[3H metyrapol and 4-[131I]iodometomidate label overlapping, but not identical, binding sites on rat adrenal membranes

Article abstract of DOI:10.1021/mp3006227

Metyrapone, metyrapol, and etomidate are competitive inhibitors of 11-deoxycorticosterone hydroxylation by 11β-hydroxylase. [ ³H]Metyrapol and 4-[¹³¹I]iodometomidate bind with high affinity to membranes prepared from bovine and rat adrenals. Here we report inhibitory potencies of several compounds structurally related to one or both of these adrenostatic drugs, against the binding of both radioligands to rat adrenal membranes. While derivatives of etomidate inhibited the binding of both radioligands with similar potencies, derivatives of metyrapone inhibited the binding of 4-[¹³¹I]iodometomidate about 10 times weaker than the binding of [³H]metyrapol. By X-ray structure analysis the absolute configuration of (+)-1-(2-fluorophenyl)-2-methyl-2-(pyridin-3-yl)-1-propanol [(+)-11, a derivative of metyrapol] was established as (R). We introduce 1-(2-fluorophenyl)-2-methyl-2-(pyridin-3-yl)-1-propanone (9; K_i = 6 nM), 2-(1-imidazolyl)-2-methyl-1-phenyl-1-propanone (13; 2 nM), and (R)-(+)-[1-(4-iodophenyl)ethyl]-1H-imidazole (34; 4 nM) as new high affinity ligands for the metyrapol binding site on 11β-hydroxylase and discuss our results in relation to a proposed active site model of 11β-hydroxylase.

Full text of DOI:10.1021/mp3006227

Article
pubs.acs.org/molecularpharmaceutics
[3H]Metyrapol and 4‑[131I]Iodometomidate Label Overlapping, but
Not Identical, Binding Sites on Rat Adrenal Membranes
Michael L. Berger,*,† Friedrich Hammerschmidt,*,‡ Renzhe Qian,‡ Stefanie Hahner,§ Andreas Schirbel,∥
Martina Stichelberger,⊥,# Roger Schibli,# Jie Yu,⊥,○ Vladimir B. Arion,∇ Anna Woschek,‡,¶
‡
⊥,¶
̈
Elisabeth Ohler, and Ilse M. Zolle
†
⊥Department of Nuclear Medicine and Center for Brain Research, Medical University of Vienna, Austria
‡Institute of Organic Chemistry and ∇Institute of Inorganic Chemistry, University of Vienna, Austria
∥
§Endocrinology & Diabetes Unit, Department of Internal Medicine I, and Department of Nuclear Medicine, University of
Wuerzburg, Germany
#Center for Radiopharmaceutical Sciences, Paul Scherrer Institute, Villigen, Switzerland
S
* Supporting Information
ABSTRACT: Metyrapone, metyrapol, and etomidate are
competitive inhibitors of 11-deoxycorticosterone hydroxyla-
tion by 11β-hydroxylase. [3H]Metyrapol and 4-[131I]-
iodometomidate bind with high affinity to membranes
prepared from bovine and rat adrenals. Here we report
inhibitory potencies of several compounds structurally related
to one or both of these adrenostatic drugs, against the binding
of both radioligands to rat adrenal membranes. While
derivatives of etomidate inhibited the binding of both
radioligands with similar potencies, derivatives of metyrapone
inhibited the binding of 4-[131I]iodometomidate about 10
times weaker than the binding of [3H]metyrapol. By X-ray
structure analysis the absolute configuration of (+)-1-(2-fluorophenyl)-2-methyl-2-(pyridin-3-yl)-1-propanol [(+)-11, a derivative
of metyrapol] was established as (R). We introduce 1-(2-fluorophenyl)-2-methyl-2-(pyridin-3-yl)-1-propanone (9; Ki = 6 nM),
2-(1-imidazolyl)-2-methyl-1-phenyl-1-propanone (13; 2 nM), and (R)-(+)-[1-(4-iodophenyl)ethyl]-1H-imidazole (34; 4 nM) as
new high affinity ligands for the metyrapol binding site on 11β-hydroxylase and discuss our results in relation to a proposed
active site model of 11β-hydroxylase.
KEYWORDS: 11β-hydroxylase, metyrapone, [3H]metyrapol, etomidate, radioligand binding, rat adrenals
radioligand may have been its easy synthesis (from metyrapone
and [3H]NaBH4).7
INTRODUCTION
■
The adrenostatic drugs metyrapone and etomidate are active
site blockers of 11β-hydroxylase (CYP11B1), a member of the
mitochondrial cytochrome P450 family responsible for the
synthesis of the stress hormone corticosterone from deoxy-
corticosterone (DOC) (for review, see Omura, 2006).1 In
contrast to drug-metabolizing P450s in microsomes, most
mitochondrial P450s show high specificity to their endogenous
substrate and have negligible activity toward xenobiotic
compounds. In addition, etomidate acts as a sedative via the
GABAA receptor,2,3 however at concentrations about 3 orders
of magnitude higher. Since 11β-hydroxylase is highly
concentrated in the adrenal cortex, high affinity radioligands
are of clinical diagnostic interest as imaging radiotracers to
visualize adrenal tissue, especially in cases of adrenocortical
cancer.4,5 Already in 1974, [3H]metyrapol was reported as a
medium affinity radioligand for beef adrenal cortex mitochon-
dria.6 Subfractionation studies localized the binding site to the
inner mitochondrial membrane, known to harbor 11β-
hydroxylase. One reason for the early application of this
Recently, we have established SARs of several etomidate
derivatives at a binding site on rat adrenal membranes with high
affinity for the radioligand 4-[131I]iodometomidate8
([131I]IMTO). Now, we tested the same and additional
compounds at a [3H]metyrapol binding site in the same
preparation. Both classes of compounds appear to address the
same or very similar binding sites, most likely on CYP11B1. In
human adrenals, the m-RNA for this hydroxylase is 3 orders of
magnitude more abundant than the m-RNA for CYP11B2,9
another potential target for both radioligands. In rat adrenals,
65 times (Wistar rats) and 52 times (Fischer rats) more
CYP11B1 than CYP11B2 mRNA is expressed10 (we used
Wistar rats). Although CYP17 is prominently expressed in
Received: October 29, 2012
Revised: January 18, 2013
Accepted: January 23, 2013
Published: January 23, 2013
© 2013 American Chemical Society
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human adrenal cortex,9 metyrapone had only weak or no effect
on CYP17 (as on CYP11A1 and CYP21) in a human
adrenocortical cell line.11 In bovine adrenal mitochondria,
metyrapone and A-phenyl-metyrapone inhibited CYP11A1
much more weakly than CYP11B1.12 Some drug metabolizing
cytochrome P450 enzymes in the liver are sensitive to
inhibition by metyrapone (IC50s from 1 to 4 μM, i.e.,
equivalent to CYP11B1).13 However, these inducible enzymes
are not involved in steroid biosynthesis, and their extrahepatic
expression is low.14 Thus, we assume that >90% of the binding
of [3H]metyrapol and of [131I]IMTO was to CYP11B1 and that
binding to other adrenal P450 enzymes did not significantly
contribute to our results. Assessing inhibitory potencies of
metyrapone-type and etomidate-type compounds at both
targets, we were interested in analogues in between the
molecular skeletons of metyrapone (two pyridinyl substituents
connected by a two-carbon atom bridge) and etomidate (a
single carbon atom bridge connecting a benzene with an
imidazole ring, the latter carrying a carbonyl side chain). We
were particularly interested in the question whether structural
features favorable for one class might be introduced with
benefit into the other class. Especially, metyrapone-type ligands
with higher affinity than metyrapol may result from such a
strategy, possibly allowing the visualization of other types of
adrenocortical neoplasms than the established radioligands
based on etomidate.
the phenyl residue of 5 have been mentioned19 but have never
been described in detail. We now add a description of their
syntheses as Supporting Information. Since the 2-fluoro
derivative 9 turned out as the most potent compound of this
series, we prepared further analogues of 9 (10−12). The
cyclopropyl derivative 10, formally derived from 9 by
connecting the two methyl groups at position 2 of the aliphatic
bridge, was prepared with 32% yield from ketone 9a (precursor
for 9, see Supporting Information) by double alkylation with
1,2-dibromoethane (Scheme 2).
a
Scheme 2. Preparation of Metyrapone Analogue 10
a
Reagents and conditions: (a) tBuOK/DMF; (b) BrCH2CH2Br.
