New selective inhibitors of steroid 11β-hydroxylation in the adrenal cortex. Synthesis and structure-activity relationship of potent etomidate analogues

Article abstract of DOI:10.1021/jm800012w

Derivatives of etomidate were evaluated as inhibitors of adrenal steroid 11β-hydroxylations. Stereoselective coupling by Mitsunobu produced chirally pure analogues to study the effect of configuration, modification of the ester, and substitution in the phenyl ring, with the aim to probe specific sites for introducing a radionuclide. Iodophenyl metomidate (IMTO) labeled with iodine-131 served as radioligand for structure-affinity relationship studies. We have characterized the kinetic parameters of specific ¹³¹I-IMTO binding on rat adrenal membranes and used the displacement of ¹³¹I-IMTO binding to evaluate functionalized MTO analogues. Our results indicated that (1) (R)-configuration is essential for high affinity, (2) highest potency resides in the ethyl, 2-propyl, and 2-fluoroethyl esters, and (3) substitution of the phenyl ring is well tolerated. The clinically used inhibitors metyrapone and ketoconazole inhibited ¹³¹I-IMTO binding with low affinity. Incubation of selected analogues with human adrenocortical NCI-h295 cells demonstrated a high correlation with the inhibitory effect on cortisol secretion.

Full text of DOI:10.1021/jm800012w

2244
J. Med. Chem. 2008, 51, 2244–2253
New Selective Inhibitors of Steroid 11ꢀ-Hydroxylation in the Adrenal Cortex. Synthesis and
Structure–Activity Relationship of Potent Etomidate Analogues
Ilse M. Zolle,*,† Michael L. Berger,‡ Friedrich Hammerschmidt,§ Stefanie Hahner,# Andreas Schirbel, and Biljana Peric-Simov§
Department of Medicinal/Pharmaceutical Chemistry and Institute of Organic Chemistry, UniVersity of Vienna, 1090 Vienna, Austria, Center for
Brain Research, Medical UniVersity of Vienna, 1090 Vienna, Austria, and Department of Medicine and Department of Nuclear Medicine,
UniVersity of Wuerzburg, 97080 Wuerzburg, Germany
ReceiVed March 2, 2007
Derivatives of etomidate were evaluated as inhibitors of adrenal steroid 11ꢀ-hydroxylations. Stereoselective
coupling by Mitsunobu produced chirally pure analogues to study the effect of configuration, modification
of the ester, and substitution in the phenyl ring, with the aim to probe specific sites for introducing a
radionuclide. Iodophenyl metomidate (IMTO) labeled with iodine-131 served as radioligand for structure-affinity
relationship studies. We have characterized the kinetic parameters of specific 131I-IMTO binding on rat
adrenal membranes and used the displacement of 131I-IMTO binding to evaluate functionalized MTO
analogues. Our results indicated that (1) (R)-configuration is essential for high affinity, (2) highest potency
resides in the ethyl, 2-propyl, and 2-fluoroethyl esters, and (3) substitution of the phenyl ring is well tolerated.
The clinically used inhibitors metyrapone and ketoconazole inhibited 131I-IMTO binding with low affinity.
Incubation of selected analogues with human adrenocortical NCI-h295 cells demonstrated a high correlation
with the inhibitory effect on cortisol secretion.
Introduction
of metyrapone were evaluated by structure–activity relationship
(SAR)studies12–15 asprecursorsforlabelingwithradionuclides.16–22
The reduced derivative 3H-metyrapol has been used as a
radiotracer for the evaluation of metyrapone derivatives.18,23,24
High-affinity binding of P-450c11 inhibitors to adrenal
membranes permits the application of in vitro binding proce-
dures. Recently, we have introduced 131I-IMTO to characterize
high-affinity binding sites on crude membranes prepared from
whole rat adrenals.25,26 Derivatives of etomidate and of
metyrapone were investigated as displacers of specific radioli-
gand binding using 131I-IMTO and 3H-metyrapol, respectively.24
Analogues of etomidate displaced both radioligands with similar
potencies, whereas metyrapol and structurally related compounds
inhibited 131I-IMTO binding with low affinity (IC50 in micro-
molar range). ETO (1) and MTO (2) were identified as
considerably more potent inhibitors of P-450c11 than metyrapone
and metyrapol.24 Selected correlates were also tested as inhibi-
tors of cortisol secretion in human adrenocortical NCI-h295 cells
supporting the validity of SAR data.24
Etomidate (R)-1-(1-phenylethyl)-1H-imidazole-5-carboxylic
acid ethyl ester (ETOa) (1) and the methyl ester metomidate
(2) are short-acting anesthetic drugs with an adrenostatic side
effect, producing low levels of plasma cortisol after long-term
infusion.1,2 Investigations of the effects of 1 on the cytochrome
P-450 species and on steroid biosynthesis in adrenal cortex
mitochondria revealed that 1 interacts selectively with the
mitochondrial cytochrome P-450 species affecting mitochondrial
11ꢀ-hydroxylations exclusively.3 Metomidate is listed in the
European Pharmacopeia as an inherent impurity of 1.4
Another inhibitor of cytochrome P-450 (P-450c11) is
metyrapone (2-methyl-1,2-bis-(3-pyridyl)-1-propanone), which
binds specifically to the inner mitochondrial membrane.5 Several
investigators have shown that the steroid 11ꢀ-hydroxylation
system and the cytochrome P-450 are located in the inner
mitochondrial membrane of adrenal cortex mitochondria.6–8 The
fact that deoxycorticosterone (DOC) hydroxylation is sensitive
to metyrapone was another strong argument that the high-affinity
binding site for metyrapone is located on the P-450 specific for
11ꢀ-hydroxylations.9,10 Clinically, specific binding at the site
of steroid 11ꢀ-hydroxylation is associated with the inhibition
of cortisol secretion. Metyrapone and etomidate are highly
selective drugs for the treatment of hypercortisolemia.2,11
Clinical application of adrenocortical enzyme inhibitors has
stimulated their investigation as radiotracers. Several derivatives
131I-IMTO was used as a radioligand to characterize high-
affinity binding sites on crude membranes prepared from whole
rat adrenals. A comparison of IC50 values obtained by the
displacement of specifically bound 131I-IMTO by structurally
related compounds offered insight into the structural require-
ments for high-affinity binding in vivo.
The superior binding characteristics of ETO (1) and MTO
(2) as adrenocortical enzyme inhibitors have promoted the
development of radiolabeled derivatives. 11C-metomidate was
introduced as the first carbon-11 labeled radiotracer for PET
investigations of the adrenal cortex and its tumors.27,28 Subse-
quent developments include 18F-FETO29 for PET and 123I-
IMTO26 for SPECT investigations.
Here, we describe the synthesis and biochemical evaluation
of a series of analogues of ETO (1) and MTO (2) and present
quantitative data on SAR studies. Derivatives reported previ-
ously had been obtained as racemic mixtures.30 The adoption
of the Mitsunobu reaction,31 which is based on coupling a chiral
precursor alcohol with commercially available methyl 1H-
* To whom correspondence should be addressed. Phone/fax: +43-1-405
71 98. E-mail: ilse.zolle@univie.ac.at.
†
Department of Medicinal/Pharmaceutical Chemistry, University of
Vienna.
‡
Medical University of Vienna.
Institute of Organic Chemistry, University of Vienna.
Department of Medicine, University of Wuerzburg.
§
#
Department of Nuclear Medicine, University of Wuerzburg.
a Abbreviations: ETO, etomidate; MTO, metomidate; IMTO, 4-iodophe-
nyl metomidate; FETO, fluoroetomidate; ETO acid, etomidate free acid;
DOC, deoxycorticosterone; SPECT, single photon emission computed
tomography; PET, positron emission tomography; P-450c11, cytochrome
P-450 11ꢀ-hydroxylase; NCI-h295, human adrenocortical cancer cell line.
10.1021/jm800012w CCC: $40.75
2008 American Chemical Society
Published on Web 03/19/2008

Etomidate Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2245
Scheme 1. Modification of the Ester Function (2, 3a, 3b)
approach, with yields varying between 25% and 67%. However,
under strictly controlled reaction conditions, racemization was
prevented and alkylation occurred selectively at N-1 with
inversion of configuration. Secondary alcohols with (S)-con-
figuration yielded derivatives of physiologically more active (R)-
MTO, whereas for the preparation of (S)-MTO (10b), the (R)-
configured alcohol (9b) was reacted. Enantiomeric excesses for
the substituted imidazoles agreed with those of the alcohols used
as starting materials, documenting inversion of configuration.36
For the synthesis of derivatives of 1 and 2 substituted in the
phenyl ring in position 3 or 4 (10g-k), synthesis started from
the 3- and 4-substituted phenylethanol, respectively, to produce
(S)-alcohols36 (9g-k), followed by Mitsunobu coupling, shown
in Scheme 5. 4-Br-MTO (10k) was obtained by halodestanny-
lation of the 4-trimethylstannyl-MTO precursor with bromine.36
The polar hydroxymethyl substituent of 11 was introduced by
palladium-catalyzed formylation of 4-iodo-MTO26,36 (10j) with
carbon monoxide and tributyltin hydride (yield 64%), followed
by reduction of the aldehyde with NaBH4 in 85% yield.39
The synthesis of I-ETO (10i) may serve as an example for
coupling (S)-configured 1-(4-iodophenyl)ethanol (9i) with ethyl
1H-imidazole-5-carboxylate by the Mitsunobu reaction (see
Experimental Procedures). For this purpose, racemic 4-iodo-
phenylethanol was transformed to a racemic ester (1-(4-
iodophenyl)ethyl chloroacetate),36 which was resolved by lipase
SAM II, an enzyme known to hydrolyze (R)-esters of secondary
benzylic alcohols.37 Transesterification of the isolated (S)-ester
yielded (9i).36
Scheme 2. Synthesis of the 2-Fluoroethyl Ester (5) and the
N-Methylamide (6)
imidazole-5-carboxylate, offered access to chiral derivatives
modified at the active center and substituted in the phenyl ring.
