Cushing's syndrome: Development of highly potent and selective CYP11B1 inhibitors of the (pyridylmethyl)pyridine type

Article abstract of DOI:10.1021/jm400240r

Potent and selective CYP11B1 inhibitors could be promising therapeutics for the treatment of Cushing's syndrome. Optimization of Ref 1 (5-((1H-imidazol-1- yl)methyl)-2-phenylpyridine) led to compound 44 (5-((5-methylpyridin-3-yl) methyl)-2-phenylpyridine) with a 50-fold improved IC₅₀ value of 2 nM toward human CYP11B1 and an enhanced inhibition of the rat enzyme (IC ₅₀ = 2440 nM) compared to Ref 1 (IC₅₀ > 10000 nM). Furthermore, selectivities over CYP11B2, CYP17, and CYP19 were observed, as well as satisfying metabolic stability not only in human and rat plasma but also in liver S9 fraction. Investigation of cytotoxicity and inhibition of hepatic CYP2A6 and CYP3A4 showed that 44 fulfills first safety criteria and can be considered for further in vivo evaluation in rats.

Full text of DOI:10.1021/jm400240r

Article
pubs.acs.org/jmc
Cushing’s Syndrome: Development of Highly Potent and Selective
CYP11B1 Inhibitors of the (Pyridylmethyl)pyridine Type
Juliette Emmerich,† Qingzhong Hu,† Nina Hanke,‡ and Rolf W. Hartmann*,†
†Pharmaceutical and Medicinal Chemistry, Saarland University, and Department of Drug Design and Optimization, Helmholtz
Institute for Pharmaceutical Research Saarland (HIPS), Campus C2-3, 66123 Saarbrucken, Germany
̈
‡Elexopharm GmbH, Campus A1, 66123 Saarbrucken, Germany
̈
S
* Supporting Information
ABSTRACT: Potent and selective CYP11B1 inhibitors could be
promising therapeutics for the treatment of Cushing’s syndrome.
Optimization of Ref 1 (5-((1H-imidazol-1-yl)methyl)-2-phenyl-
pyridine) led to compound 44 (5-((5-methylpyridin-3-yl)methyl)-
2-phenylpyridine) with a 50-fold improved IC50 value of 2 nM
toward human CYP11B1 and an enhanced inhibition of the rat
enzyme (IC50 = 2440 nM) compared to Ref 1 (IC50 > 10000 nM).
Furthermore, selectivities over CYP11B2, CYP17, and CYP19 were
observed, as well as satisfying metabolic stability not only in human
and rat plasma but also in liver S9 fraction. Investigation of
cytotoxicity and inhibition of hepatic CYP2A6 and CYP3A4 showed that 44 fulfills first safety criteria and can be considered for
further in vivo evaluation in rats.
this might lead to hypokalemia.3 Consequently, novel and
selective CYP11B1 inhibitors are urgently needed. However,
selective inhibition of CYP11B1 or CYP11B2 is very
INTRODUCTION
■
Cortisol is involved in many important physiological processes.
However, excessive cortisol leads to Cushing’s syndrome that is
characterized by visceral obesity, hypertension, and diabetes.
Nearly all cases of endogenous Cushing’s syndrome are caused
challenging due to the high degree of homology (93%)
between them.4 In spite of this difficulty, we have developed
selective CYP11B2 inhibitors.5 A CYP11B1 inhibitor Ref 16a,b
by tumors in the hypothalamus, the adrenals or, most
was also identified recently, which is potent (IC50 = 107 nM)
frequently, the pituitary. Cases of the last origin are specifically
and selective over 17α-hydroxylase-17,20-lyase (CYP17),
termed Cushing’s disease, in which a pituitary adenoma
aromatase (CYP19), and in particular CYP11B2, for which a
overproduces adrenocorticotropic hormone (ACTH), leading
selectivity factor (SF = IC50 CYP11B2/IC50 CYP11B1) of 13 was
to an elevation of plasma cortisol concentrations. Surgical
removal of the tumor is therefore the first-line treatment. If the
achieved. In comparison to the most selective inhibitor in
clinical use, metyrapone (IC50 = 15 nM, SF = 4.8), selectivity
tumor recurs or resection is not feasible due to size or location
was improved. Still, our inhibitors require further enhancement
of the tumor, radiotherapy is applied. However, the inherent
of inhibitory potency to decrease the doses which have to be
administered, thus reducing the risks of off-target effects.