Reductive removal of the carbonyl group of ketone 9 in three
steps furnished the 2-methylpropane derivative 12 (Scheme 3):
Scheme 3. Synthesis of the 2-Methylpropane Derivative 12
a
from 9
CHEMICAL SYNTHESES
■
Radioligands. [3H]Metyrapol ([3H]1) was custom-labeled
by Amersham (12 Ci/mmol) and purified before use by TLC
with CHCl3/CH3OH (90/10), revealing another agreeable
property of this radioligand (in addition to the ease of its
synthesis): Upon purification, we never detected any other
radioactive products than [3H]1 itself (autoradiography of the
TLC plate gave a single band comigrating with unlabeled 1). As
reviewed by Morton (2001)15 radioactive decay of a 3H-labeled
compound leaves a positive charge at the former position of the
tritium. From this, the easiest way to form a stable product is
the generation of a carbonyl group, resulting in (non-
radioactive) metyrapone (2) (Scheme 1). Simple TLC on
silica gel was sufficient to remove this radiolysis product (Rf for
1 0.22, for 2 0.50). The synthesis of [131I]IMTO has been
described previously.16
a
Reagents and conditions: (a) NaBH4/EtOH; (b) nBuLi, CS2, MeI;
(c) (TMS)3SiH, AIBN.
Metyrapone Derivatives. The syntheses of 4′-Br- (3) and
1′-O-2 (4, the N-oxide) with substituents in the pyridinyl
residue close to the quaternary carbon (ring B),17 and of A-
phenyl-metyrapone [2-methyl-1-phenyl-2-(pyridin-3-yl)-1-
propanone, 5]18 have been described. The syntheses of 2-Br-
(6), 2-MeO- (7), 2-HO- (8), and 2-F-5 (9) with substituents in
At first, the ketone was reduced with NaBH4 to racemic alcohol
( )-11, which was transformed into xanthogenate ( )-11a by a
standard procedure.20 The lithium alkoxide formed on addition
of nBuLi to the alcohol was added to carbon disulfide to give an
O-alkyl dithiocarbonate, which was alkylated with MeI. Radical
deoxygenation of ( )-11a with (TMS)3SiH/AIBN in dry
toluene at 90 °C furnished the desired derivate 12 in 55%
yield.20,21
Scheme 1. Suggested Mechanism for Radiolysis of
a
[3H]Metyrapol, Yielding Metyrapone as the Main Product
Metyrapone is biotransformed in part to metyrapol,22−24
preferentially to the (−)-enantiomer (in the rat). At CYP11B1
both enantiomers show similar inhibitory activities.24,25
However, the absolute configurations are not known. Here
we describe optical resolution of the metyrapol derivative
( )-11 with (+)-Noe-lactol dimer (MBF-O-MBF) (Scheme
4).26 (+)-Camphor-10-sulfonic acid-catalyzed acetalization
furnished the two preferentially formed axial diastereomers A
and B, which were separated by flash chromatography in equal
1
a
yields of 32% and found by H NMR spectroscopy to be
The process can be interpreted as oxidation (although the electron is
of nuclear origin).
homogeneous (ee 99%). They were deblocked in MeOH/
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a
Scheme 4. Resolution of ( )-11 via Acetals A and B
Figure 1. X-ray structure of 11b
Etomidate Derivatives. Metomidate (14) was commer-
cially available; the syntheses of its derivatives 15−23 (see
Table 2) have been described;8,29 only the fluoro derivative 24
was newly prepared for this study by a reported method,29
starting from (S)-1-(4-fluorophenyl)ethanol of 99% ee.
Etomidate (25) was obtained commercially; the syntheses of
its derivatives 26−32 (see Table 2) have been described.8,29
During a first attempt to convert 14 in one step to the N-
methylamide 30, unexpectedly the ester group was almost
quantitatively lost, opening an easy synthetic access to
etomidate analogues without ester group. Consequently, we
prepared 33 and 34 from 25 and 20, respectively (Scheme 6).
a
Reagents and conditions: (a) DCM/H+; (b) MeOH/H+.
DCM/(+)-camphor-10-sulfonic acid to give (+)- and (−)-11,
respectively. To determine the ee of the (+)-enantiomer by a
second method, ( )- and (+)-11 were converted to (R)-α-
methoxy-α-trifluoromethylphenylacetic acid esters [(R)-
Scheme 6. Synthesis of Etomidate Derivatives without Ester
Mosher esters]27 and investigated by H NMR spectroscopy.
1
a
Group
Again, the (+)-enantiomer was found to have an ee of 99%.
To assign the absolute configurations to the enantiomers, the
dextrorotary alcohol was converted to the lithium alkoxide at
−78 °C and reacted with excess (S)-1-phenylethylisocyanate
(Scheme 5). Addition of two molecules of the isocyanate
Scheme 5. Preparation of Derivative 11b from (+)-11 for X-
ray Structure Analysis
a
a
Reagents and conditions: (a) EtOH/MeNH2, 180 °C.
As side products, the amides were formed in low yields, but the
main products were 33 and 34. This simple synthetic method is
new, and its mechanisms should be investigated, since the
decarboxylation of imidazole carboxylic acids in general
requires harsher reaction conditions (e.g., refluxing in diphenyl
ether at 260 °C).30 The preparation of (S)-33 from 1-
phenylethylamine has been described.30 Finally, we tested
pyridin-3-yl derivatives of 14 and 168,29 (36 and 37) and the
2,2′-bipyridin-5-yl derivatives 38−41.31,32
a
Reagents and conditions: (a) nBuLi/THF, −78 °C; (b) (S)-
Ph(Me)CHNCO.
EXPLORATION OF [3H]METYRAPOL BINDING
CONDITIONS
■
resulted in the urea derivative 11b. The crystals obtained by
slow evaporation of the solvent (DCM/hexanes) were suitable
for X-ray structure analysis. The (+)-alcohol had (R)-
configuration at the stereogenic center (C7), based on the
known (S)-configuration of the derivatizing chiral isocyanate
(Figure 1). Consequently, the (−)-alcohol has (S)-config-
uration. 2-(1-Imidazolyl)-2-methyl-1-phenyl-2−1-propanone
(A-phenyl-B-imidazolyl-metyrapone, 13), a known compound,
was prepared by the literature procedure28 in 47% yield, mp
104−105 °C (DCM/hexanes).
In our first approach to label adrenal membranes with [3H]1
we followed the protocol of Satre and Vignais (1974),6 using
mitochondrial membranes from beef adrenal cortex. Our results
(KD = 23 nM, BM = 513 pmol/mg protein)33 were similar to
those of Satre and Vignais (KD = 10−18 nM, BM = 400−600
pmol/mg protein). However, material obtained from slaughter-
house bulls may be of variable quality. Therefore, we tested the
possibility to switch from beef to rat adrenals. We explored
possible species differences by direct comparison of [3H]1
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affinities to 20 μm slices cut from shock-frozen beef adrenal
cortex and rat whole adrenals, resulting in KD = 60 nM for the
beef and in KD = 225 nM for the rat material.33 Thus, we saw
differences in the affinities of beef and rat adrenal binding sites
for [3H]1. For the other radioligand 4-[131I]IMTO we observed
similar KD values for beef adrenocortical (9.6 2.2 nM, n = 6)
and for rat adrenal membranes (11.6 2.5 nM, n = 8).8 Given
our previous results8 with rat membranes and the higher affinity
ligand [131I]IMTO (and in light of better accessibility of rat
adrenals), we decided to improve the reliability of [3H]1
binding to the rat material.
of IC50 values of our test compounds against [3H]1 binding, we
kept to the more reliable two-wash protocol, since IC50 values
do not depend on absolute values.
INHIBITION OF BINDING BY METYRAPONE
■
ANALOGUES
Both 1 and 2 blocked [131I]IMTO binding to rat adrenal
membranes with medium potency (Ki near 1 μM, Table 1).