Modified esters were generally obtained by transesterification
of commercially available etomidate.
Synthesis of Radioligand. The radioligand 131I-IMTO was
derived from (R)-IMTO (10j), which was converted to the
4-trimethylstannyl-MTO precursor.26,36 The precursor facilitates
labeling at room temperature, producing 131I-IMTO with high
specific activity. The product was obtained with high RCY, high
radiochemical purity, and high in vitro stability (Table 1). 131I-
IMTO was formulated in 1 mL of saline, and in vitro stability
was analyzed by HPLC, showing a slow linear loss of
radioiodine with time.
Chemistry
Synthesis of MTO Analogues. Ester derivatives included
the methyl ester (2) and the n-propyl and 2-propyl esters.
Transesterification of commercially available (R)-enantiomer 1
at ambient temperature in dry methanol, n-propanol, or 2-pro-
panol in the presence of the corresponding sodium alkoxide
yielded 2 and the n-propyl32 and 2-propyl esters (3a and 3b),
in yields of 71–80% (Scheme 1).
The 2-fluoroethyl ester (5) was prepared from ETO acid (4)
(derived from 1) and 2-fluoroethanol using triphenylphosphine
(Ph3P) and di-tert-butyl azodicarboxylate (DtBAD) in toluene
(Scheme 2).31,33
Results and Discussion
Binding of MTO Analogues. Kinetics of 131I-IMTO
Binding. In pilot studies the association of 131I-IMTO to adrenal
membranes was followed for 75 min, using the original
radioligand solution without addition of carrier (final concentra-
tion 0.1 nM). Specific binding reached a high maximum after
10–20 min that decreased steadily at later time points; i.e., no
stable saturation was reached. If the radioligand concentration
was increased by addition of carrier IMTO (10j) to a total
concentration of 2 nM, a stable plateau was reached within 10
min (Figure 1a). Thus, a small amount of 131I-IMTO was
progressively lost during the incubation process, presumably
because of degradation, uptake, or nonspecific binding to
surfaces. This small loss could be neglected if the total
concentration was chosen to be sufficiently high.
In an attempt to increase the in vivo stability of MTO
analogues, the N-methylamide (6) and its N-methoxy derivative
(7) were synthesized. The methyl ketone (8) was also considered
as a suitable replacement.
Synthesis of 6 started from ETO acid (4), which was
converted to the acid chloride30 and then reacted with excess
methylamine in THF (Scheme 2). Reaction of the acid chloride
with N,O-dimethylhydroxylamine in pyridine yielded crystalline
Weinreb amide (7), formed in 57% yield, which was trans-
formed to the methyl ketone (8) by adding CH3Li at low
temperature.34 The reaction sequence is shown in Scheme 3.
Modifications Affecting Chirality and the Substitution
of the Phenyl Ring. Derivatives with different chirality (10a-f)
and also with various substituents in the phenyl ring (10g-k)
were produced by enantioselective synthesis (Scheme 4). The
Mitsunobu reaction is based on coupling a chiral benzylic
alcohol(9a-k)withmethyl1H-imidazole-5-carboxylate.26,31,33,35,36
Two of these (9a and 9d) are achiral. The chiral precursor
alcohols were obtained separately with high enantiomeric
excesses (95–99%) by lipase-catalyzed enantioselective hy-
drolysis of their racemic esters37,38 and by preparative HPLC
on a chiral stationary phase in the case of (()-9h. Mitsunobu
coupling of chiral alcohols with commercially available methyl
1H-imidazole-5-carboxylate offered a direct and stereoselective
The association time course of 131I-IMTO to rat adrenal
membranes is given in Figure 1a (data pooled from seven
experiments). Computerized curve fitting to the association
function B(t) ) B0[1 - exp (-νt)] resulted in an observable
association rate constant ν of 0.65 ( 0.06 min-1 ((SD) at a
ligand concentration L ) 2 nM, corresponding to an association
half-time of 1.1 min. For dissociation experiments, membranes
were first fully equilibrated with 2 nM radioligand, and
dissociation was initiated by the addition of excess unlabeled
IMTO and stepwise filtration at 15 s intervals. Data pooled from
four experiments (Figure 1b) demonstrated fast reversibility of

2246 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Zolle et al.
Scheme 3. Synthesis of the N-Methoxyamide (7) and the Methyl Ketone (8)
Scheme 4. Enantioselective Synthesis of N-1 Derivatives Affecting the 1-Phenylethyl Moiety (10a-k)a
a Synthesis of 10i shown in Scheme 5.
Scheme 5. Enantioselective Synthesis of (R)-4-Iodophenyl
Etomidate (10i)
binding and resulted in a dissociation constant b ) 0.36 ( 0.03
Figure 1. Association (a) and dissociation kinetics (b) of total 131I-
IMTO binding to rat adrenal membranes. Data are the mean ( SD
pooled from four experiments performed in duplicate. Dotted lines
follow the best fit functions, and dotted horizontal lines indicate
nonspecific binding.
min-1 ((SD) and a derived dissociation half-time of 1.93 min.
Once b is known, the true association rate constant (a) can be
calculated from the relationship ν ) La + b, giving a ) 0.10
min-1 nM-1. Thus, a first rough estimate for the dissociation
equilibrium constant KD was obtained from KD ) b/a ) (0.36
min-1)/(0.10 min-1 nM-1) ) 3.6 nM. A more reliable KD value
should be accessible by saturation analysis at equilibrium
conditions.
(a) and reciprocal plots (b), the linearity of the latter suggesting
a single class of binding sites. As a mean of eight analyses, we
obtained KD ) 11.6 ( 2.5 nM and Bmax ) 12.1 ( 0.4 pmol/mg
tissue. This KD value appears to be more reliable than the
estimate from the rate constants.
For saturation analysis, adrenal membranes were incubated
with five concentrations of 131I-IMTO ranging from 2 to 200
nM for 20–30 min. At the lowest concentration, 0.06 mg of
tissue bound approximately 10% of the free ligand (∼0.1 pmol).
Figure 2 gives representative examples for saturation isotherms
Displacement of 131I-IMTO Binding by MTO
Analogues. SAR studies of MTO analogues are based on the
high affinity of the radiotracer 131I-IMTO. Test compounds were
evaluated by the displacement of specific 131I-IMTO binding
on rat adrenal membranes; the concentration of the radioligand
was 2 nM. All effective inhibitors of 131I-IMTO binding yielded
monophasic displacement curves, with steepness close to unity
(nH ∼1.0). Figure 3 shows displacement curves for potent
P-450c11 inhibitors ETO (1), I-ETO (10i), and FETO (5), in
comparison with metyrapol.
Table 1. Characteristics of 131I-IMTO
radiochemical yield
radiochemical purity
specific activity
concentration
in vitro stability (dec rate)
93.7 ( 1.2% (15)
97.3 ( 1.3% (15)
50 GBq/µmol
30–160 MBq/mL
0.27%/h

Etomidate Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2247
Table 3. Inhibition of Specific 131I-IMTO Binding on Rat Adrenal
Membranes by Analogues with Modifications of the Estera
Figure 2. Dependence of specific 131I-IMTO binding (“bound”) on
free 131I-IMTO concentration (“free”), with two representative examples
of eight experiments performed in duplicate: (a) saturation isotherms;
(b) reciprocal transformations (Eadie-Hofstee plots). In most experi-
ments, reciprocal transformation resulted in linear correlations.
a IC50 and Hill coefficients (nH) expressed as mean values ( SD (n,
number of experiments). If n ) 2, both values are indicated. (/) Significantly
more potent than other compounds shown, p < 0.05. (//) Significantly
less potent than other compounds shown, p < 0.001. (///) Practically
inactive. n.d.: not determined (nH was fixed to 1.0 for computer analysis).
Figure 3. Inhibition of specific 131I-IMTO binding by 1 (ETO, circles),
10i (4-I-ETO, squares), 5 (FETO, triangles), and metyrapol (diamonds).
Data were pooled from 4 to 11 experiments. Dotted graphs were
constructed from the parameters given in Tables 2, 3, and 5.
