq saturated NaHCO (30 mL) and NaCl (30 mL) solu-
3
5.1.4. Synthesis procedure D
tion and dried (Na SO ). The solvent was evaporated
2
5.1.4.1. 2-(2-(Naphthalen-2-yl)ethyl](propyl)amino]-1-
(pyridin-3-yl)ethanol dihydrobromide (16). (2-Naphtha-
len-2-yl)-acetonitrile (5.0 g, 30.0 mmol) in dry THF
(15 mL) was heated to reflux and 2 M solution of bor-
ane-dimethyl sulfide complex in diethyl ether (16.5 mL,
33.0 mmol) was added. The dimethyl sulfide liberated
in the course of the reaction was allowed to distill off.
The heating was continued for 5 h. The THF solution
was then hydrolyzed during addition of 6 N HCl
(14 mL, 84 mmol). After 30 min, the clear solution ob-
tained was cooled to rt and neutralized with 6 N NaOH
(21 mL, 126 mmol). The reaction mixture was stirred at
rt for another 1 h. EtOAc (30 mL) was added and the
organic layer was washed with aq saturated NaHCO3
(30 mL) and NaCl (30 mL) solution and dried (Na SO ).
4
under reduced pressure to give a product which was
chromatographed on silica (MeOH/EtOAc, 2:10) to give
dark yellow oil 9b; yield: 6.60 g, 85%.
The solution of compound 9b (1.38 g, 4.5 mmol) in dry
THF (10 mL) was heated to reflux and 2 M solution
of borane-dimethyl sulfide complex in diethyl ether
(
8.0 mL, 17.0 mmol) was added in drops over the period
of 15 min, allowing dimethyl sulfide to distill off. The
reaction mixture was refluxed for 10 h. The THF solu-
tion was then hydrolyzed during addition of 6 N HCl
(
3.0 mL, 18.0 mmol). After 30 min, the clear solution
obtained was cooled to rt and neutralized with 6 N
NaOH (4.5 mL, 25.0 mmol). The reaction mixture was
stirred at rt for another 1 h. EtOAc (20 mL) was added
and the organic layer was washed with aq saturated
2
4
The solvent was evaporated under reduced pressure to
give a product which was further chromatographed on
silica (MeOH/EtOAc, 2:10 to gradient elution MeOH/
EtOAc, 10:2) to give a pale yellow solid 13. Final com-
pound 16 was further prepared by reaction steps de-
NaHCO (20 mL) and NaCl (20 mL) solution and dried
3
(
Na SO ). The solvent was evaporated under reduced
2 4
pressure to give a product which was further chromato-
graphed on silica (MeOH/EtOAc, 2:10) to give colorless
oil 10b; yield: 0.87 g, 65%.
1
scribed in procedure B. Yield: 7%. H NMR d 0.94
(3H, t, J = 7.5 Hz), 1.75–1.83 (2H, m) 3.15–3.59 (8H,
m), 5.43 (1H, bs), 7.45–8.03 (8H, m), 8.56 (1H, d,
J = 7.9 Hz), 8.87 (1H, d, J = 5.3 Hz), 8.99 (s, 1H, H -
Py); C NMR d 10.89, 16.30 or 16.47, 26.55, 52.83,
Secondary amine 10b (0.30 g, 1.2 mmol) and
PhCH CH CHO (0.27 mL, 2.5 mmol) were dissolved
2
1
3
2
2

T. Koro sˇ ec et al. / Bioorg. Med. Chem. 16 (2008) 209–221
219
5
1
1
3.52, 54.19, 5.13, 56.94 or 57.27, 64.39 or 64.48, 123.61,
25.69, 125.93, 126.46 or 126.54, 127.57, 128.75, 131.29,
33.07, 140.13, 141.99, 143.30; mp 130–132 ꢁC; IR
DK-2100 Copenhagen, Denmark); atorvastatin (Lek
d. d.)
(
KBr) 3166, 2953, 1626, 1538, 1453, 1350, 1217, 1094,
5.1.5.3. Cell culture incubation with new compounds
and atorvastatin. Human hepatoma cell line (HepG2-
ATCC No. HB-8065) was split in the recommended ra-
À1
07, 755, 681, 621 cm ; MS (FAB): M + H : 335; Anal.