Table 1. Inhibition of [3H]1 and [131I]IMTO Binding to Rat
Adrenal Membranes by Metyrapone-Type Compounds (Ki
SD, or Both Values if n = 2)
Attempts to detect specific [3H]1 binding to whole
membranes from rat adrenals with the filtration technique
failed. However, the centrifugation technique yielded robust
specific binding (if at least 0.3 mg rat adrenal tissue per vial had
been invested), with less than 10% nonspecific binding. In fact,
the centrifugation technique is the method of choice for
radioligands with medium affinity and fast dissociation rate.34
Incubation on ice yielded better results than incubation at room
temperature (rt) (Figure 2). Saturation analysis (on ice)
[3H]1
Ki (nM)
[
131I]IMTO
X
R
R′
n
Ki (nM)
n
1
N
N
C
C
C
C
C
C
C
C
C
C
HO
O
O
O
O
O
O
O
HO
HO
H
H
98
36
9
2
7
3
4
3
2
3
5
2
3
3
3
6
809 315
804 637
143 60
77, 85
9
7
9
2
4
2
2
3
3
3
2
3
2
H
5
H
13.7 4.5
11.2 5.7
9.9, 11.1
6
Br
MeO
HO
F
7
96 26
179, 128
102, 77
8
9.3 2.6
5.84 1.60
38.1, 22.6
235 56
9
10
F
215 146
527 233
154 28
57, 49
(R)-11
(S)-11
12
F
F
50
8
F
20.6 2.7
13
O
H
2.05 0.70
20
6
However, 1 and 2 were much more potent in displacing bound
[3H]1. The SARs of metyrapone-like compounds exhibited
some similarities at both sites (Table 1): (1) Replacing the
pyridinyl substituent adjacent to the carbonyl group (the A-
ring) by a phenyl substituent (a first step toward the molecular
skeleton of etomidate) led to a considerable (more than factor
5) increase in potency (compounds 5−9); (2) additional
replacement of the other pyridine ring by an imidazole ring (a
second step toward etomidate) increased the potency still
further (by at least another factor 5; compound 13). Structure
13 bears already remarkable resemblance to the structure of
etomidate without ester group (33). Since 9, the 2-F derivative
of 5, turned out as one of the most potent displacers of [3H]1,
we subjected this structure to closer exploration. Connecting
the methyl substituents (10) was of disadvantage at both
binding sites (Table 1). After reduction of the keto group and
steric resolution of the secondary alcohol, we observed a
slightly higher potency of (S)-(−)-11 as compared to (R)-
(+)-11 (for 1 itself, no such steric preference has been
described24,25). However, even the more potent isomer (S)-
(−)-11 was weaker than unreduced 9. Leaving out the oxygen
resulted in 12, a compound of considerable potency at both
binding sites. Already Hays et al.18 had observed potent
inhibition of 11β-hydroxylase by a derivative of A-phenyl-
metyrapone without oxygen.
Figure 2. Representative results of experiments exploring conditions
for [3H]1 binding. Increasing concentrations of the radioligand (upper
panel) yielded saturation isotherms that were linearized by reciprocal
transformation (lower panel). The slopes of the reciprocal plots
yielded the highest affinity (112 nM in this example) for binding and
centrifugation at 0 °C, if pellets were rinsed twice (filled circles).
Affinities were weaker at 20 °C (213 nM; open squares), and (at 0 °C)
if pellets were rinsed only once (230 nM; open triangles).
resulted in the following parameters (mean SD, n): KD = 108
20 nM (5); BM = 4.9 1.2 pmol/mg tissue (5). Since this
standard protocol included two rinses of the pelleted
membranes (some textbooks34 even suggest three rinses), we
reduced the number of rinses to one, to scrutinize the reliability
of the observed BM value. In two such experiments (one of
them shown in Figure 2), we observed the following
parameters: KD = 230, 289 nM; BM = 11.0, 14.9 pmol/mg
tissue (nonspecific binding 2.6 times higher than after two
washes). With the high affinity radioligand [131I]IMTO, we had
observed in the same membrane preparation (rat adrenals,
filtration assay) a BM = 12.1 pmol/mg tissue,8 close to our
single wash results with [3H]1. Nevertheless, for the estimation
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Table 2. Inhibition of [3H]1 and [131I]IMTO Binding to Rat Adrenal Membranes by Etomidate-Type Compounds (Ki SD, or
both values if n = 2)
[3H]1
Ki (nM)
[
131I]IMTO
RI
RC
RP
n
Ki (nM)
n
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Et
(R)-Me
(S)-Me
H
3.03 1.46
161, 157
4
2
3
3
3
3
3
3
3
3
3
4
2
2
3
3
3
2
4
5
3
3
4.02 1.87
9
502 226
6
4
a
10.6 3.9
2.20 1.52
2.71 0.93
11.1 4.4
11.3 1.6
2.83 0.33
3.04 1.32
2.49 1.37
6.80 2.24
3.02 1.82
14.0, 18.2
25
9
a
a
a
(R)-Et
5.31 0.82
2.78 0.60
3.79 1.52
8.7 3.2
3
(see structure)
(R)-Me
(R)-Me
(R)-Me
(R)-Me
(R)-Me
(R)-Me
(R)-Me
(R)-Me
(R)-Me
(R)-Me
(R)-Me
(R)-Me
(R)-Me
(R)-Me
(R)-Me
(R)-Me
(R)-Me
3
3-I
4-I
4
24
4
4-Br
8.8 2.4
4-Me
4-HOMe
4-F
3.88 1.80
2.81 0.77
7.3, 9.0
4
a
4
2
0.97 0.39
3.73 0.98
2.47 0.47
2.47 0.47
1.21 0.28
12
5
a
a
a
a
CO2Et
4-I
CO2CH2CH2F
CO2Pr
1.76, 2.12
4
4.05 2.12
4.99 1.38
4
CO2iPr
5
a
C(O)NHMe
C(O)N(Me)OMe
C(O)Me
H
47
6
183 78
6
a
4.92, 3.84
14.2, 11.9
2
a
7.91 3.40
57 14
51, 53
2
686 139
31, 26
3
H
4-I
4.34 1.92
38830 16180
2
a
CO2H
104910 35310
3
a
From ref 8.
INHIBITION OF BINDING BY ETOMIDATE
ANALOGUES
■
With the exception of 24, 33, and 34, we have already described
syntheses, properties, and potencies as inhibitors of
[
131I]IMTO binding of the etomidate analogues 14−35.8
Comparison of the SARs at both sites (Table 2) allows the
following general conclusions: (1) At both sites, (R)-isomers
are much more potent than (S)-isomers (by about 2 orders of
magnitude, compare 14 to 15); (2) the free acid (35) is at both
sites almost inactive; (3) most etomidate derivatives are highly
potent inhibitors at both sites, with no obvious preference for
one or the other. Apart from these more general observations,
the two sites seem to follow their own SARs (see Figure 3).
Interestingly, 33 and 34 without ester group displace [3H]1
more potently than [131I]IMTO, possibly because of their
structural resemblance to metyrapone-type compounds.
INHIBITION OF BINDING BY
PYRIDINYL-CONTAINING ETOMIDATE
ANALOGUES
■
Figure 3. Comparison of inhibitory potencies at binding sites labeled
with [3H]metyrapol to those at sites labeled with [131I]IMTO, for
various structural classes; filled circles, etomidate analogues; open
circles, metyrapone analogues; open diamonds, pyridinyl etomidates;
open squares, etomidates without ester group; crosses, DOC and
ketoconazole (keto); compound numbers inscribed to the symbols
(some of them occluding each other). The dotted line indicates
identical potency at both sites. All values are from Tables 1−4.
While the replacement of one of the pyridine rings by a
benzene ring took the structure of metyrapone one step closer
to that of etomidate (strengthening the affinities to both
binding sites), we now explored these relationships from the
other side, by introducing a pyridinyl substituent into the
molecular skeleton of etomidate. Remarkably, all pyridinyl
analogues of etomidate acted more potently as inhibitors of
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Table 3. Inhibition of [3H]1 and [131I]IMTO Binding to Rat Adrenal Membranes by Pyridinyl-Metomidate and Structural
Analogues (Ki SD, or Both Values if n = 2)
[3H]1
[
131I]IMTO
X
RC
R
Ki (nM)
n
Ki (nM)
n
14
36
16
37
38
39
40
41
C
N
C
N
N
N
N
N
Me
Me
H
H
H
H
H
3.03 1.46
4.96, 4.81
10.6 3.9
90, 109
4
2
3
2
3
3
2
3
4.02 1.87
9
a
17.7 3.2
4
4
a
25
9
a
H
742 205
154 56
981 341
3
Me
H
pyridin-2-yl
pyridin-2-yl
pyridin-2-yl
pyridin-2-yl
9.6 3.0
92 22
3
10
3
Me
Me
3895, 5754
1268 448
21580 9550
10170 12860
2
a
From ref 8.
[3H]1 binding than as inhibitors of [131I]IMTO binding (Table
3; although introduction of pyridinyl always led to some loss in
potency). Direct comparison was possible for the phenyl/
pyridinyl pairs 36/14 and 37/16 (Table 3), with losses by
factors 4.4 and 30 for inhibition of [131I]IMTO binding, but
only by factors 1.6 and 9.4, respectively, for the inhibition of
[3H]1 binding.
labeled with [3H]1 (Table 4), a preference probably related to
the presence of a two-carbon bridge connecting two aromatic
Table 4. Inhibition of [3H]1 and [131I]IMTO Binding to Rat
Adrenal Membranes by Deoxycorticosterone (DOC) and
Ketoconazole (Ki SD)
Surprising to us was the small influence of a second pyridinyl
substituent attached to the first one on the inhibition of [3H]1
binding (weaker by factor 2), in contrast to the more
pronounced effect of this modification on the inhibition of
[
131I]IMTO binding (factor 9; Table 3, compare 36 to 38).