Table 4. Inhibition of Specific 131I-IMTO Binding to Rat Adrenal
Membranes by MTO Analogues with Modifications at the Chiral
Centera
Table 2. Inhibition of Specific 131I-IMTO Binding to Rat Adrenal
Membranes by ETO (1) and MTO (2) Analogues with Modifications of
the Phenyl Ringa
compd
R
X
IC50 (nM) (SD
n
nH
0.92
0.90, 0.85
0.88
0.83, 067
1.07, 1.01
0.80
(SD
n
2
(R)-CH3 CH 3.69
(1.92
(10.9
(281
(0.96
(240
(3.81
(0.70
6
4
4
3
3
4
3
(0.24
4
2
4
2
2
4
3
10a
10b
10c
10d
10e
10f
H
CH 28.8**
CH 492***
compd
R
R′
X
IC50 (nM) (SD
n
nH
(SD
n
(S)-CH3
(R)-C2H5 CH 6.23*
(0.18
1
2
C2H5
CH3
CH3
CH3 4-CH3
CH3 3-I
C2H5 4-I
CH3 4-I
CH3 4-Br
CH3 4-CH2OH CH
CH
CH
N
CH
CH
CH
CH
CH
1.08* (0.42 11 0.86 (0.16
6
3.69
(1.92
6
4
3
4
5
0.92 (0. 24
0.80 (0.16
0.73 (0.05
0.84 (0.04
1.17 (0.21
4
4
3
3
4
H
N
N
870***
20.7**
3.26
10e
10g
10h
10i
10j
10k
11
20.7** (3.81
(R)-CH3
1-indanyl
(0.16
(0.17
3.58
4.44
4.37
8.98
8.95
3.29
(1.03
(1.78
(1.15
0.91
a IC50 and Hill coefficients (nH) expressed as mean values ( SD (n,
number of experiments). If n ) 2, both values are indicated. (/) Significantly
less potent than 2, p < 0.05. (//) Significantly less potent than 2, p <
0.01. (///) Significantly less potent than all other compounds shown.
(3.72 15 1.04 (0.26 13
(0.48
(0.90
3
4
0.90 (0.11
0.74 (0.18
3
4
a IC50 and Hill coefficients (nH) expressed as mean values ( SD (n,
number of experiments). (/) Significantly more potent than all other
compounds in the table, p < 0.01. (//) Significantly less potent than other
compounds shown, p < 0.001.
experiments indicated that substitution with iodine in position
4 (para) resulted in a slight decrease in potency, whereas iodine
in position 3 (meta) was tolerated (compare 2 and 10h). In
contrast, 4-methyl-MTO (10g), and 4-hydroxymethyl-MTO (11)
showed no negative effect of substitution. Thus, the slight shift
in potency of 4-I-MTO (10j), also observed in case of (10i), is
unlikely to be due to steric hindrance. Replacing the phenyl
ring by pyridine yielded 3-pyridyl-MTO (10e), with slightly
reduced potency.
The effect of phenyl substitution was studied in detail in order
to facilitate regioselective labeling with radiohalogen (Table 2,
Figure 4). The inhibitory potencies of halogenated derivatives
3-I-MTO (10h), 4-I-MTO (10j), and 4-Br-MTO (10k) were
compared with MTO (2). Statistical tests based on several

2248 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Zolle et al.
Figure 4. Displacement of specific 131I-IMTO binding by 2 (MTO,
dotted line; n ) 6) and 10j (I-MTO,full line; n ) 13). Lines follow
best fit functions.
Table 5. Inhibition of Specific 131I-IMTO Binding on Rat Adrenal
Membranes by Compounds Known To Interact with Steroid
Hydroxylationa
inhibitor
ketoconazole
11-deoxycorticosterone 0.89
IC50 (µM)
(SD
n
nH
(SD
n
Figure 5. Correlation of log IC50 values obtained by the inhibition of
cortisol secretion and by 131I-IMTO displacement. The dotted line
indicates identity (R ) 0.954, p < 0.001).
0.71
(0.49
(0.33
(0.39
(0.79
7
5
7
5
1
2
1.19 (0.22
0.86 (0.16
0.88 (0.13
0.85 (0.12
n.d.
6
4
5
3
metyrapol
1.02
1.16
82.9
137, 142
were derived from racemic mixtures.30 Differences in hypnotic
potency among various esters were minor, and yet the presence
of the ester moiety was essential.
metyrapone
aminoglutethimide
proadifen
a IC50 and Hill coefficients (nH) expressed as mean values ( SD (n,
number of experiments). If n ) 2, both values are indicated. n.d.: not
determined (nH was fixed to 1.0 for computer analysis).
n.d.
It is of considerable interest that the structural requirements
of ETO analogues postulated for sustaining hypnotic activity
correlated well with our quantitative SAR data based on their
inhibitory potencies as competitive displacers of 131I-IMTO
binding. Ester modification had almost no effect, yet hydrolysis
of the ester abolished inhibitory potency (Table 3). Chirality
determined the potency of selected substituents at the carbon
bridge (Table 4). Studies with intact adrenocortical mitochondria
reported previously have also demonstrated that the interaction
of etomidate with cytochrome P-450c11 was highly selective
and stereospecific.3 Both biological functions of etomidate
(inhibition of P-450c11 and hypnotic activity) require the (R)-
configuration.
Studies of the effect of ester size on the binding potency
demonstrated that the ethyl and 2-propyl esters (1 and 3b),
showed the highest potency, followed by the n-propyl (3a),
2-fluoroethyl (5), and methyl ester (2) (Table 3, Figure 3). The
methyl ester was chosen as a radioligand. Further structural
modifications at position 5 of the imidazole ring were performed
to control enzymatic hydrolysis in vivo. The ester was replaced
by amide (6) and by methyl ketone (8), resulting in a
considerable loss of affinity. Additional substitution of 6 with
N-methoxy partly restored affinity (7). However, amide nitrogen
in this position of the molecule was considered unfavorable.
Thus, attempts to increase the in vivo stability by replacing the
ester had a negative effect on the binding potency. Hydrolysis
of the ester resulted in a total loss of inhibitory potency (4)
(Table 3).
Derivatives listed in Table 4 differ in their geometries at their
chiral centers. The potency of (S)-MTO (10b) was found to be
2 orders of magnitude weaker than that of (R)-MTO (2). If the
methyl substituent at the center of chirality was omitted (10a),
the effect was moderate and additional replacement of phenyl
by pyridyl caused a drop in potency (10d and 10e). Replacing
methyl by ethyl did not affect potency (10c). Integrating ethyl
into a bicyclic indanyl system40 was well tolerated, on the basis
of IC50 values (2 and 10f).
The importance of a one-carbon bridge with alkyl branching
between N-1 of imidazole and the phenyl ring had been
emphasized for sustaining the hypnotic activity of etomidate
derivatives.30 Lengthening of the side chain to include two
carbon atoms, with or without branching, or omission of
branching or direct attachment of the aryl group to the nitrogen
resulted in a total loss of hypnotic properties.30 Omission of
the alkyl substituent described as compound 10a in this study
showed reduced potency. These early investigations of the
structural requirements of ETO analogues as hypnotic agents
led to the conclusion that the nature of the N-substituent was
critical, anticipating the influence of chirality, although results
Besides the structurally derived analogues of MTO, other
compounds known to suppress P-450c11 activity were also
tested (Table 5.) These included metyrapone, metyrapol, and
ketoconazole, with IC50 values in the micromolar range (Figure
4).41 The natural substrate 11-deoxycorticosterone displaced
specific 131I-IMTO binding at low micromolar concentrations.
Very weak inhibition of 131I-IMTO binding was observed with
4-aminoglutethimide (an inhibitor of 20R-hydroxylation of
cholesterol) and with proadifen (an inhibitor of oxidative drug
metabolism). Despite the fact that metyrapone is known to bind
to cytochrome P-450 enzymes in adrenocortical mitochondria
and in liver microsomes3 and that ketoconazole is an inhibitor
of many cytochrome P-450 enzyme reactions42 including steroid
11ꢀ-hydroxylations,43 displacement of specific 131I-IMTO bind-
ing by these inhibitors and by DOC strongly suggested that
P-450c11 is the site of interaction.
Direct Measurement of Cortisol Secretion. A comparison
of the inhibitory potencies obtained by radioligand displacement
with measurements of the inhibitory effect of etomidate and
selected derivatives on cortisol secretion is shown in Figure 5.
The corresponding IC50 values subjected to regression analysis
are presented in Table 6. Direct measurements of cortisol
secretion in a living cell culture correlated well with the
inhibitory potencies of analogues observed by 131I-IMTO
displacement.

Etomidate Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2249
Table 6. Comparison of the Inhibitory Potency of ETO Derivatives Obtained by Displacement of 131I-IMTO Binding and by Inhibition of Cortisol
Secretion
displacement of 131I-IMTO
IC50(nM) log IC50
0.033
inhibition of cortisol secretiona
IC50(nM) log IC50
0.66
compd
inhibitor
1
2
10b
10a
10e
10j
5
6
7
8
(R)-etomidate
1.08 ( 0.42
3.69 ( 1.92
492 ( 281
28.8 ( 10.9
20.7 ( 3.81
9.0 ( 3.72
2.9 ( 0.55
215 ( 91
4.57 ( 1.41
13.9 ( 10.2
592.9
(R)-metomidate
(S)-metomidate
demethyl-MTO
3-pyridyl-MTO
4-iodo-MTO
2-fluoroethyl ester
N-methylamide
N-methoxyamide
methyl ketone
0.57
2.69
1.46
1.32
0.954
0.46
2.33
1.18
1.78
1.14
2.77
1.51
1.72
0.84
1.11
2.21
1.45
1.96
32.1
52.2
6.95
8.92, 17.1
135.3, 191
19.5, 37.0
59.0, 125
16.7; 14.0
59.8; 62.1
a Maximum inhibition exceeded 90%.