+
8
Calcd for C H N OBr : C, 53.24, H, 5.69, N, 5.64.
Found C, 52.87, H, 5.63, N, 5.15.
2
2
28
2
2
2
tio (1:2-3), 75 cm cell flasks, using four flasks per exper-
imental condition. Cells were incubated at 37ꢁC with 5%
5
.1.5. Synthesis procedure E
.1.5.1. 1-(Phenethyl(propyl)amino)-3-(pyridin-3-yloxy)
propan-2-ol (19). To a solution of NaOH (1.0 g,
5.0 mmol) in water (15 mL) was added pyridin-3-ol
2.38 g, 25.0 mmol), followed by 2-(chloromethyl)oxi-
CO in DMEM high containing 5% calf serum and 1%
2
5
L-glutamine. After 24 h culturing the medium was re-
placed with the one supplemented with 10 lM concen-
tration of a selected compound. 10 lM solution of
atorvastatin served as a positive control.
2
(
rane, keeping the reaction temperature below 35 ꢁC.
The mixture was then stirred at room temperature for
3
After 24 h the growth medium was replaced and [ H]
1
2 h. After cooling, the solvent was evaporated under re-
acetate added in a concentration of 40 lCi per ml of
the medium (400 lCi per flask). After 4 h cells were har-
vested using 2 mL of trypsin and the cell pellet resus-
pended in 1 mL of distilled water. Cells were
homogenized using the freeze and thaw method. From
cell homogenate sterols were extracted and protein con-
centration determined using the Bio-Rad protein assay.
duced pressure to give a product which was further
chromatographed on silica (MeOH) to give a brown
oil 17, yield: 1.134 g, 30%.
To a solution of compound 17 (1.5 g, 10.0 mmol) in an-
hyd EtOH (30 mL) were added phenethylamine
(
KI (0.015 g, 0.1 mmol). The mixture was stirred at
1.25 mL, 10.0 mmol), K CO (1.38 g, 3.3 mmol), and
2 3
5.1.5.4. Sterol extraction. Homogenates were trans-
ferred into 4 mL glass vials. Internal standard ergosterol
(0.25 mg/mL) was added prior to extraction process.
Sterols were extracted in 3 mL of extraction solution
(75% n-heptane: 25% isopropanol (vol./vol.)), with addi-
tion of 100 lL of 0.3 M NaH PO (pH 1.0). Closed vials
8
0 ꢁC for 4 h. After cooling, the solvent was evaporated
under reduced pressure to give a product which was fur-
ther chromatographed on silica (MeOH) to give a
brown oil 18, yield: 1.252 g, 46%.
2
4
Secondary amine 18 (0.982 g, 3.6 mmol) and CH CH
3
CHO (0.8 mL, 7.4 mmol) were dissolved in 1,2-dichloro-
ethane (30 mL) and then treated with NaBH(OAc)3
were vigorously shaken (1800 rpm) for 2 h. After extrac-
tion procedure vials were centrifuged (2000g, 10 min)
and organic phase transferred to fresh conical glass
tubes. Extraction procedure was repeated using 1 ml of
extraction solution for 15 min. Organic phases were
pooled.
2
(
1.33 g, 6.3 mmol). The mixture was stirred at rt under
an Ar atmosphere overnight. The reaction mixture was
quenched by adding aq saturated NaHCO3 (30 mL)
solution and the product was extracted with EtOAc
(
30 mL). The EtOAc extract was dried (Na SO ) and
2
Primary extracts were dried in vacuum centrifuge, re-
dissolved in 2 ml of n-heptane, and incubated for
10 min at the room temperature with mild shaking.
After centrifugation (10 min, 2000g) extracts were trans-
ferred into fresh glass tubes and stored in dark and cold.
4
the solvent was evaporated under reduced pressure to
give the crude free base, which was chromatographed
on silica (MeOH) to give a brown oil 19; yield:
0
.226 g, 20% (95% pure by area% HPLC analysis).