Synthesis of the 2,2-bipyridinyl derivative 38 was inspired by
the option to form a complex with radioactive technetium.35
Preliminary autoradiographic results obtained with a conjugate
of the demethyl analogue 39 with a 99mTc(I)−tricarbonyl
complex turned out discouraging, with no significant difference
between total and nonspecific binding.36 Nevertheless, we
continued by synthesizing 41, the complex of the 2,2-bipyridyl
analogue of metomidate 38 with the Tc analogue rhenium.31
Although this rather bulky conjugate inhibited [3H]1 binding
eight times more potent than [131I]IMTO binding, the
observed Ki (1.27 μM) precluded any use as an in vivo
imaging tracer.
[3H]1
Ki (nM)
[
131I]IMTO
n
Ki (nM)
n
a
a
DOC
1009 261
56 18
3
3
759 281
606 418
5
7
ketoconazole
a
From ref 8.
nuclei, although the number of carbon atoms connecting two
aromatic rings was not a general predictor of site preference.
Although all two-carbon-bridged aromatic compounds
inhibited [3H]1 binding better than [131I]IMTO binding (by
factors 2.2−23, Table 1, open circles in Figure 3), the converse
was not true. Several one-carbon-bridged compounds also
exhibited preference for [3H]1-labeled sites, especially 33 and
34 (open squares in Figure 3), and all etomidate derivatives
with a pyridinyl ring (open diamonds in Figure 3), notably the
2,2-bipyridine derivative 38. In these latter compounds,
particular structural features of etomidate had been modified
into the direction of closer resemblance with metyrapone-type
compounds (either absence of a carbonyl group-containing
substituent or replacement of a benzene with a pyridine ring).
The most potent inhibitor of [3H]1 binding without a carbonyl
group as substituent was the imidazole derivative 13 (Ki against
[3H]1 binding 2 nM). It may be of interest to develop this new
compound as a radioactive probe to study more efficiently
metyrapone-type binding sites on adrenal membranes. The
fluoro derivative 9 may be another new option to label with
high affinity (Ki 5.8 nM) these sites, in addition to the 4-
In fact, application of a 99mTc(I) conjugate with 38 to mice
did not result in any selective accumulation of radioactivity in
the adrenals.31 As an unintended byproduct of the synthesis of
38 we obtained its isomer 40 (with the carbonyl substituent at
position 4 instead of 5). Although this modification might
appear unimportant on first glance, its consequences (a
reduction in potency at both radioligand binding sites by
more than 2 orders of magnitude) were dramatic. For a better
understanding of this effect, it would be interesting to study the
4-carbonyl analogues of other etomidate derivatives.
SPECIFIC STRUCTURAL LEADS?
■
Structurally unrelated substances known to interact with 11β-
hydroxylase inhibited the binding to both sites. [131I]IMTO and
[3H]1 binding were inhibited by similar concentrations of
DOC, the main substrate of the enzyme. The fungicide
ketoconazole, however, was 11 times more potent at the site
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iodophenyl derivative 34 (Ki 4.3 nM). At least 9, 13, and 34
appear more appropriate for this purpose than radiolabeled 1.
In vivo studies may be warranted to further investigate these
compounds as new tracers.
Much weaker correlation is seen comparing inhibition of
cortisol secretion to inhibition of [3H]1 binding (Figure 4B
without triangles; R = 0.65, P < 0.05). Most of the tested
compounds had no recognizable effect on enzyme activity at
concentrations that displaced 50% of bound [3H]1. The
triangles in Figure 4B reproduce data obtained with beef
adrenocortical mitochondria33 demonstrating, for a series of
metyrapone-type compounds, that their potencies as enzyme
inhibitors were much weaker (by a mean factor 16) than their
potencies as inhibitors of [3H]1 binding. Binding to the
“metyrapone site” may not be enough to achieve enzyme
inhibition. On the other hand, binding to the “etomidate site”
appears sufficient to inhibit enzyme activity (although this
inhibition may be of only minor clinical relevance, since it is of
short duration).
RELATION TO ENZYME ACTIVITY
■
Since metyrapone and etomidate block 11β-hydroxylase by
directly binding to the active catalytic center, including
interaction of an aromatic nitrogen atom with the sixth ligating
position of the prosthetic group heme iron atom,37 the affinities
of these and structurally related compounds to adrenal
membranes might correlate with their potencies as inhibitors
of 11β-hydroxylase enzymatic activity. We have recently
obtained such a correlation for several etomidate derivatives,
measuring their effects on the secretion of cortisol by NCI-
h295 cells and on the binding of [131I]IMTO to rat adrenal
membranes.8 In the upper panel of Figure 4 we present once
CONCLUSIONS
■
Our results may indicate that etomidate- and metyrapone-type
compounds share overlapping binding sites at the catalytic
center of 11β-hydroxylase. On the basis of our observations, we
propose a hypothetical active site model illustrated schemati-
cally in Figure 5. In this model, the interaction site involved in
Figure 5. Tentative arrangement of etomidate (a) and metyrapone (b)
in a schematic binding pocket close to the reactive center of CYP11B1
(amino acids refer to human sequence). Note that both occupy the 6th
ligating position of the heme iron atom (in gray), but only etomidate
reaches directly to the catalytic T318 (bold). Adapted from Belkina et
al. (2001)39 and Roumen et al. (2007).37
the binding of [3H]1 is also involved in the binding of
[
131I]IMTO, but vice versa only a part of the site involved in
the binding of [131I]IMTO contributes to [3H]1 binding.
This model was chosen in analogy to the three-dimensional
model proposed by Roumen et al. (2007),37 based on docking
and dynamics simulations applied to CYP11B1 and on the
known crystal structure of bacterial cytochromes. In this model,
metyrapone and etomidate are proposed to provide one
aromatic ring for π-stacking with arg110 and phe130. A
nitrogen atom of the second aromatic ring ligates the catalytic
heme iron. Only etomidate forms an additional hydrogen bond
with thr318, investing its carbonyl group (see Figure 7 of the
reference; the model is incomplete inasmuch as the high degree
of stereospecificity of etomidate remains unexplained). Radio-
tracers actually in use for adrenocortical tumor imaging are
exclusively derived from etomidate,4,5 including the catalytic
site in their binding mode. Here we presented with 9, 13, and
34 three potential tracers with similar affinities, but slightly
differing binding modes. The clinical value of this new type of
tracer should be the subject of further studies. We would
predict that the labeling achieved with the two types of tracers
should be similar, but with less enzyme inhibition by
metyrapone-type than with etomidate-type tracers.
Figure 4. Comparison of inhibitory potencies against steroid 11β-
hydroxylation (ordinate) with those against binding of [131I]IMTO (a)
and against binding of [3H]1 (b), respectively, for test compounds
structurally related to etomidate (filled symbols) or to metyrapone
(open symbols). The dotted line indicates identical potencies at both
targets. Data on cortisol secretion by NCI-h295 cells in a and b (left
ordinate) taken from ref 8; data on production of [14C]corticosterone
from [14C]DOC by beef adrenocortical mitochondria in b (open
triangles, right ordinate) taken from ref 33.
more these data, complemented with data on three further
compounds: 2 (IC50 = 3.5 μM),38 13 (IC50 = 663 139 nM; n
= 3), and 32 (IC50 = 1220, 1590 nM; n = 2). While 2 and 32
fitted well with the other compounds, the imidazole derivative
13 had a much weaker effect on cortisol secretion than on
[
131I]IMTO binding. Nevertheless, correlationeven including
13was high (R = 0.93, P < 0.001).
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DCM (2 × 5 mL). The combined organic layers were washed
with brine and dried (Na2SO4), and solvents were removed on
a rotary evaporator. The residue was purified by flash
chromatography (DCM/EtOAc, 3/1, Rf = 0.27) to yield
alcohol ( )-11 (0.051 g, 79%) as colorless crystals; mp 130−
131 °C (DCM/hexanes).
EXPERIMENTAL SECTION
■
General Procedures. 1H NMR spectra were recorded on a
Bruker DRX-400 spectrometer at 400.13 MHz, in CDCl3 using
the residual solvent peak as internal reference [δ(CHCl3) =
7.24]. 13C NMR spectra (mainly J modulated) were recorded
on the same spectrometer operating at 100.61 MHz [δ(CDCl3)
= 77.00]. Chemical shifts δ are given in parts per million,
coupling constants J in Hz. IR spectra were recorded as films on
a silicon disk on a Perkin-Elmer 1600 FT-IR or a Bruker
VERTEX 70 IR spectrometer. Optical rotations were measured
at 20 °C on a Perkin-Elmer 341 polarimeter in a 1 dm cell.