Clinical Application. Etomidate is known as a short-acting
hypnotic used parenterally for the induction and maintenance
of anesthesia and for sedation. Prolonged sedation, however,
revealed a potent side effect of this drug as an inhibitor of
adrenal steroidogenesis.44,45 The intravenous induction dose for
anesthesia in adults was reported as 0.3 mg/kg, whereas adrenal
suppression has been observed with doses of 0.04 mg/kg.46
Etomidate potentiates the action of GABA at the GABAA
receptor complex at concentrations around 1 µM,47,48 whereas
its affinity for 131I-IMTO binding sites on adrenal membranes
lies around 1 nM (this study). Very weak uptake of radioactivity
in the brain was observed after injection of 131I-IMTO for
imaging, indicating that binding of trace amounts of IMTO to
GABA receptors was insignificant.49
ment of specific 131I-IMTO binding demonstrated that the
inhibitory potency resided exclusively in the (R)-configured
analogues and that the intact ester was essential for binding.
Detailed SAR studies revealed that none of the esters or
substituents in the phenyl ring affected binding affinity.
Compounds known to suppress adrenocortical P-450c11, e.g.,
metyrapone and ketoconazole, were also tested. Since specific
binding of 131I-IMTO was inhibited by concentrations of
metyrapone and metyrapol known to block P-450c11 (more
specifically so than ketoconazole) and also by low micromolar
concentrations of the natural substrate DOC, the most likely
target of specific 131I-IMTO binding was the enzyme P-450c11.
Additionally, measurements based on steroid secretion in a living
cell culture indicated a dose-dependent inhibition of 11ꢀ-
hydroxylations. The inhibitory potency of selected etomidate
derivatives measured by cortisol secretion and by the displace-
ment of 131I-IMTO binding showed a high correlation and
provided further evidence that specific binding of inhibitors
occurred at the site of cortisol synthesis. Infact, analogues of
ETO (1) and MTO (2) showing high binding affinity, have been
identified as potent inhibitors of cortisol secretion.1–3,24,45,46,49
Derivatization offered valuable guidance for precursor syn-
thesis. New synthetic methods have been developed to label
MTO analogues with high specific activity,26,27,29 which is a
prerequisite for receptor imaging. Results further indicated that
halogenation of the phenyl ring may offer considerable versatil-
ity for labeling with SPECT and PET radionuclides (i.e., 123I,
124I, 76Br, 18F). Some (R)-configured substituents might fulfill
the strict steric requirements for introducing a radioactive label
at the chiral center (see Table 4). Labeled derivatives may also
gain importance for in depth evaluation of the pharmacology
of this versatile molecule as a potent P-450c11 enzyme inhibitor
and as a valued anesthetic.
Labeled etomidate derivatives have shown excellent charac-
teristics as adrenal imaging agents.26,28,50 These properties are
based on high-affinity binding on tissue rich in P-450c11 enzyme
activity and on excellent selectivity, restricting specific uptake
to adrenocortical tissue.
Ample experience with 11C-metomidate has been documented
in patients with adrenal pathology,51–54 and preliminary results
with 18F-FETO were obtained in healthy subjects.55 Since safe
application devoid of any pharmacological effect is dose-related,
trace amounts of the radiotracer warrant a wide safety margin
(SF). This is accomplished by carrier-free labeling of PET and
SPECT radiotracers with high specific activities so that a
negligible mass of an ETO derivative will provide an adequate
amount of radioactivity in a single intravenous dose. Actually,
the PET tracer 11C-MTO was generally produced with a specific
activity of 60 GBq/µmol, and the SPECT tracer 123I-IMTO is
now available with 1000 GBq/µmol (27 Ci/µmol). External
scintigraphy thus is performed with a microdose (0.06–5 µg)
of the PET or SPECT tracer avoiding a hypnotic effect (SF g
21.000) as well as cortisol suppression (SF g 2.800).
Substitution with a radiohalogen in the phenyl ring offered
access to diagnostic as well as therapeutic MTO derivatives,
all derived from a stannylated precursor. Radionuclide therapy
is based on ꢀ- and R-emitting isotopes, e.g., 131I, 82Br, and 211At,
while 123I-IMTO is being evaluated for diagnostic purposes.
Experimental Procedures
Chemical Synthesis. Chemicals were obtained from Aldrich,
Lancaster, and Sigma and were generally used without further
purification. Lipase SAM II (Amano Enzyme Europe Ltd., England)
was stored at +4 °C and used as supplied.
1H NMR spectra were recorded on a Bruker DRX400 spectrom-
eter in CDCl3 using the residual solvent peak as internal reference
[δ(CHCl3) ) 7.24] at 400.13 MHz. 13C NMR spectra (in part
J-modulated) were recorded on the same spectrometer operating
at 100.61 MHz (internal reference CDCl3; δ ) 77.00). Chemical
shifts, δ, are given in parts per million. Coupling constants, J, are
given in Hz. IR spectra were recorded as films on a silicon disk
using a Perkin-Elmer 1600 FT-IR spectrometer.56 Optical rotations
were measured at 20 °C on a Perkin-Elmer 341 polarimeter in a 1
dm (10 cm) cell, and concentration is expressed as g/100 mL. TLC
Conclusions
Derivatization of ETO (1) and MTO (2) at three distinct sites
provided information on the structural requirements for high-
affinity binding. The choice of substituents gave an indication
of which labeling approach would produce suitable radioligands.
Structural modifications concerned the ester moiety, the chiral
center, and the phenyl ring.
The radioligand 131I-IMTO displayed fast on- and off-kinetics
and bound with high affinity to a single class of binding sites
on rat adrenal membranes. IC50 values obtained by the displace-
was performed on 0.25 mm thick Merck plates, silica gel 60 F254
Flash chromatography was performed with Merck silica gel 60
.

2250 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Zolle et al.
(230–400 mesh). Spots were visualized by UV and/or with iodine
IMTO 10j (1.91 g, 67%, 99% ee); [R]20D +76.0 (c 1.09, acetone).
Anal. (C13H13IN2O2) C, H, N.
or dipping of 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. A Metrohm
702 SM Titrino instrument was used as an autotitrator.
Analytical HPLC was performed on a Jasco System (PU-980
pump, UV 975 and RI 930) using a Chiracel OD-H column, 0.46
cm × 25 cm. Preparative HPLC was performed on a Rainin system
(Dynamax SD-1 pump, equipped with a model UV-1 absorbance
detector) using a Chiracel OD column, 5 cm × 50 cm. Retention
times (tR) were measured on an analytical column using 5%
2-propanol-hexane, 1 mL/min, UV detection (254 nm). Tetrahy-
drofuran (THF) was distilled from potassium, and Et2O was distilled
from LiAlH4.
General Transesterification Procedure. A solution of 1 (0.10
g, 0.405 mmol) in dry MeOH, n-PrOH or PrOH (5 mL) and the
i
respective sodium alkoxide (1 M) was kept for 5 h at room
temperature. After neutralization with glacial acetic acid, the
solution was concentrated under reduced pressure, diluted with
water (2 mL), and extracted three times with EtOAc. The combined
organic layers were dried (MgSO4) and concentrated under reduced
pressure to leave a residue, which was purified by flash chroma-
tography on silica gel to give the desired ester.
(R)-(+)-Methyl
1-[1-Phenylethyl]-1H-imidazole-5-
carboxylate (2). Rf ) 0.65 (Et2O/iPr2NH 10/1); yield 71% as a
viscous oil; [R]20D +77.09 (c 1.1, acetone). Infrared spectrum was
superimposable with an authentic sample and with the (S)-isomer
10b.26
Mitsunobu Reaction of Benzylic Alcohols 9 with Methyl
1H-Imidazole-5-carboxylate.
(R)-(+)-Propyl
1-[1-Phenylethyl]-1H-imidazole-5-
carboxylate (3a). Rf ) 0.36 (hexane/Et2O/iPr2NH, 5/10/1); yield
78% as a viscous oil; [R]20 +60.06 (c 1.57, acetone). The
D
racemate30 and (R)-3a32 are known compounds. Anal. (C15H18N2O2)
C, H, N.
(R)-(+)-2-Propyl
1-[1-Phenylethyl]-1H-imidazole-5-
carboxylate (3b). Rf ) 0.36 (hexane/Et2O/iPr2NH, 5/10/1); yield
80% as a crystalline solid; mp 56 °C (hexane); [R]20 +44.52 (c
D
0.93, acetone). The racemate is a known compound.30 Anal.
(C15H18N2O2) C, H, N.
(R)-(+)-2-Fluoroethyl
1-[1-Phenylethyl]-1H-imidazole-5-
carboxylate (5). A solution of DtBAD (0.128 g, 0.554 mmol) in
dry toluene (2 mL) was added to a stirred mixture of Ph3P (0.145
g, 0.554 mmol), methyl 1H-imidazole-5-carboxylate (0.100 g, 0.462
mmol) and 2-fluoroethanol (44 mg, 0.040 mL, 0.681 mmol; handle
with care!) in dry toluene (2 mL) under an atmosphere of argon.
After 18 h, water (two drops) was added and the mixture was
concentrated under reduced pressure to give a residue, which was
purified by flash chromatography (first column, 60 g of silica gel,
hexane/Et2O/iPr2NH, 5/10/1, Rf ) 0.25, 98 mg of mixture of
2-fluoroethyl ester and hydrazo ester; second flash chromatography,
40 g of silica gel, Et2O as eluent, Rf ) 0.30) to give 5 (38 mg,
31%) as a crystalline solid, mp 51 °C (hexane); [R]20D +106.29 (c
0.72, acetone). Anal. (C14H15FN2O2) C, H, N.