side effects of irradiation make a safe medication therapy
desirable.1a In addition, blockade of steroidogenesis with drugs
Because the introduction of substituents at the methylene
before surgery lowers circulating cortisol levels, leading to a
bridge of a similar compound class resulted in a very potent and
reversion of the metabolic consequences of cortisol excess and
selective inhibitor Ref 26c (IC50 = 2 nM, SF = 11), various
groups, in particular alkyl moieties, were introduced onto the
methylene bridge of the current scaffold (Chart 1).
Furthermore, the central pyridine ring was replaced by other
heterocycles, preserving the aromatic N because it was deemed
to be important for selectivity.6b After the most suitable core
had been identified, the heme-complexing imidazolyl7 was
exchanged by various heterocycles containing an sp2 hybrid N.
Moreover, isosteric exchange of the methylene bridge by O or
NH, as well as the introduction of substituents at the distal N-
containing heterocycle, was also performed. These efforts led to
thus reduces the surgical risks.1b In the case of metabolic
syndrome, which shares some symptoms with Cushing’s
syndrome and is also accompanied by excessive cortisol, albeit
in adipose and liver tissue rather than in circulation, 11β-
hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitors
are promising therapeutics.2 However, it is doubtful whether
11β-HSD1 inhibitors are able to reduce elevated cortisol
plasma levels. By contrast, metyrapone, which is in clinical use
for the treatment of Cushing’s syndrome, can normalize plasma
cortisol levels by inhibiting 11β-hydroxylase (CYP11B1).1a This
enzyme catalyzes the last step of cortisol biosynthesis: the
hydroxylation of 11-deoxycortisol to cortisol. However,
metyrapone is not selective enough over aldosterone synthase
(CYP11B2), which is crucial in aldosterone production, and
Received: November 15, 2012
© XXXX American Chemical Society
A
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Chart 1. Metyrapone and the Concept of Inhibitor Design
a
Scheme 1. Synthesis of Compounds 15−24
a
Reagents and conditions: (a) n-BuLi, Et2O, −80 °C, 1 h; (b) DMF, −80 °C, 1 h, HCl; (c) method A, R1B(OH)2, Pd(PPh3)4, Na2CO3, toluene/
EtOH/H2O, reflux, 5 h; (d) method B, R2 MgCl or R2 MgBr, THF; (e) method C, CDI, NMP, 170 °C.
a
28 novel compounds (Chart 1), which were evaluated for
inhibition of CYP11B1 and selectivities over CYP11B2, CYP17,
and CYP19. Selective compounds were further evaluated for
metabolic stability, inhibition of hepatic CYP3A4 and CYP2A6,
and cytotoxicity. Although Ref 1 was potent toward human
CYP11B1 (IC50 = 107 nM), it showed a poor inhibition of the
rat enzyme (IC50 > 10000 nM), which makes it impossible to
use rats for pharmacodynamic experiments and proof of
concept. Therefore, the most potent and selective compound of
this series was evaluated for rat CYP11B1 inhibition and
metabolic stability in rat plasma and S9 fraction to identify a
candidate for further in vivo studies.