Melting points were measured on a Reichert Thermovar
instrument and are uncorrected. Analytical HPLC was
performed on a Jasco system (PU-980 pump, UV 975, and
RI 930) using as chiral stationary phase a Chiracel OD-H
column, 0.46 × 25 cm, 1 mL/min, UV (254 nm). TLC was
performed on 0.25 mm Merck plates, silica gel 60 F254. Flash
(column) chromatography was performed with Merck silica gel
60 (230−400 mesh). Spots were visualized by UV, by exposing
to iodine vapor, or by dipping the plate into a solution of 23 g
of (NH4)6Mo7O24·4H2O and 1 g of Ce(SO4)2·4H2O in 500 mL
of 10% H2SO4 in water, followed by heating with a heat gun.
THF was distilled from potassium, and Et2O from LiAlH4 prior
to use.
Syntheses. 1-(Pyridin-3-yl)cyclopropyl 2-Fluorophenyl
Ketone (10). Potassium t-butoxide (0.317 g, 2.83 mmol, 1.2
equiv) was added to a stirred solution of ketone 9a (0.508 g,
2.36 mmol; see Supporting Information) in dry DMF (10 mL)
at 0 °C under argon, followed by 1,2-C2H4Br2 (0.666 g, 0.31
mL, 3.54 mmol, 1.5 equiv) after 25 min. The mixture was
stirred for 2.5 h at 0 °C. Then another portion of the butoxide
(0.292 g, 2.60 mmol, 1.1 equiv) was added at rt, and stirring
was continued for 18 h. Solvents were removed on a rotary
evaporator. The residue was dissolved in EtOAc (10 mL) and
extracted with water (10 mL). The aqueous phase was
separated and extracted with EtOAc (2 × 10 mL). The
combined organic layers were dried (Na2SO4) and solvents
removed on a rotary evaporator. The residue was purified by
flash chromatography (hexanes/EtOAc, 2/1, Rf 0.33) and bulb-
to-bulb distillation (110 °C/0.3 mbar) to yield cylopropyl
compound 10 (0.180 g, 32%) as a colorless oil.
1H NMR: δ 8.51 (d, J = 2.1, 1H, Hpyr), 8.34 (dd, J = 4.8, 1.5,
1H, Hpyr), 7.64 (ddd, J = 7.8, 2.0, 1.5, 1H, Hpyr), 7.23−7.16 (m,
1H, Har), 7.16 (dd, J = 7.8, 4.8, 1H, Hpyr), 7.07−6.98 (m, 2H,
Har), 6.93 (ddd, J = 10.5, 8.3, 1.0, 1H, Har), 5.12 (s, 1H, CHO),
2.79 (br. s, 1H, OH), 1.36 (d, J = 1.3, 3H, CH3), 1.33 (s, 3H,
CH3). 13C NMR: δ 159.93 (d, JFC = 245.4, CF), 148.86
(HCpyr), 147.22 (HCpyr), 141.18 (Cpyr), 135.05 (HCpyr), 129.22
(d, JFC = 3.9, Car), 129.02 (d, JFC = 8.5 Hz, Car), 128.27 (d, JFC
= 2.9, Car), 123.57 (d, JFC = 3.4, Car), 122.61 (Cpyr), 114.82 (d,
J
FC = 23.0, Car), 73.87 (CHO), 42.49 (C(CH3)2), 25.44 (d, JFC
= 1.2, CH3), 22.25 (CH3). IR (Si): νmax 3.315, 2974, 1484,
1453, 1417, 1222, 1102, 1057, 913 cm−1. Anal. Calcd for
C15H16FNO (245.29): C, 73.45; H, 6.57; N, 5.71. Found: C,
73.22; H, 6.72; N, 5.73.
( )-O-1-(2-Fluorophenyl)-2-methyl-2-(pyridin-3-yl)propyl
S-Methyl Dithiocarbamate (11a). n-BuLi (0.30 mL, 0.48
mmol, 1.5 equiv) was added dropwise to a stirred solution of
alcohol ( )-11 (0.080 g, 0.32 mmol) at −78 °C under argon,
followed by CS2 (0.251 g, 0.20 mL, 3.3 mmol, 10 equiv) at −45
°C after 20 min and MeI (0.468 g, 0.31 mL, 3.3 mmol, 10
equiv) at −30 °C 30 min later. The reaction mixture was stirred
for 40 min at −30 °C, and then solvents were removed on a
rotary evaporator. The residue was diluted with EtOAc (15
mL) and washed with water (15 mL). The organic phase was
separated and the aqueous one extracted with EtOAc (2 × 15
mL). The combined organic layers were dried (Na2SO4), and
solvents were removed on a rotary evaporator. The residue was
purified by flash chromatography (hexanes/EtOAc, 1/1, Rf
0.56) to yield xanthogenate ( )-11a (0.103 g, 94%) as a
colorless oil.
1H NMR: δ 8.56 (m, 1H, Hpyr), 8.46 (dd, J = 4.6, 1.5, 1H,
Hpyr), 7.62 (ddd, J = 8.1, 2.3, 1.5, 1H, Hpyr), 7.24−7.17 (m, 2H,
Har, Hpyr), 6.98−6.91 (m, 2H, Har), 6.82 (s, 1H, CHO), 6.74
(td, J = 7.6, 1.5, 1H, Har), 2.45 (s, 3H, SCH3), 1.46 (s, 6H,
CH3). 13C NMR: δ 214.23 (CS), 160.22 (d, JFC = 247.7,
CF), 148.84 (HCpyr), 147.82 (HCpyr), 139.46 (HCpyr), 134.82
(HCpyr), 129.81 (d, JFC = 8.5, HCar), 128.76 (d, JFC = 3.4,
HCar), 123.63 (d, JFC = 12.9, Car), 123.62 (d, JFC = 3.6, HCar),
122.63 (HCpyr), 115.03 (d, JFC = 22.6, HCar), 83.56 (CHO),
42.00 (Cq), 25.27 (CH3), 23.32 (CH3), 19.00 (SCH3). IR (Si):
νmax 2977, 1490, 1234, 1203, 1062 cm−1. Anal. Calcd for
C17H18FNOS2 (335.46): C, 60.87; H, 5.41; N, 4.18. Found: C,
60.57; H, 5.15; N, 4.16.
1H NMR: δ 8.47−8.43 (m, 1H, Hpyr), 8.34 (dd, J = 4.8, 1.5
Hz, 1H, Hpyr), 7.53−7.48 (m, 1H, Har), 7.35−7.29 (m, 1H,
Hpyr), 7.28−7.20 (m, 1H, Har), 7.08 (dd, J = 7.7, 4.8, 1H, Hpyr),
7.03 (td, J = 7.3, 0.8, 1H, Har), 6.85−6.78 (m, 1H, Har), 1.88−
1.77 (m, 2H, Ccyclo), 1.43−1.32 (m, 2H, Hcyclo). 13C NMR: δ
200.55 (d, JFC = 1.6, CO), 158.45 (d, JFC = 250.8, CF),
151.14 (d, J = 1.1, HCpyr), 148.11 (HCpyr), 137.03 (d, JFC = 1.2,
HCpyr), 135.37 (Car), 132.57 (d, JFC = 8.3, HCar), 129.07 (d, JFC
= 3.5, HCar), 127.72 (d, JFC = 16.2, Car), 124.18 (d, JFC = 3.2,
HCar), 122.86 (HCpyr), 115.76 (d, JFC = 21.8, HCar), 35.69
(Ccyclo), 18.12 (2C, H2Ccyclo). IR (Si): νmax 1681, 1611, 1483,
1452, 1416, 1299, 1224, 1195, 989 cm−1. Anal. Calcd for
C15H12FNO (241.26): C, 74.67; H, 5.01; N, 5.81. Found: C,
74.39; H, 4.84; N, 5.76.
( )-1-(2-Fluorophenyl)-2-methyl-2-(pyridin-3-yl)-1-propa-
nol (11). NaBH4 (0.010 g, 0.26 mmol, 1 equiv) was added to a
stirred solution of ketone 9 (0.064 g, 0.26 mmol) in ethanol (5
mL) at rt. After 30 min a drop of glacial acetic acid was added
and solvents were removed on a rotary evaporator. The residue
was dissolved in DCM (5 mL) and extracted with water (5
mL). The aqueous phase was separated and extracted with
1-(2-Fluorophenyl)-2-methyl-2-(pyridin-3-yl)propane (12).