(R)-(+)-N-Methyl-1-[1-phenylethyl]-1H-imidazole-5-
carboxamide (6). A solution of 4 (27 mg, 0.125 mmol) derived
from 1 and DMF (1 drop) in thionyl chloride30 (3 mL) was refluxed
for 1 h. After cooling, the reaction mixture was concentrated under
reduced pressure. The residue was dissolved in a solution (2 M, 4
mL) of methylamine in THF and stirred for 18 h. The mixture was
concentrated under reduced pressure. The residue was purified by
flash chromatography (EtOAc, Rf ) 0.09) to give the amide 6 (20
mg, 70%) as a gum; [R]20D +85.40 (c 2.52, acetone). Anal. Calcd
for C13H15N3O (229.12): C, 68.10; H, 6.59. Found: C 68.13; H
6.70.
(R)-(+)-N-Methoxy-N-methyl-1-[1-phenylethyl]-
1H-imidazole-5-carboxamide (7). A solution of 1 (0.247 g, 1.01
mmol) in ethanol (2.5 mL) and a solution of NaOH (0.048 g, 1.2
mmol) in water (5.4 mL) were combined and refluxed for 30 min
(TLC). The cooled mixture was concentrated under reduced pressure
and dried in a vacuum desiccator over KOH. Thionyl chloride (5
mL) was added to the residue. The mixture was stirred vigorously
and refluxed for 30 min. Removal of SOCl2 under reduced pressure
gave a residue, which was dissolved in dry CH2Cl2 (10 mL). N,O-
Dimethylhydroxylamine hydrochloride34 (0.11 g, 1.21 mmol) was
added, followed by dry pyridine (0.32 g, 0.33 mL, 4.04 mmol) after
cooling to 0 °C. Stirring was continued for 2.5 h, and then the
reaction mixture was concentrated under reduced pressure. Brine
(30 mL) was added to the residue, and the product was extracted
with ethyl acetate (3 × 20 mL). The combined organic layers were
dried (Na2SO4) and concentrated. The residue was purified by flash
chromatography (EtOAc/EtOH, 10/1, TLC Rf ) 0.52) to give 7
(0.148 g, 57%) as a crystalline product; mp 120–122 °C (CH2Cl2/
hexane); [R]20D +107.38 (c 0.75, acetone). Anal. (C14H17N3O2) C,
H, N.
General Procedure.36 A solution of (S)-alcohol (1.1 mmol,
>98% ee) in dry THF (2 mL) was added dropwise to a stirred
solution of methyl 1H-imidazole-5-carboxylate (0.139 g, 1.1 mmol)
and triphenylphosphine (0.345 g, 1.3 mmol) in dry THF (3.0 mL)
in an atmosphere of argon at -30 °C. Then a solution of di-tert-
butyl azodicarboxylate (0.304 g, 1.32 mmol) in dry THF (2 mL)
was added, and the reaction mixture (while stirring) was allowed
to warm up from -30 to 0 °C within 2.5 h. No alcohol could be
detected by TLC (diethyl ether/diisopropylamine, 10:1). The
reaction mixture was concentrated under reduced pressure. The
residue was mixed with diethyl ether (5 mL) and stirred for 2 h.
The crystals (Ph3PO and hydrazo ester) were collected and washed
with diethyl ether (3 × 2 mL). The filtrate was evaporated under
reduced pressure to yield a residue, which was purified by flash
chromatography (n-hexane/diethyl ether/diisopropylamine, 60:30:
1) on silica gel to give a chirally pure product.
The enantiomeric excesses of (R)- and (S)-alcohols were
determined by HPLC on a chiral stationary phase (Chiracel OD-
H, column size 5 cm × 50 cm). (R)-Configuration was assigned to
the alcohol formed by enzymatic saponification, since lipase SAM
II is known to hydrolyze preferentially the (R)-esters of secondary
benzylic alcohols.
(R)-(+)-Methyl 1-[1-(4-Iodophenyl)ethyl]-1H-imidazole-5-
carboxylate (10j).26 The (S)-alcohol 9j (1.98 g, 7.98 mmol, 98%
ee) in dry THF (14.5 mL) was added to a stirred solution of methyl
1H-imidazole-5-carboxylate (1.008 g, 7.98 mmol) and triph-
enylphosphine (2.503 g, 9.43 mmol) in dry THF (22.0 mL) in an
atmosphere of argon at -30 °C. Then a solution of di-tert-butyl
azodicarboxylate (2.204 g, 9.57 mmol) in dry THF (14.5 mL) was
added and the stirred reaction mixture was allowed to warm up
from -30 to 0 °C within 2.5 h. No alcohol could be detected by
TLC (diethyl ether/diisopropylamine, 10:1). The reaction mixture
was concentrated under reduced pressure. The residue was mixed
with diethyl ether (36 mL) and stirred for 2 h. The crystals (Ph3PO
and hydrazo ester) were collected and washed with diethyl ether
(3 × 15 mL). The filtrate was evaporated under reduced pressure
to yield a residue, which was purified by flash chromatography (n-
hexane/diethyl ether/diisopropylamine, 50:30:1) on silica gel to give

Etomidate Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2251
(R)-(+)-5-Acetyl-[1-phenylethyl]-1H-imidazole (8). A solution
of MeLi34 (0.34 mL, 0.54 mmol, 1.6 M in Et2O, 0.4% LiCl) was
added to a solution of 7 (0.108 g, 0.42 mmol) in dry THF (7.6
mL) at -55 °C under argon. Stirring was continued until the starting
material was consumed (TLC, 2.5 h). Water (5 mL) was added,
and the mixture was extracted with ethyl acetate (3 × 20 mL).
The combined organic layers were dried (Na2SO4) and concentrated
under reduced pressure. The residue was flash-chromatographed
is a known compound.40 Anal. (C14H14N2O2) (242.27) C, H. N:
calcd, 69.41; found, 68.95.
(R)-(+)-Methyl 1-[1-(4-Methylphenyl)ethyl]-1H-imidazole-5-
carboxylate (10g). (S)-(-)-1-(4-Methylphenyl)ethanol37 [0.136 g,
1.0 mmol, 95% ee by 1H NMR of Mosher ester, [R]20D -29.74 (c
1.14, MeOH)57 [R]25 -37.30 (c 1.02, MeOH)] was transformed
D
into 10g according to the general procedure for the Mitsunobu
reaction. Flash chromatography (Et2O/iPr2NH, 10/1; TLC Rf ) 0.66
in Et2O/iPr2NH, 10/1) gave 10g (0.104 g, 44%) as a viscous oil;
[R]20D +82.53 (c 1.47, acetone). The hydrochloride of the racemate
is a known compound. Anal. Calcd for C14H16N2O2 (244.29): C,
68.83; H, 6.60. Found: C, 69.11; H, 6.93.
(EtOAc, TLC Rf ) 0.49) to yield 8 (0.066 g, 74%) as an oil; [R]20
D
+71.98 (c 0.94, acetone). Anal. (C13H14N2O) C, H, N.
Methyl
1-Phenylmethyl-1H-imidazole-5-
carboxylate (10a). Phenylmethanol (0.216 g, 2 mmol, 0.21 mL)
was transformed into 10a according to the general procedure for
the Mitsunobu reaction; Rf ) 0.78 (Et2O/iPr2NH, 10/1); yield 47%
as a viscous oil. Crystallization from hexane furnished colorless
needles, mp 58 °C. Anal. Calcd for C12H12N2O2 (216.24): C, 66.65;
H, 5.59; N, 12.96. Found: C, 66.84; H, 5.76; N, 13.02.
(R)-(+)-Methyl 1-[1-(3-Iodophenyl)ethyl]-1H-imidazole-5-
carboxylate (10h). (S)-(-)-1-(3-Iodophenyl)ethanol {0.248 g, 1
mmol, 98% ee by HPLC; obtained by preparative HPLC on chiral
stationary phase (Chiracel OD-H, 5 cm × 50 cm, 5% 2-propanol/
hexane, 240 mL/min, 10 °C) of racemic mixture, (S)-alcohol tR )
(S)-(-)-Methyl
1-[1-Phenylethyl]-1H-imidazole-5-
7.7 min, (R)-alcohol tR ) 9.1 min, (S)-alcohol [R]20 -12.92 (c
D
carboxylate (10b). (R)-(+)-1-Phenylethanol37 (0.244 g, 2 mmol,
99% ee by HPLC) was transformed into 10b according to the
general procedure for the Mitsunobu reaction; Rf ) 0.61 (Et2O/
iPr2NH, 9/1); yield 51% as viscous product; [R]20D -78.36 (c 2.08,
acetone). Infrared spectrum was superimposable with an authentic
sample and with the (R)-enantiomer 2.
1.18, MeOH); (R)-alcohol [R]20 +48.4 (c 1.25, acetone)} was
D
transformed into 10h according to the general procedure for the
Mitsunobu reaction. Flash chromatography (Et2O/iPr2NH 10/1; TLC
Rf ) 0.64, Et2O/iPr2NH, 10/1) gave 10h (0.136 g, 38%) as a viscous
oil; mp 64–66 °C (hexane/trace of CH2Cl2) ([R]20D +44.31 (c 0.77,
acetone). Anal. (C13H13IN2O2) C, H, N.
(R)-(+)-Methyl
1-[1-Phenylpropyl]-1H-imidazole-5-
carboxylate (10c). (S)-(-)-1-Phenylpropanol37 (0.272 g, 2 mmol,
97% ee by HPLC) was transformed into 10c according to the
general procedure for the Mitsunobu reaction; Rf ) 0.78 (Et2O/
iPr2NH, 10/1); yield 61% as viscous product; [R]20D +93.47 (c 0.95,
acetone). The hydrochloride of the racemate is a known com-
pound.30 Anal. (C14H16N2O2) C, H, N.