Scheme 2. Synthesis of Compounds 33−36
CHEMISTRY
■
a
Reagents and conditions: (a) method A, PhB(OH)2, Pd(PPh3)4,
Final compounds 15−24 were synthesized from commercially
available 2,5-dibromopyridine (Scheme 1). After the trans-
formation of one bromine substituent into a formyl group via
lithium−halogen exchange followed by a nucleophilic for-
mylation, the aromatic ring R1 was introduced via a Suzuki
coupling reaction, leading to the intermediates 2−4. Grignard
reactions were employed to insert various substituents on the
methylene bridge, and the resulting alcohols were subsequently
converted to compounds 15−24 via a nucleophilic substitution
by reacting with 1,1′-carbonyldiimidazole (CDI). For the
synthesis of compounds with different central rings (33−36),
the heterocyclic intermediates 25−28 were obtained via
different routes (Scheme 2). Pyridazine compound 25 was
synthesized from commercially available 3-chloro-6-methylpyr-
idazine and phenylboronic acid via a Suzuki reaction, whereas
Na2CO3, toluene/EtOH/H2O, reflux, 5 h; (b) propane-1,2-diamine,
ethanol, KOH; (c) NaOMe, MeOH, (E)-3-ethoxy-2-methylacrylalde-
hyde, H2O, RT; (d) (methoxymethyl)triphenylphosphonium chloride,
KOt-Bu, THF; (e) n-BuLi, PhCN; (f) method D, NBS, DBPO, CCl4,
reflux, 12 h; (g) method E, imidazole, K2CO3, DMF, 120 °C, 2 h.
pyrazine intermediate 26 was yielded by nucleophilic addition
of propane-1,2-diamine to phenylglyoxal. A condensation
between benzamidine and (E)-3-ethoxy-2-methylacrylaldehyde
led to pyrimidine compound 27. The isoquinoline intermediate
28, in contrast, was built via a Wittig reaction of o-
bromoacetophenone followed by a bromine−lithium exchange
and a subsequent nucleophilic substitution by benzonitrile for
the ring closure. These methyl intermediates 25−28 were
subsequently brominated under Wohl−Ziegler conditions and
B
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a
Scheme 3. Synthesis of Compounds 39−49 and 52
a
Reagents and conditions: (a) method A, corresponding boronic acid, Pd(PPh3)4, Na2CO3, toluene/EtOH/H2O, reflux, 5 h; (b) method D, NBS,
DBPO, CCl4, reflux, 12 h; (c) method F, corresponding boronic acid, PdCl2(dppf), Cs2CO3, DME/H2O/EtOH, 150 °C, 150 W, 18 bar, 20 min,
microwave; (d) HBr, 130 °C, 12 h.
a
Scheme 4. Synthesis of Compounds 55 and 56
a
Reagents and conditions: (a) method A, PhB(OH)2, Pd(PPh3)4, Na2CO3, toluene/EtOH/H2O, reflux, 5 h; (b) CuI, N,N′-dimethylethylenedi-
amine, NaI, dioxane, 110 °C; (c) CuI, 2-picolinic acid, 3-hydroxypyridine, K3PO4, DMSO; (d) Pd(OAc)2, ( )BINAP, 3-aminopyridine, Cs2CO3,
toluene.
modified with imidazolyl via a nucleophilic substitution with
CDI to yield the desired final compounds 33−36. Moreover, a
common building block 38 was synthesized from 2-bromo-5-
methylpyridine using a Suzuki reaction with phenylboronic acid
followed by a Wohl−Ziegler bromination (Scheme 3). Further
Suzuki coupling of 38 with the boronic acids of the
corresponding N-containing heterocycles yielded final com-
pounds 39−49. An exception was compound 52, where m-
bromopyridyl was inserted first, followed by simultaneous
Suzuki couplings (method A) at both pyridyl moieties to
introduce the phenyl groups. It should be noted that in the
synthesis of compounds 45−49, PdCl2(dppf) was used as the
catalyst instead of Pd(PPh3)4 and microwave irradiation was
employed to facilitate the reaction (method F) because of the
low yields resulting from the conventional Suzuki coupling
conditions with thermal heating (method A). The hydroxy
compound 49 was obtained from ether cleavage of the methoxy
precursor 48. To obtain the O (55) and NH (56) isosters of
the methylene compound (39), intermediate 53 was prepared
via selective Suzuki coupling at the 2-position of 5-bromo-2-
iodopyridine. A halogen exchange converting 5-bromo to iodo
(54) was performed, and the latter compound was
subsequently coupled with 3-hydroxypyridine using CuI,
picolinic acid, and K3PO4 as catalysts to obtain the final
compound 55. For the amination of 54 with 3-aminopyridine,
Pd-BINAP and excessive Cs2CO3 as a base had to be employed
instead to give compound 56 (Scheme 4).