A solution of AIBN (0.011 g) and (TMS)3SiH (0.207 g, 0.26
mL, 0.83 mmol, 3 equiv) in dry toluene (1 mL) was added to a
stirred solution of xanthogenate ( )-11a (0.093 g, 0.28 mmol)
in dry toluene (5 mL) at 90 °C under argon. After 2 h volatile
components were removed under reduced pressure (up to 80
°C, 1 mbar; bulb to bulb distillation apparatus). The residue
was purified by flash chromatography (hexanes/EtOAc, 2/1, Rf
= 0.29) to yield 12 (0.035 g, 55%) as a colorless oil.
1H NMR: δ 8.57 (br, s, 1H, Hpyr), 8.43 (br. d, J = 4.8, 1H,
Hpyr), 7.56 (td, J = 8.1, 2.0, 1H, Hpyr), 7.18 (dd, J = 8.1, 4.8, 1H,
Hpyr), 7.15−7.07 (m, 1H, Har), 6.96−6.83 (m, 2H, Har), 6.67
(td, J = 7.6, 1.8, 1H, Har), 2.90 (d, J = 0.9, 2H, CH2), 1.36 (d, J
= 0.8, 6H, CH3). 13C NMR: δ 161.40 (d, JFC = 244.5, CF),
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147.97 (HCpyr), 147.03 (HCpyr), 143.76 (Cpyr), 133.77 (HCpyr),
132.49 (d, JFC = 4.0, HCar), 128.08 (d, JFC = 8.0, HCar), 124.95
(d, JFC = 15.1, Car), 123.26 (d, JFC = 3.0, HCar), 122.77 (HCpyr),
115.12 (d, JFC = 23.1, HCar), 42.90 (CH2), 38.06 (Cq), 27.51
(2C). IR (Si): νmax 2973, 2938, 1656, 1615, 1492, 1454, 1384,
1231, 986 cm−1. HRMS (ESI): Calcd for C15H16FNH
(230.1345). Found: 230.1339 5 ppm.
(+)-Camphor-10-sulfonic acid (0.322 g, 1.388 mmol, 3 equiv)
was added to a stirring solution of diastereomer A in dry
methanol (5 mL) at rt. After stirring for 2 h the reaction
mixture was neutralized with a saturated aqueous solution of
NaHCO3, filtered, and concentrated under reduce pressure.
Water was added and the mixture extracted with EtOAc (3 ×
15 mL). The combined organic layers were dried (Na2SO4)
and solvents removed on a rotary evaporator. The residue was
purified by flash chromatography (DCM/EtOAc, 2/1, Rf =
0.41) to yield (R)-(+)-11 (0.113 g, 98%) as colorless crystals;
mp 78−79 °C (DCM/hexanes); [α]2D0 +30.18 (c 0.56, acetone).
Similarly, diastereomer B (0.119 g, 0.281 mmol) was converted
to (S)-(−)-11 (0.064 g, 92%) as colorless crystals; mp 79−81
Resolution of ( )-11. Reaction of Noe-lactol Dimer
[(+)-MBF-O-MBF] with ( )-11. A mixture of alcohol ( )-11
́
(0.368 g, 1.5 mmol) and molecular sieves (1.5 g, 4 Å) in dry
DCM (15 mL) was stirred for 15 min at rt before Noe’s reagent
[0.280 g, 075 mmol, 0.5 equiv, (+)-MBF-O-MBF] and
(+)-camphor-10-sulfonic acid (0.492 g, 2.1 mmol, 1.4 equiv)
were added at 0 °C. After 2 h the mixture was filtered and the
eluate washed with a saturated aqueous solution of NaHCO3
and dried (Na2SO4), and solvents were removed on a rotary
evaporator. The residue was purified by flash chromatography
(DCM/EtOAc, 7/1) to furnish diastereomer A (0.205 g, 32%,
Rf = 0.40) as a colorless viscous oil, and diastereomer B (0.205
g, 32%, Rf = 0.33) as colorless crystals, mp 78−80 °C.
1
°C; [α]2D0 −34.80 (c 0.64, acetone). The H and 13C NMR
spectra of (R)-(+)- and (S)-(−)-11 were identical to those of
( )-11.
Preparation of (R)-Mosher Ester of ( )- and (R)-(+)-11. A
mixture of alcohol ( )-11 (0.020 g, 0.082 mmol) in dry DCM
(0.5 mL), dry pyridine (1 mL), and Mosher’s acid chloride (S)-
(+)-MTPACl (0.3 mL, 0.159 mmol, 1.9 equiv, 0.53 M in dry
1,4-dioxane) was left at rt for 18 h. Water (0.5 mL) and HCl
(0.5 mL, 2 M) were added. After 15 min solvents were
removed on a rotary evaporator. The residue was mixed with
water (5 mL) and extracted with DCM (3 × 5 mL). The
combined organic layers were dried (Na2SO4) and solvents
removed on a rotary evaporator. The residue was purified by
flash chromatography (DCM/EtOAc, 7/1, Rf = 0.62) to yield a
mixture of diastereomeric (R)-Mosher esters (S)-(−)- and (R)-
(+)-11·MTPA-(R) as a colorless oil. Similarly, alcohol (R)-
(+)-11 (0.011 g, 0.045 mmol) was converted to (R)-
(+)-11·MTPA-(R) as a colorless oil; ee ≥99%.
1
Diastereomer A. H NMR: δ 8.65 (dd, J = 2.3, 0.5, 1H,
Hpyr), 8.42 (dd, J = 4.8, 1.5, 1H, Hpyr), 7.68 (ddd, J = 8.1, 2.3,
1.5, 1H, Hpyr), 7.24−7.16 (m, 1H, Har), 7.19 (ddd, J = 8.1, 4.8,
0.5, 1H, Hpyr), 7.10−7.01 (m, 2H, Har), 6.98 (ddd, J = 11.1, 8.3,
1.0, 1H, Har), 5.06 (s, 1H, CHPh), 4.86 (dd, J = 3.3, 2.0, 1H,
OCHO), 3.08 (dd, J = 9.6, 1.5, 1H, CHO), 2.71−2.61 (m, 1H,
CH(CH2)2), 1.65−1.60 (m, 2H, CH2(CH)2), 1.53−1.35 (m,
3H), 1.30 (s, 3H, CH3), 1.27 (d, J = 2.0, 3H, CH3), 1.24−1.16
(m, 1H, CCH2CH2), 1.05−0.96 (m, 1H), 0.82 (s, 3H, CH3),
0.81 (s, 3H, CH3), 0.70 (s, 3H, CH3). 13C NMR: δ 161.52 (d,
JFC = 246.2, CF), 149.29 (HCpyr), 147.04 (HCpyr), 142.54
(HCpyr), 134.57 (HCpyr), 129.79 (d, JFC = 4.1, HCar), 128.99
1H NMR: diagnostic signals for (R)-(+)-11·MTPA-(R), δ
8.44 (dd, J = 4.8, 2.3, 1H, Hpyr), 8.38 (d, J = 2.3, 1H, Hpyr), 6.64
(td, J = 7.8, 1.5, 1H, Har), 6.39 (s, 1H, PhCHO), 3.28 (q, J =
1.0, 3H, OCH3), 1.37 and 1.30 (two s, each 3H, CH3); for (S)-
(−)-11·MTPA-(R), 8.54 (d, J = 2.3, 1H, Hpyr), 8.48 (dd, J =
4.8, 2.3, 1H, Hpyr), 6.57 (td, J = 7.8, 1.5, 1H, Har), 6.32 (s, 1H,
PhCHO), 3.24 (q, J = 1.0, 3H, OCH3), 1.40 and 1.34 (two s,
each 3H, CH3).
(R)-1-(2-Fluorophenyl)-2-methyl-2-(pyridin-3-yl)propyl N-
[(S)-1-Phenylethyl]-N-[(S)-1-phenylethyl-carbamoyl]-
carbamate (11b). n-BuLi (0.060 mL, 0.096 mmol, 0.34 equiv,
1.6 M in hexanes) was added to a stirred solution of (+)-11
(0.069 g, 0.280 mmol) in dry THF (2 mL) under argon at −78
°C. After stirring for 10 min freshly distilled (S)-(−)-1-
phenylethyl isocyanate (0.080 mL, 0.56 mmol, 2 equiv) was
added slowly, and stirring was continued for 1 h at −78 °C.
Water (5 mL) was added at rt, and the mixture was stirred for
10 min before it was concentrated at reduced pressure. Water
and EtOAc (5 mL each) were added. The aqueous layer was
removed and extracted with EtOAc (2 × 5 mL). The three
combined organic layers were dried (Na2SO4) and solvents
removed on a rotary evaporator. The residue was flash-
chromatographed (hexanes/EtOAc, 1/1, Rf = 0.51) to give
carbamate 11b (0.072 g, 48%) as colorless crystals. Crystals
suitable for X-ray structure analysis were obtained by slow
evaporation of solvent from a solution in hexanes/DCM at +4
°C; mp 140−143 °C; [α]D20 −50.93 (c 0.49, acetone).