(R)-(+)-Methyl 1-[1-(4-(Hydroxymethyl)phenylethyl]-1H-imidazole-5-
carboxylate (11). Bu3SnH (2.358 g, 2.18 mL, 8.1 mmol, 2.1 equiv,
dissolved in 40 mL of dry toluene) was added to a degassed and
stirred solution of 10j (1.425 g, 4.0 mmol) and Pd(Ph3P)4 (0.184
g, 0.16 mmol) in dry toluene (20 mL) slowly (5 h, syringe pump)
at 50 °C under an atmosphere of carbon monoxide.39 After the
addition of the Bu3SnH, the reaction mixture was stirred for another
16 h, then cooled and concentrated under reduced pressure. The
residue was flash-chromatographed (Et2O/MeOH, 100/1, TLC Rf
) 0.28 for aldehyde) to give starting material (0.328 g, 23%) and
the aldehyde (0.781 g, 75%) containing a small amount of an
Methyl
1-[3-Pyridylmethyl]-1H-imidazole-5-
carboxylate (10d). At 0 °C and under argon a solution of
3-pyridylmethanol (0.164 g, 1.5 mmol) in dry THF (3 mL) was
added to a stirred suspension of Ph3P (0.465 g, 1.77 mmol, 1.18
equiv) and methyl 1H-imidazole-5-carboxylate (0.189 g, 1.5 mmol,
1.0 equiv) in dry THF (5 mL). After the addition of DtBAD (0.414
g, 1.8 mmol, 1.2 equiv) the reaction mixture was stirred for 3 h at
0 °C. The solvent was removed under reduced pressure. HCl (2
M) was added to the viscous oil, and the acidic solution was
extracted twice with CH2Cl2. Then 2 M NaOH was added to the
aqueous phase, and the basic solution was extracted three times
with CH2Cl2. The combined organic solutions were dried (MgSO4)
and concentrated under reduced pressure to leave a residue, which
was purified by flash chromatography (MeOH/Et2O, 1/5, TLC Rf
) 0.38) to yield 10d as a crystalline solid (0.l96 g, 60%); mp 52–53
°C (hexane). Anal. (C11H11N3O2) C, H, N.
1
impurity. Immediately after recording the H NMR spectrum the
compound was reduced. A solution of the aldehyde (0.746 g, 2.89
mmol) dissolved in dry MeOH (6 mL) was added to a stirred
suspension of NaBH4 (0.055 g, 1.45 mmol) in dry MeOH (3 mL)
at room temperature under argon. When the reduction was finished
(15 min, TLC Et2O/MeOH, 50/1), the solvent was removed under
reduced pressure. Water (10 mL) was added to the residue, and
the mixture was extracted with CH2Cl2 (2 × 20 mL). The combined
organic layers were dried (MgSO4) and concentrated. The residue
was flash-chromatographed (first Et2O/MeOH, 100/1, then 25:1,
TLC Rf ) 0.28 for 25/1) to give 11 (0.639 g, 85% from aldehyde,
64% from 10j) as a colorless gum; [R]20D +96.24 (c 1.25, acetone).
Anal. (C14H16N2O3) C, H, N.
(R)-(+)-Methyl
1-[1-(3-Pyridyl)ethyl]-1H-imidazole-5-
carboxylate (10e). (S)-(-)-1-(3-Pyridyl)ethanol [0.096 g, 0.78
Radiochemistry. 131I-NaI was obtained from GE Healthcare
Buchler, Braunschweig, Germany. Analytical HPLC was performed
on a C18 column, Nucleosil 100-7 250 mm × 4.6 mm, CS
Chromatographie Service, Langerwehe, Germany.
mmol, prepared by a literature procedure,38 96% ee (by H NMR
1
of Mosher ester); [R]20D -33.21 (c 1.33, acetone), [R]20D -18.13
(c 1.24, CHCl3), [R]20 -101.9 (c 1.21, CHCl3)], methyl 1H-
D
imidazole-5-carboxylate, triphenylphosphane, and di-tert-butyl
azodicarboxylate were reacted according to the general procedure
for the Mitsunobu reaction. The reaction mixture was concentrated
in vacuo. The residue was taken up in 2 N HCl. The mixture was
extracted twice with CH2Cl2. The pH of the aqueous phase was
brought to 7–8 by adding solid K2CO3. The product was extracted
three times with CH2Cl2. These three organic phases were com-
bined, dried (MgSO4), and concentrated in vacuo. The residue was
purified by flash chromatography (MeOH/Et2O, 1/5, TLC Rf ) 0.38)
to give 10e (0.045 g, 25%) as a viscous oil; [R]20D +70.70 (c 1.14,
acetone). Anal. (C12H13N3O2) C, H, N.
To a sealed conical glass vial containing 30 µg of stannylated
precursor in 30 µL of ethanol, radioiodine corresponding to 37–185
MBq nca in 1–10 µL of 0.02 N NaOH and 6 µL of 1 N HCl were
added. A solution of 15 µg of chloramine-T trihydrate in 10 µL of
water (1.5 mg/mL) was added last to initiate labeling. The sample
was mixed and allowed to proceed for 3 min at room temperature
before adding 7 µL of 1 N NaOH. 131I-IMTO 10j was isolated
from the reaction mixture by analytical HPLC using a C18 phase
and CH3OH/H2O/diethylamine 60/40/0.2 as eluent with a flow rate
of 1.5 mL/min. After evaporation of the solvent under reduced
pressure the tracer was formulated in 1 mL of saline. In vitro
stability in this vehicle was analyzed by HPLC in daily intervals
for 1 week, demonstrating a slow linear loss of radioiodine with
time. The specific activity of radiolabeled IMTO was determined
by HPLC using a calibration curve generated by linear regression
analysis of recorded mass from solutions of known concentrations
of IMTO versus peak area of UV absorption at 254 nm.
(R)-(-)-Methyl
1-[Indan-1-yl]-1H-imidazole-5-
carboxylate (10f). (S)-(+)-1-Indanol37 {0.134 g, 1.0 mmol, >95%
ee by HPLC, [R]20 +29.57 (c 1.15, distilled CHCl3)} was
D
transformed into 10f according to the general procedure for the
Mitsunobu reaction. Flash chromatography (Et2O/iPr2NH, 10/1;
TLC Rf ) 0.77 in Et2O/iPr2NH, 10/1) gave 10f (0.077 g, 32%);
mp 93 °C (hexane), ([R]20D -67.86 (c 1.54, acetone). The racemate

2252 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Zolle et al.
Biochemistry. Whole adrenals from adult male Wistar rats were
stored at -80 °C. Membranes obtained by homogenization were
stored as aliquots at -80 °C.
was reduced to 2%. Test compounds were added to cell suspensions
of 1 million cells in 1 mL at concentrations ranging from 10 nM
to 100 µM and incubated for 48 h. Experiments were performed in
triplicate. At the end of the incubation period, cells were centrifuged
and aliquots (50 µL) of the supernatant were analyzed using a
cortisol RIA kit. Cortisol values (µg/dL) were expressed as percent
of controls. The control value was based on six determinations
without addition of inhibitor. IC50 values were calculated using
PRISM Graph Pad, version 3.0, San Diego, CA.
Membrane Preparation. For preparation of total membranes,
adrenals were thawed and homogenized in 100 parts of homog-
enization buffer (10 mM K2HPO4/10 mM HEPES, pH 7.1) with a
glass/Teflon homogenizer (Potter-type). The homogenate was
centrifuged at 35.000g for 10 min. The pellet was resuspended in
fresh buffer and centrifuged. Membranes were washed two more
times, including a 10 min warming period in a 37 °C water bath,
and stored as aliquots at -80 °C. On the day of the experiment, an
aliquot was thawed, diluted with incubation buffer (containing NaCl;
see below), and centrifuged and the pellet resuspended in incubation
buffer.
Acknowledgment. This work was supported by the Öster-
reichische Nationalbank, Jubiläumsfondsproject No. 8680. The
authors gratefully acknowledge the contribution of Dr. Herbert
Kvaternik at the Austrian Research Centers, Seibersdorf, the
donation of SAM II by Amano Enzyme Europe Ltd. (England),
and expert assistance by S. Felsinger for recording the NMR
spectra, S. Schneider for preparative HPLC separation of an
(S)-alcohol, Dr. A. Woschek and Dr. F. Wuggenig for chemical
syntheses.
131I-MTO Binding Procedure. Glass vials containing a final
volume of 0.5 mL of incubation buffer (10 mM K2HPO4/10 mM
HEPES, 150 mM NaCl, pH 7.1), 20.000–40.000 cpm 131I-IMTO
together with IMTO carrier (2 nM, i.e., 1 pmol/vial, having a
specific activity of 9–18 Ci/mmol) and the adrenal membrane
suspension corresponding to 0.06–0.1 mg original tissue/vial were
immersed in a 23 °C water bath for 20–30 min. Membranes with
bound radioligand were isolated by filtration over glass fiber filters
presoaked in incubation buffer (conventional presoaking in poly-
ethylene imine resulted in elevated background values). Filters were
washed twice with buffer, transferred into vials, and measured in
a γ counter.