BIOLOGICAL RESULTS AND DISCUSSION
■
Inhibition of CYP11B1 and CYP11B2. Compounds 15−
24, 33−36, 39−49, 52, 55, and 56 were evaluated for their
inhibitory activities in V79 MZh cells expressing either human
CYP11B1 or CYP11B2. The IC50 values (n ≥ 3, relative
standard deviations <25%) are presented in Tables 1−3 in
comparison to metyrapone. As expected, the introduction of
alkyl groups onto the methylene bridge of Ref 1 (IC50 = 107
nM) significantly increased CYP11B1 inhibition up to 9-fold
(compounds 15−18; IC50 values ranging from 12 to 33 nM).
Here, the trend can be observed that with increased bulkiness
of the alkyl groups (Me < Et < c-prop < i-prop), inhibition of
CYP11B1 is enhanced. This is probably caused by occupation
of an additional hydrophobic pocket near the heme. Similarly,
introduction of alkyl substituents onto the methylene bridge of
Ref 3 (IC50 = 181 nM) led to significant increases in CYP11B1
inhibition as well (21, Me, IC50 = 21 nM; 22, c-prop, IC50 = 11
nM). By contrast, the naphthalene analogues showed similar
(23, Me, IC50 = 60 nM) or reduced (24, c-prop, IC50 = 151
nM) potency compared to the nonsubstituted Ref 4 (IC50 = 68
nM), probably indicating different binding modes of the phenyl
or thienyl compounds. Interestingly, introduction of aromatic
substituents onto the methylene bridge of Ref 1, such as 2-
furan (19, IC50 = 69 nM) and phenyl (20, IC50 = 124 nM),
entailed weaker inhibition of the target enzyme compared to
the alkyl analogues, as was similarly observed in the compound
class of Ref 2.6c However, the improvement of selectivity over
CYP11B2 via introduction of substituents onto the methylene
bridge, as seen in the compound class of Ref 2,6c was not
observed. Instead, these modifications increased CYP11B2
C
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inhibition by 3−28-fold, thus leading to a reduction of
selectivity (Table 1).
Table 2. Inhibition of CYP11B1 and CYP11B2 by
Compounds 33−36
Table 1. Inhibition of CYP11B1 and CYP11B2 by
Compounds 15−24
a
Mean value of at least three experiments. The deviations were <25%.
b
Hamster fibroblasts expressing human CYP11B1 or CYP11B2;
c
substrate 11-deoxycorticosterone, 100 nM. SF: IC50 CYP11B2
IC50 CYP11B1
sources.
/
d
.
See ref 6a, IC50 values differ due to different enzyme
reduced by the withdrawing effect of the second N. Although
both 3-pyridine and isoquinoline enhanced inhibition, only the
3-pyridyl analogue 39 preserved a similar selectivity factor of 10
compared to Ref 1 (SF = 13), whereas the isoquinoline
compound 41 exhibited a reduced selectivity of 4.
Moreover, the isosteric exchange of the methylene bridge by
O (55) or NH (56) led to a dramatic reduction of activity (IC50
values of 1015 and >5000 nM, respectively). This probably
indicates a hydrophobic region around the bridge, where these
polar moieties are not tolerated.
a
Mean value of at least three experiments. The deviations were <25%.
b
Hamster fibroblasts expressing human CYP11B1 or CYP11B2;
c
substrate 11-deoxycorticosterone, 100 nM. SF: IC50 CYP11B2
IC50 CYP11B1
sources.
/
Therefore, optimizations of Ref 1 (IC50 = 107 nM, SF = 13)
via substitution on the methylene bridge, replacement of the
central pyridine moiety by other N-containing heterocycles,
and isosteric exchange of the methylene bridge led to an
increase of CYP11B1 inhibition in some cases. However,
selectivity over CYP11B2 decreased. In contrast, the exchange
of the heme-complexing imidazolyl by 3-pyridyl resulted in a
more potent but similarly selective compound 39 (IC50 = 32
nM, SF = 10), which was regarded as a suitable scaffold for
further optimization. Additional substituents with various
profiles in terms of bulkiness, electronic properties, and H-
bond acceptor and donor characteristics were introduced to the
3-pyridyl ring. The electron withdrawing groups 5-CONH2 and
5-F (compounds 45 and 46) strongly reduced CYP11B1
inhibition by 13- and 4-fold to 427 and 125 nM, respectively,
compared to the nonsubstituted compound 39 (IC50 = 32 nM).