(d, JFC = 8.4, HCar), 125.94 (d, JFC = 13.2, Car), 123.47 (d, JFC
=
3.3, HCar), 122.45 (HCpyr), 115.01 (d, JFC = 23.0, HCar),
104.88 (OCHO), 88.80 (CHO), 75.48 (PhCHO), 52.67 (Cq),
48.11 (Cq), 47.08 (CH), 41.72 (Cq), 39.79 (CH), 32.08 (CH2),
26.09 (CH2), 25.68 (d, JFC = 2.1, PhC(CH3)2), 21.37
(PhC(CH3)2), 20.91 (CH3), 20.46 (CH2), 18.70 (CH3),
14.64 (CH3). IR (Si): νmax 2952, 1486, 1416, 1224, 1093,
1077, 1034, 1008, 990 cm−1. [α]2D0: +143.27 (c 1.07, acetone).
Diastereomer B. 1H NMR: δ 8.60 (d, J = 2.2, 1H, Hpyr), 8.43
(dd, J = 4.8, 1.7, 1H, Hpyr), 7.60 (ddd, J = 8.1, 2.3, 1.7, 1H,
Hpyr), 7.17 (dd, J = 8.1, 4.8, 1H, Hpyr), 7.17−7.10 (m, 1H, Har),
6.96−6.88 (m, 3H, Har), 5.03 (br. d, J = 3.3, 1H, OCHO), 4.81
(s, 1H, PhCHO), 3.29 (dd, J = 9,6, 1.3, 1H, CHO), 2.68−2.57
(m, 1H, CH(CH2)2), 1.68−1.60 (m, 2H, CH2(CH)2), 1.50−
1.35 (m, 3H), 1.35 (s, 3H, CH3), 1.31 (s, 3H, CH3), 1.27−1.18
(m, 1H), 1.02−0.93 (m, 1H), 0.77 (s, 3H, CH3), 0.73 (s, 3H,
CH3), 0.45 (s, 3H, CH3). 13C NMR: δ 159.87 (d, JFC = 244.5,
CF), 149.23 (Cpyr), 147.11 (HCpyr), 141.41 (Cpyr), 134.95
(HCpyr), 129.68 (d, JFC = 4.1, HCar), 128.37 (d, JFC = 8.4,
HCar), 128.12 (d, JFC = 13.7, Car), 122.91 (d, JFC = 3.2, HCar),
122.35 (HCar), 114.46 (d, JFC = 23.0, HCar), 109.70 (OCHO),
89.36 (CHO), 78.15 (PhCHO), 52.49 (Cq), 47.86 (Cq), 47.13
(CH), 42.07 (Cq), 39.76 (CH), 32.20 (CH2), 26.02 (CH2),
25.34 (ArC(CH3)2), 22.55 (ArC(CH3)2), 20.79 (CH3), 20.44
(CH2), 18.45 (CH3), 14.18 (CH3). IR (Si): ν
2927, 1485,
max
1413, 1388, 1222, 1186, 1099, 1077, 1030, 1007, 990, 954
cm−1. [α]D20 +52.55 (c 1.02, acetone). Anal. Calcd for
C27H34FNO2 (423.56): C, 76.56; H, 8.11; N, 3.31. Found: C,
76.52; H, 7.90; N, 3.28.
Deprotection of Diastereomers A and B To Get
Enantiomerically Pure (+)-11 and (−)-11, Respectively.
Further Syntheses. (R)-(+)-Methyl 1-[1-(4-Fluorophenyl)-
ethyl-1H-imidazole-5-carboxylate (24). Enantioselective re-
duction of 4-fluorophenyl methyl ketone with (R)-(+)-2-
methyl-oxazaborolidine/BH3·Me2S by a literature procedure40
gave 60% (S)-(−)-1-(4-fluorophenyl)ethanol, [α]2D0 −39.86 (c
1127
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2.23, acetone), ee 96% by (R)-Mosher ester. The ee of the
alcohol was increased to >99% by enantioselective esterifica-
tion41 with lipase Candida antarctica on acrylic resin (Sigma):
(S)-Alcohol of 96% ee (5 mmol), vinyl acetate (20 mmol),
tBuOMe (15 mL) and lipase (50 mg) were shaken for 18 h at
rt. The enzyme was removed and the filtrate was concentrated
on a rotary evaporator. The residue was purified by flash
chromatography (hexanes/EtOAc, 4/1) to yield 90% (S)-
alcohol of ee >99%, [α]2D0 −41.19 (c 2.18, acetone). The (R)-
Mosher esters27 of ( )- and (S)-alcohol were prepared and
137.99 (2 CHar), 135.96 (CHhet), 129.68 (CHhet), 127.84 (2
CHar), 117.76 (CHhet), 93.55 (CI), 56.03 (NCHPh), 21.84
(CH3). IR (Si): νmax 2980, 1488, 1404, 1226, 1079, 1006 cm−1.
Anal. Calcd for C11H11IN2 (298.12): C, 44.32; H, 3.72; N, 9.40.
Found: C, 44.34; H, 3.85; N, 9.17.
Binding Procedures. Whole membranes were prepared
from rat adrenals as described.8 Similarly, whole membranes
were prepared from the adrenal cortex of slaughterhouse bulls.
An aliquot of this preparation sufficient to obtain at least 0.3 mg
of original tissue per vial was added to robust polypropylene
vials (Biovials, Beckman Instruments) containing a solution of
12−18 nM [3H]metyrapol (12 Ci/mmol, Amersham) in 10
mM K2HPO4/HEPES (pH 7.2), 150 mM NaCl, with or
without various concentrations of test compounds, in a final
volume of 0.5 mL. Nonspecific binding was estimated in the
presence of 10 μM etomidate. Vials were left standing in icy
water (0 °C) for 20 min and centrifuged for 15 min at 40000g,
the supernatant was discarded, and the pellets were rinsed twice
(for a few seconds) with ice cold buffer. Finally, 1.8 mL of
toluene-based scintillation cocktail was added, the vials were
agitated for 15 min, and radiation was quantified in a beta
scintillation counter. Data were evaluated as described.8
Cortisol Secretion. Cortisol secretion by NCI-h295 cells
was estimated as described.8
1
investigated by H NMR spectroscopy; significant signals of
(R)-Mosher ester of (R)-alcohol [δ 3.44 (q, J = 1.2, 3H,
OCH3), 1.55 (d, J = 6.5, 3H, CH3)] and of (S)-alcohol [3.54
(q, J = 1.2, 3H, OCH3), 1.60 (d, J = 6.5, 3H, CH3)]. (S)-
Alcohol (0.167 g, 1.19 mmol, ee >99%) was transformed into
(R)-24 (0.165 g, 56%) as a colorless oil by a literature
procedure29 (−30 °C to rt in 7 h) except that flash
chromatography was performed with hexanes/Et2O/iPr2NH,
6/6/1, Rf = 0.31; ee ≥98% [by analytical HPLC on Chiracel
OD-H, 1 mL/min, iPrOH/hexanes, 7.5/92.5; (R)-24: tR 14.2
min, (S)-24: tR 12.0 min]; [α]2D0 +71.92 (c 1.3, acetone).
Similarly, ( )-24 was prepared in 60% yield from racemic
alcohol.
1H NMR: δ 7.75 (s, 1H, Him), 7.71 (s, 1H, Him), 7.18−7.11
(m, 2H, Har), 7.03−6.96 (m, 2H, Har), 6.31 (q, J = 7.1, 1H,
CHAr), 3.78 (s, 3H, OCH3), 1.83 (d, J = 7.1, 3H, CH3). 13C
NMR: δ 162.27 (d, J = 247.0, CF), 160.63 (CO), 139.58
(CHim), 138.27 (CHim), 136.93 (d, J = 2.1, Car), 127.99 (d, J =
8.4, 2CHar), 122.26 (Cim), 115.75 (d, J = 21.4, 2CHar), 54.72
(ArCH), 51.47 (OCH3), 22.27 (CH3). IR (Si): νmax 2951,
1717, 1512, 1363, 1221, 1133 cm−1. Anal. Calcd for
C13H13FN2O2 (248.25): C, 62.90; H, 5.28; N, 11.28. Found:
C, 63.06; H, 5.27; N, 10.99.