1
Overall yield: 3%. H NMR d 0.93 (3H, t, J = 7.2 Hz),
1
5.1.5.5. HPLC analysis. The organic phase was dried,
reconstituted in mobile phase for HPLC reversed phase
separation, and loaded onto a Prism-RPN, 5 lm,
250 · 4.6 mm HPLC column running in 100% acetoni-
trile at 1.00 mL/min at 40 ꢁC temperature. Scintillation
liquid was added after UV detection at 30 mL/h, at the
room temperature, to evaluate tritium labeled sterols
on the radiodetector.
.52 (2H, m), 1.53–1.61 (4H, m), 2.78 (4H, s), 3.91
2H, m), 4.29 (1H, d, J = 10.5 Hz), 7.15–7.58 (9H, m);
C NMR d 12.21, 21.20, 34.39, 57.61, 57.75, 59.14,
5.98, 70.15, 127.09, 128.01, 128.40, 130.03, 134.61,
36.03, 142.10, 169.13; IR (NaCl) 3380, 2970, 1726,
(
1
3
6
1
1
À1
+
563, 1462, 1369 cm ; MS (FAB): M + H : 315;
HRMS m/z Calcd for C H N O [MH ] 313.191603.
1
À
9
25
2
2
Found 313.192350.
Sterol’s retention times were determined by comparing
eluted peaks with runs of the following standards:
lathosterol, zymosterol, 7-dehydrocholesterol, desmos-
terol, lanosterol, cholesterol, and FF-MAS. The non-
5
.1.5.2. Reagents and solvents for the biological
3
TM
section. [ H] acetate (NEN Life Science Products);
trypsin, calf serum, L-glutamine, Dulbecco’s modified
Eagle’s medium (DMEM high) (Sigma); Bio-Rad re-
agent (Bio-Rad Lab); ketoconazole (Sigma); commer-
cial sterol standards (Steraloids): lathosterol,
zymosterol, 7-dehydrocholesterol, desmosterol, lanos-
terol, ergosterol, and cholesterol; laboratory standards:
FF-MAS (Laboratory of Reproductive Biology, The
Juliane Marie Center for Children, Women and
Reproduction, University Hospital of Copenhagen,
3
availability of H-labeled sterol standards disables the
exact quantification of H-labeled sterols. Thus, radio-
3
detected sample peak areas were compared to normal
medium peak areas resulting in the relative levels of
sterols.
Results were also normalized on ergosterol quantity and
protein concentration. Ergosterol served as an internal

220
T. Koro sˇ ec et al. / Bioorg. Med. Chem. 16 (2008) 209–221
standard for the extraction procedure. Bradford assay
has been used to determine the protein concentration.
Protein concentration is not a direct measure of cell via-
bility, however, it correlates to the cell number.
gen) for FF-MAS and T-MAS and Prof. Dr. M.R.
Waterman (Institute of Biochemistry, Medical Faculty,
Vanderbilt University, Nashville, TN) for the human
CYP51 expression plasmid.
Two separate experiments with two replicas were done.
Each replicon of compound was calculated with each
non-treated value resulting in four calculations of one
experiment. Results are presented as a mean of 8 calcu-
lated ratios and a corresponding standard deviation.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2007.10.001.
5.1.5.6. Determination of the apparent dissociation
constant on purified human CYP51. CYP51 was ex-
pressed and purified by the methods described previ-
3
9
References and notes
ously (see SDS page in the supplementary data).
Binding of nine novel compounds to the enzyme was
determined spectrophotometrically. Human CYP51
was titrated with an inhibitor and the resulting absor-
bance difference spectra were plotted as a function of
the inhibitor concentration. Spectra were taken at the
room temperature in buffer containing 50 mM phos-
phate buffer, 100 mM NaCl, 0.1 mM EDTA, and 10%
glycerol (buffer A) on dual-beam UV spectrophotome-
ter. Ketoconazole and 4m were dissolved in DMSO.
Other compounds were dissolved in water. Inhibitor in-
duced spectral changes were monitored as type II spec-
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2
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