Supporting Information Available: Elemental analysis results,
spectroscopic data, and cortisol inhibition data. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
Inhibition Studies Assay. Compounds to be evaluated as
competitive inhibitors of 131I-IMTO binding were incubated at
concentrations between 0.1 nM and 300 µM. Constant ligand
concentration (2 nM) was maintained in all experiments by the
addition of IMTO carrier to the 131I-IMTO radiotracer solution
immediately before use. Glass vials contained a final volume of
0.5 mL of the incubation buffer (10 mM K2HPO4/10 mM HEPES,
150 mM NaCl, pH 7.1), 20.000–40.000 cpm 131I-IMTO radiotracer
solution (2 nM), and the test compounds competing with specific
radioligand binding. ETO carrier (10 µM) was used to determine
nonspecific binding. The adrenal membrane suspension correspond-
ing to 0.06–0.1 mg of the original tissue per vial was added last.
The reaction vials were immersed in a 23 °C water bath for 20–30
min. After incubation, membranes with bound radioligand were
separated by filtration and filters were washed twice and measured
in a γ counter. The cpm (total binding) values were corrected by
subtraction of cpm for nonspecific binding. Binding potency was
expressed as the IC50 value.
(1) Fellows, I. W.; Bastow, M. D.; Byrne, A. J.; Allison, S. P. Adreno-
cortical suppression in multiply injured patients: a complication of
etomidate treatment. Br. Med. J. 1983, 287, 1835–1837.
(2) Drake, W. M.; Perry, L. A.; Hinds, C. J.; Lowe, D. G.; Reznek, R. H.;
Besser, G. M. Emergency and prolonged use of intravenous etomidate
to control hypercortisolemia in a patient with Cushing‘s syndrome
and peritonitis. J. Clin. Endocrinol. Metab. 1998, 83, 3542–3544.
(3) Vanden Bossche, H.; Willemsens, G.; Cools, W.; Bellens, D. Effects
of etomidate on steroid biosynthesis in subcellular fractions of bovine
adrenals. Biochem. Pharmacol. 1984, 33, 3861–3868.
(4) The European Pharmacopeia 5.0; Etomidate, Monograph 1514;
Council of Europe, Maisonneuve: Sainte-Ruffine, France, 2005.
(5) Satre, M.; Vignais, P. V.; Idelman, S. Distribution of the steroid 11ꢀ-
hydroxylase and the cytochrome P-450 in membranes of beef adrenal
cortex mitochondria. FEBS Lett. 1969, 5, 135–140.
(6) Satre, M.; Vignais, P. V. Steroid 11ꢀ-hydroxylation in beef adrenal
cortex mitochondria. Binding affinity and capacity of specific [14C]-
steroids and for [3H]metyrapol, an inhibitor for the 11ꢀ-hydroxylation
reaction. Biochemistry 1974, 13, 2201–2209.
(7) Yago, N.; Ichii, S. Submitochondrial distribution of components of
the steroid 11ꢀ-hydroxylase and cholesterol sidechain-cleaving enzyme
systems in hog adrenal cortex. J. Biochem. (Tokyo) 1969, 65, 215–
224.
(8) Dodge, A. H.; Christensen, A. K.; Clayton, R. B. Localization of steroid
11 beta-hydroxylase in the inner membrane subfraction of rat adrenal
mitochondria. Endocrinology 1970, 87, 254–261.
(9) Young, D. G.; Hall, P. F. Steroid hydroxylation in bovine adrenocor-
tical mitochondria. Competition between side-chain cleavage of
cholesterol and 11ꢀ-hydroxylation. Biochemistry 1971, 10, 1496–1502.
(10) Wilson, L. D.; Harding, B. W. Oxidation of 20R-hydroxycholesterol
by adrenal cortex mitochondria. J. Biol. Chem. 1973, 248, 9–14.
(11) Engelhardt, D. Steroid biosynthesis inhibitors in Cushing’s syndrome.
Clin. InVest. 1994, 72, 481–488.
(12) Napoli, J. L.; Counsell, R. E. New inhibitors of steroid 11ꢀ-
hydroxylase. Structure-activity relationship studies of metyrapone-
like compounds. J. Med. Chem. 1977, 20, 762–766.
(13) Hays, S. J.; Tobes, M. C.; Gildersleeve, D. L.; Wieland, D. M.;
Beierwaltes, W. H. Structure-activity relationship study of the
inhibition of adrenal cortical 11ꢀ-hydroxylase by new metyrapone
analogues. J. Med. Chem. 1984, 27, 15–19.
(14) Zolle, I.; Woloszczuk, W.; Höfer, R. Synthesis and in Vitro Evaluation
of Metyrapone Derivatives as Potential Inhibitors of 11ꢀ-Hydroxylase
Activity. In Radiopharmaceuticals and Labelled Compounds 1984,
Proceedings Series, IAEA-CN-45/67; International Atomic Energy
Agency: Vienna, Austria, 1985; pp 337–342.
(15) Damani, L. A.; Mitterhauser, M.; Zolle, I.; Lin, G.; Oehler, E.; Ho,
Y.-P. Metabolic and pharmacokinetic considerations in the design of
2-phenyl substituted metyrapone derivatives: 2-methoxyphenyl-
metyrapone as a radioligand for functional diagnosis of adrenal
pathology. Nucl. Med. Biol. 1995, 22, 1067–1074.
(16) Wieland, D. M.; Ice, R. D.; Beierwaltes, W. H. Synthesis of
radiolabeled enzyme inhibitors of the adrenal cortex. J. Nucl. Med.
1976, 17, 525.
Mathematical Analysis. Data from association and dissociation
experiments were computer-fitted to the appropriate monoexpo-
nential functions. Data from saturation experiments were recipro-
cally transformed to Eadie-Hofstee plots and subjected to linear
correlation analysis. Data from inhibition experiments were computer-
nH
nH
fitted to the function B(x) ) (B0)(IC50 )/(IC50 + xnH), where B0
is specific binding in the absence of inhibitor, x the inhibitor
concentration (nM), and nH the Hill coefficient. Specific binding
(B0) was obtained by subtracting nonspecific from total binding.
Ki values, as an independent measure of the ligand concentration
L, can be obtained with the equation of Cheng and Prusoff: Ki )
IC50/(1 + L/KD); thus, IC50 values have to be divided by 1 + 2/11.6
) 1.17. As a measure of reproducibility, mean values of repeated
measurements are given with standard deviations (SD). Statistical
comparison between two mean values is done by the Student’s t
test and between more than two mean values by ANOVA and post
hoc Newman-Keuls test.
Cortisol Assay. The human adrenocortical cancer cell line NCI-
h29558 was obtained from the American type Culture Collection
(ATCC, Rockville, MD). Cell medium RPMI 1640 and fetal calf
serum (FCS) were supplied by Invitrogen (Eggenstein, Germany).
Insulin, transferrin, and selenium were obtained from Sigma
(Deisenhofen, Germany). Cortisol radioimmunoassay kits were
obtained from DPC Biermann, Bad Nauheim, Germany.
Cells were grown in RPMI 1640 medium supplemented with
transferrin, insulin, selenium, and 5% fetal calf serum (FCS) at 37
°C under an atmosphere of 5% CO2/95% air. For experiments, FCS

Etomidate Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2253
(17) Beierwaltes, W. H.; Wieland, D. M.; Ice, R. D.; Seabold, J. E.; Sarkar,
S. D.; Gill, S. P.; Mosley, S. T. Localization of radiolabeled enzyme
inhibitors in the adrenal gland. J. Nucl. Med. 1976, 17, 998–1002.
(18) Beierwaltes, W. H.; Wieland, D. M.; Mosley, S. T.; Swanson, D. P.;
Sarkar, S. D.; Freitas, J. E.; Thrall, J. H; Herwig, K. R. Imaging the
adrenal glands with radiolabeled inhibitors of enzymes: concise
communication. J. Nucl. Med. 1978, 19, 200–203.
(19) Robien, W.; Zolle, I. Synthesis of radioiodinated metyraponesa
potential adrenal imaging agent. Int. J. Appl. Radiat. Isot. 1983, 34,
907–914.
(39) Baillargeon, V. P.; Stille, J. K. Palladium-catalyzed formylation of
organic halides with carbon monoxide and tin hydride. J. Am. Chem.
Soc. 1986, 108, 452–461.
(40) Godefroi, E. F.; Van Cutsem, J.; Van der Eycken, C. A. M.; Janssen,
P. A. J. 1-(1-Indanyl)- and 1-(1-tetralyl)imidazole-5-carboxylate esters,
a novel type of antifungal agents. J. Med. Chem. 1967, 10, 1160–
1161.
(41) Lamberts, S. W. J.; Bons, E. G.; Bruining, H. A.; De Jons, F. H.
Differential effects of imidazole derivatives etomidate, ketoconazole
and miconazole and of metyrapone on the secretion of cortisol and
its precursors by human adrenocortical cells. J. Pharmacol. Exp.
Therap. 1987, 240, 259–264.
(42) Murray, M.; Reidy, G. F. Selectivity in the inhibition of mammalian
cytochromes P-450 by chemical agents. Pharmacol. ReV. 1990, 42,
85–100.
(20) Hays, S. J.; Mangner, T. J. Synthesis of iodine-125 labelled analogues
of metyrapone and metyrapol. J. Labelled Compd. Radiopharm. 1983,
XX, 1373–1381.
(21) Zolle, I.; Halldin, C.; Yu, J.; Berger, M. L. [11C]-2-Methoxy-
phenylmetyrapone for PET. J. Labelled Compd. Radiopharm. 1993,
XXXII, 547–549.