(R)-(−)-[1-Phenylethyl]-1H-imidazole (33). In a sealed tube
under argon, a solution of 25 (0.040 g, 0.16 mmol) in 3 mL of
dry ethanol with 1.5 M methylamine was heated for 18 h at 180
°C (oven temperature). After cooling, the solvents were
removed on a rotary evaporator. The residue was flash
chromatographed (EtOAc, N-substituted imidazole, Rf = 0.26;
N-methyl amide, Rf = 0.13) to give as a side product the N-
methyl amide 30 (7 mg, 19%) as a gum, and the desired N-
Inhibition of [14C]Corticosterone Formation. The
enzyme 11β-hydroxylase catalyzes the formation of [14C]-
corticosterone from [14C]DOC. The influence of several
metyrapone derivatives on the Vmax of this reaction was studied
as described.33,42 In brief, 0.5 μCi of [14C]DOC (NEN, 60
mCi/mmol, final concentration 13.6 μM) was incubated at rt
for 1−60 min in 80 mM Tris.HCl (pH 7.4), containing (final
concentrations): NADP (2.5 mM), glucose-6-phosphate (25
mM), glucose-6-phosphate dehydrogenase (Sigma; 1 unit), and
mitochondria prepared from beef adrenal cortex (0.35 mg
protein), in a final volume of 250 μL. For isolation of the
reaction product [14C]corticosterone, a 10 μL aliquot was
spotted (together with carriers) on a silica gel plate and
developed with benzene/Et2O/MeOH (40/58/2). Bands were
visualized under UV (Rf for corticosterone 0.17, for DOC
0.42), and radioactivity of the respective segments was
quantified in a liquid scintillation counter. In the absence of
inhibitors, Vmax was 0.136 μmol/min, and the KM was estimated
to 12.4 μM.
Crystallographic Structure Determination. X-ray dif-
fraction measurements for 11b were performed on a Bruker X8
APEXII CCD diffractometer. A single crystal was positioned at
35 mm from the detector, and 2542 frames were measured,
each for 30 s over 1° scan width. The data were processed using
SAINT software.43 The structures were solved by direct
methods and refined by full-matrix least-squares techniques.
Non-H atoms were refined with anisotropic displacement
parameters; H atoms were inserted in calculated positions and
refined with a riding model. The following programs were used:
structure solution, SHELXS-97;44 refinement, SHELXL-97;45
molecular diagrams, ORTEP-3;46 computer, Intel CoreDuo.
Crystal data, data collection parameters, and structure refine-
ment details for 11b are given in Table S1 in the Supporting
Information. Crystallographic data have been deposited at the
Cambridge Crystallographic Data Center with the number
CCDC 848059. A copy of the data can be obtained, free of
charge, on application to CCDC, 12 Union Road, Cambridge
CB2 1EZ, U.K. (deposit@ccdc.com.ac.uk).
substituted imidazole 33 (18 mg, 65%) as a liquid: [α]D20
=
−5.95 (c 1.66, acetone); [α]2D0 −4.62 (c 1.45, CHCl3) lit.29
1
[α]2D0 +5.20 (c 3.84, CHCl3) for (S)-(+)-enantiomer. The H
NMR data were identical with those in the literature.30
13C NMR: δ 141.46 (Car), 136.01 (CHhet), 129.29 (CHhet),
128.84 (2 CHar), 128.02 (CHar), 125.92 (2 CHar), 117.88
(CHhet), 56.51 (NCHPh), 21.95 (CH3). IR (Si): νmax 2981,
1496, 1455, 1226, 1082, 1018 cm−1.
(R)-(+)-[1-(4-Iodophenyl)ethyl]-1H-imidazole (34). In a
sealed tube under argon, a solution of 20 (0.178 g, 0.5
mmol) in 3 mL of dry ethanol with 1.5 M methylamine was
heated for 24 h at 180 °C (oven temperature). After cooling,
the solvents were removed on a rotary evaporator. The residue
was flash chromatographed (EtOAc/EtOH 10/1, 34, Rf = 0.30;
N-methyl amide, Rf = 0.24) to give as a side product the N-
methyl amide (11 mg, 6%) and 34 (128 mg, 86%) as an oil,
which solidified after bulb to bulb distillation (bp 120 °C/0.1
mbar); mp 36−38 °C; [α]2D0 +5.91 (c 2.3, acetone).
1H NMR (400.13 MHz, CDCl3): δ 7.64 (m, 2H, Har), 7.55
(t, J = 1.1 Hz, 1H, Hhet), 7.06 (t, J = 1.1 Hz, 1H, Hhet), 6.87 (t, J
= 1.1 Hz, 1H, Hhet), 6.85 (m, 2H, Har), 5.27 (q, J = 7.1, 1H,
NCH), 1.81 (d, J = 7.1, 3H, CH3). 13C NMR: δ 141.32 (Car),
1128
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expression in aldosterone-producing adenomas. Eur. J. Endocrinol.
2002, 147, 795−802.
ASSOCIATED CONTENT
* Supporting Information
Syntheses of compounds 6−9 and of their precursors and table
of crystal data and details of data collection of compound 11b.
This material is available free of charge via the Internet at
http://pubs.acs.org.
■
S
(10) Ye, P.; Kenyon, C. J.; MacKenzie, S. M.; Jong, A. S.; Miller, C.;
Gray, G. A.; Wallace, A.; Ryding, A. S.; Mullins, J. J.; McBride, M. W.;
Graham, D.; Fraser, R.; Connell, J. M. C.; Davies, E. The aldosterone
synthase (CYP11B2) and 11β-hydroxylase (CYP11B1) genes are not
expressed in the rat heart. Endocrinology 2005, 146, 5287−5293.
(11) Johansson, M. K.; Sanderson, J. T.; Lund, B.-O. Effects of 3-
MeSO2-DDE and some CYP inhibitors on glucocorticoid steroido-
genesis in the H295R human adrenocortical carcinoma cell line.
Toxicol. In Vitro 2002, 16, 113−121.
AUTHOR INFORMATION
Corresponding Author
*Tel: (+431) 40160 34081 (M.L.B.); (+431) 4277 52105
(F.H.). E-mail: michael.berger@meduniwien.ac.at (M.L.B.);
friedrich.hammerschmidt@univie.ac.at (F.H.).
■
(12) Tobes, M. C.; Hays, S. J.; Gildersleeve, D. L.; Wieland, D. M.;
Beierwaltes, W. H. Adrenal cortical 11β-hydroxylase and side-chain
cleavage enzymes. Requirement for the A- or B-pyridyl ring in
metyrapone for inhibition. J. Steroid Biochem. 1985, 22, 103−110.
(13) Turpeinen, M.; Nieminen, R.; Juntunen, T.; Taavitsainen, P.;
Raunio, H.; Pelkonen, O. Selective inhibition of CYP2B6-catalized
bupropion hydroxylation in human liver microsomes in vitro. Drug
Metab. Dispos. 2004, 32, 626−631.
(14) Pavek, P.; Dvorak, Z. Xenobiotic-induced transcriptional
regulation of xenobiotic metabolizing enzymes of the cytochrome
P450 superfamily in human extrahepatic tissues. Curr. Drug Metabol.
2008, 9, 129−143.
(15) Morton, T. H. Neutral products from gas phase rearrangements
of simple carbocations. Adv. Gas-Phase Ion Chem. 2001, 4, 213−256.
(16) Schirbel, A.; Zolle, I.; Hammerschmidt, F.; Berger, M. L.;
Schiller, D.; Kvaternik, H.; Reiners, Chr. [123/131I]Iodometomidate as a
radioligand for functional diagnosis of adrenal disease: synthesis,
structural requirements and biodistribution. Radiochim. Acta 2004, 92,
297−303.
(17) Robien, W.; Zolle, I. Synthesis of radioiodinated metyrapone −
a potential adrenal imaging agent. Int. J. Appl. Radiat. Isot. 1983, 34,
907−914.
Present Address
○Central Laboratory, Peking University Shenzhen Hospital,
China.
Notes
The authors declare no competing financial interest.
¶Deceased.
ACKNOWLEDGMENTS
Parts of this study had been supported by the Osterreichische
■
̈
Nationalbank, Jubilaumsfondsprojekt 8680 and by the
̈
Wilhelm-Sander-Stiftung (Grant 2003.175.1 to S.H. and A.S.).
We thank S. Felsinger and P. Unteregger for recording the
NMR and MS spectra, respectively, and S. Schneider and J.
Theiner for performing the HPLC and combustion analyses,
respectively. The following students contributed to the binding
data: Gunar Stemer, Matthias Gassner, Sybille Gruber, Isabella
Pungel, Michael Papik, Corina Gruner, Jurgen Baldinger. We
also are indebted to Alexander Roller for X-ray data collection.
̈
̈
̈
(18) Hays, S.; Tobes, M. C.; Gildersleeve, D. L.; Wieland, D. M.;
Beierwaltes, W. H. Structure-activity relationship study of the
inhibition of adrenal cortical 11β-hydroxylase by new metyrapone
analogues. J. Med. Chem. 1984, 27, 15−19.
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