(22) Yu, J.; Mertens, J.; Rakias, F.; Zolle, I. Synthesis of radio-2-iodo-
phenylmetyrapone using Cu(I)-assisted nucleophilic exchange label-
ling: study of the reaction kinetics. Nucl. Med. Biol. 1995, 22, 257–
262.
(43) Weber, M. M.; Lang, J.; Abedinpour, F.; Zeilberger, K.; Adelmann,
B.; Engelhardt, D. Different inhibitory effect of etomidate and
ketoconazole on the human adrenal steroid biosynthesis. Clin. InVest.
1993, 71, 933–938.
(23) Satre, M.; Lunardi, J.; Vignais, P. V. [3H]Metyrapol as a tool for studies
of interactions of deoxycorticosterone with adrenal cortex mitochon-
dria. FEBS Lett. 1972, 26, 193–196.
(44) Ledingham, I. M.; Watt, I. Influence of sedation on mortality in
critically ill multiple trauma patients. Lancet 1983, 1, 1270.
(45) Wagner, R. L.; White, P. F.; Kan, P. B.; Rosenthal, M. H.; Feldman,
D. Inhibition of adrenal steroidogenesis by the anesthetic etomidate.
N. Engl. J. Med. 1984, 310, 1415–1421.
(46) Diago, M. C.; Amado, J. A.; Otero, M.; Lopez-Cordovilla, J. J. Anti-
adrenal action of subanaesthetic dose of etomidate. Anaesthesia 1988,
43, 644–645.
(24) Berger, M. L.; Hahner, S.; Hammerschmidt, F.; Schirbel, A.; Yu, J.;
Zolle, I. Inhibitors of Adrenal P-450c11 Hydroxylase Characterized
by Two Radioligands and Steroid Hormone Secretion. Presented at
the International Symposium on Trends in Radiopharmaceuticals
(IAEA), Vienna, Austria, November 14–18, 2005.
(25) Berger, M. L.; Schirbel, A.; Hammerschmidt, F.; Zolle, I. Structural
requirements of metomidate for high affinity binding to adrenal
membranes. Eur. J. Nucl. Med. Mol. Imaging 2002, 29 (Suppl. 1),
S380.
(26) Schirbel, A.; Zolle, I.; Hammerschmidt, F.; Berger, M. L.; Schiller,
D.; Kvaternik, H.; Reiners, Chr. [123/131I]Iodometomidate as a radio-
ligand for functional diagnosis of adrenal disease: synthesis, structural
requirements and biodistribution. Radiochim. Acta 2004, 92, 297–303.
(27) Bergström, M.; Bonasera, T. A.; Lu, L.; Bergström, E.; Backlin, C.;
Juhlin, C.; Långström, B. In vitro and in vivo primate evaluation of
carbon-11-etomidate and carbon-11-metomidate as potential tracers
for PET imaging of the adrenal cortex and its tumors. J. Nucl. Med.
1998, 39, 982–989.
(47) Belelli, D.; Lambert, J. J.; Peters, J. A.; Wafford, K.; Whiting, P. J.
The interaction of the general anesthetic etomidate with the γ-ami-
nobutyric acid type A receptor is influenced by a single amino acid.
Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 11031–11036.
(48) Franks, N. P. Molecular targets underlying general anaesthesia. Br. J.
Pharmacol. 2006, 147, S72–S81.
(49) Hahner, S.; Stuermer, A.; Kreissl, M.; Reiners, C.; Fassnacht, M.;
Hänscheid, H.; Beuschlein, F.; Zink, M.; Lang, K.; Zolle, I.; Allolio,
B.; Schirbel, A. Unpublished results.
(50) Mitterhauser, M.; Wadsak, W.; Wabnegger, L.; Sieghart, W.; Viern-
stein, H.; Kletter, K.; Dudczak, R. In vivo and in vitro evaluation of
18F-FETO with respect to the adrenocortical and GABAergic system
in rats. Eur. J. Nucl. Med. Mol. Imaging 2003, 30, 1398–1401.
(51) Eriksson, B.; Bergström, M.; Sundin, A.; Juhlin, C.; Örlefors, H.;
Öberg, K.; Långström, B. The role of PET in localization of
neuroendocrine and adrenocortical tumors. Ann. N.Y. Acad. Sci. 2002,
970, 159–169.
(28) Bergström, M.; Juhlin, C.; Bonasera, T. A.; Sundin, A.; Rastad, J.;
Åkerström, G.; Långström, B. PET imaging of adrenal cortical tumors
with the 11ꢀ-hydroxylase tracer 11C-metomidate. J. Nucl. Med. 2000,
41, 275–282.
(29) Wadsak, W.; Mitterhauser, M. Synthesis of [18F]FETO, a novel
potential 11ꢀ-hydroxylase inhibitor. J. Labelled Compd. Radiopharm.
2003, 46, 379–388.
(52) Khan, T. S.; Sundin, A.; Juhlin, C.; Långström, B.; Bergström, M.;
Eriksson, B. 11C-metomidate PET imaging of adrenocortical cancer.
Eur. J. Nucl. Med. Mol. Imaging 2003, 30, 403–410.
(53) Minn, H.; Salonen, A.; Friberg, J.; Roivainen, A.; Viljanen, T.; Långsjö,
J.; Salmi, J.; Välimäki, M.; Någren, K.; Nuutila, P. Imaging of adrenal
incidentalomas with PET using 11C-metomidate and 18F-FDG. J. Nucl.
Med. 2004, 45, 972–979.
(30) Godefroi, E. F.; Janssen, P. A. J.; Van der Eycken, C. A. M.; Van
Heertum, A. H. M. T.; Niemegeers, C. J. E. D,L-1-(1-Arylalkyl)imi-
dazole-5-carboxylate esters. A novel type of hypnotic agents. J. Med.
Chem. 1965, 8, 220–223.
(31) Mitsunobu, O. The use of diethyl azodicarboxylate and triphenylphos-
phine in synthesis and transformation of natural products. Synthesis
1981, 1–28.
(32) Roevens, L. F. C.; Heykants, J. J. P.; Helsen, W. A. M. (Janssen
Pharmaceutica, N.V.) 1-1-Phenylethyl)-1H-imidazole-5-carboxylic
Acid Esters, Their Enantiomers and Acid Salts. Ger. 2,609,573
(C1.C07D233/90), September 30, 1976; U.S. Appl. 557,194, March
10, 1975; Chem. Abstr. 1976, 85, 192739x.
(33) Hughes, D. L. The Mitsunobu Reaction. Org. React. 1992, 42, 335–
656.
(34) Nahm, S.; Weinreb, S. M. N-Methoxy-N-methylamides as effective
acylating agents. Tetrahedron Lett. 1981, 22, 3815–3818.
(35) Corelli, F.; Summa, V.; Brogi, A.; Monteagudo, E.; Botta, M. Chiral
azole derivatives. 2. Synthesis of enantiomerically pure 1-alkylimi-
dazoles. J. Org. Chem. 1995, 60, 2008–2015.
(36) Hammerschmidt, F.; Peric Simov, B.; Schmidt, S.; Schneider, S.; Zolle,
I. Chemoenzymatic synthesis of stannylated metomidate as a precursor
for electrophilic radiohalogenationssregioselective alkylation of meth-
yl 1H-imidazole-5-carboxylate. Monatsh. Chem. 2005, 136, 229–239.
(37) Laumen, K.; Schneider, M. P. A highly selective ester hydrolase from
Pseudomonas sp. for the enzymatic preparation of enantiomerically
pure secondary alcohols; chiral auxiliaries in organic synthesis.
J. Chem. Soc., Chem. Commun. 1988, 598–600.
(54) Hennings, J.; Lindhe, Ö.; Bergström, M.; Långström, B.; Sundin, A.;
Hellman, P. 11C-metomidate positron emission tomography of adreno-
cortical tumors in correlation with histopathological findings. J. Clin.
Endocrinol. Metab. 2006, 91, 1410–1414.
(55) Wadsak, W.; Mitterhauser, M.; Rendl, G.; Schuetz, M.; Mien, L. K.;
Ettlinger, D. E.; Dudczak, R.; Kletter, K.; Karanikas, G. 18F-FETO
for adrenocortical PET imaging: a pilot study in healthy volunteers.
Eur. J. Nucl. Med. Mol. Imaging 2006, 33, 669–672.
(56) Mikenda, W. Solid films and layers deposited on siliconsa versatile
infrared sampling technique. Vib. Spectrosc. 1992, 3, 327–330.
(57) Kitamura, M.; Oka, H.; Noyori, R. Asymmetric addition of dialkylzincs
to benzaldehyde derivatives catalyzed by chiral ꢀ-amino alcohols.
Evidence for the monomeric alkylzinc aminoalkoxide as catalyst.
Tetrahedron 1999, 55, 3605–3614.
(58) Mueller-Vieira, U.; Angotti, M.; Hartmann, R. W. The adrenocortical
tumor cell line NCI-H295R as an in vitro screening system for the
evaluation of CYP11B2 (aldosterone synthase) and CYP11B1 (steroid-
11ꢀ-hydroxylase) inhibitors. J. Steroid Biochem. Mol. Biol. 2005, 96,
259–270.
(38) Seemayer, R.; Schneider, M. P. Preparation of optically pure pyridyl-
1-ethanols. Tetrahedron: Asymmetry 1992, 3, 827–830.
JM800012W