Similar effects have also been observed in other classes of
CYP11B inhibitors.6c,8 By contrast, compound 47 (5-CF3), as
d
. See ref 6a, IC50 values differ due to different enzyme
Replacement of the central pyridine moiety by other
heterocycles, with the N remaining at the same position to
retain selectivity6b (Table 2), reduced the potency compared to
Ref 1 (IC50 = 107 nM), as was observed with the pyridazine
(33), pyrazine (34), and pyrimidine (35) compounds. This
might be due to the electron withdrawing property of the
additional nitrogens altering the electrostatic potential of the
whole molecule. Interestingly, annulation of a benzene moiety
resulting in isoquinoline (36) slightly increased inhibition (IC50
= 87 nM) at the expense of selectivity, which was reduced to
3.5. Therefore, the pyridine moiety was considered as the most
suitable central ring balancing potency and selectivity and was
retained in the further optimization process.
Subsequently, the exchange of the heme-complexing
imidazolyl by other N-containing heterocycles was investigated
as well (Table 3). 3-Pyridine (39) and isoquinoline (41)
significantly enhanced the inhibitory potency to 32 and 6 nM,
respectively, compared to the imidazole Ref 1 (IC50 = 107 nM),
whereas the 4-pyridine compound 40 showed a similar
inhibition of CYP11B1 with an IC50 value of 98 nM. By
contrast, the exchange of imidazolyl by pyrimidine (42) led to a
reduction of inhibition (IC50 = 240 nM), probably due to the
fact that the electron density on the heme-coordinating N was
an exception, showed a similar inhibition of CYP11B1 (IC50
=
38 nM) as the nonsubstituted compound 39. However, the
electron-rich groups significantly improved the inhibitory
potency, resulting in IC50 values lower than 10 nM. The 4-
Me compound 43 showed an IC50 value of 8 nM but a reduced
selectivity of 2.4 compared to the nonsubstituted compound 39
(SF = 10). Shifting of the Me group into the 5-position
(compound 44) further elevated the inhibitory potency, leading
to an IC50 value of 2 nM, which is over 50-fold more potent
D
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QSAR model using the software Open3DQSAR.10 Compounds
17, 35, 41, 44, and 46 were used as the test set to validate the
predicting ability of the model, whereas the remaining 26
compounds including Ref 1, Ref 3, and Ref 4 were employed
as the training set. Molecular interaction fields (MIF) regarding
steric effects and electrostatic potential were calculated for all
compounds within a grid box around the molecules with a 0.5
Å step size and a 5.0 Å margin. The MIF parameters were
subsequently regressed with inhibitory potency (pIC50) using
partial least-squares analysis (PLS). The model was further
improved by using smart region definition (SRD) and fractional
factorial design (FFD) methods. The cross validation of the
model using the leave-many-out paradigm and challenging the
Table 3. Inhibition of CYP11B1 and CYP11B2 by
Compounds 39−49, 52, 55, and 56
model with the test set compounds revealed a q2
value of
L20%O
0.836 with standard deviation of error of calculation (SDEC) of
0.315 and an r2 value of 0.958 with standard deviation of error
of prediction (SDEP) of 0.207, indicating a high accuracy of
prediction (Figure 1). The 3D-QSAR model revealed that in
area 1, which is located around the distal pyridyl and opposite
to the N atom, bulky groups are advantageous (Figure 2A, area
1, green). Above the methylene bridge, medium-sized groups
are favored (Figure 2A, area 2, green), whereas right outside of
these regions, bulky substituents are prohibited (Figure 2A, area
3, yellow). Areas beneath the methylene bridge and near the sp2
hybrid N also do not tolerate bulky groups as well (Figure 2A,
area 3, yellow). Moreover, discrete regions around the heme-
coordinating pyridyl, the methylene bridge, and the core show
preference for groups with positive partial charges (Figure 2B,
cyan). In contrast, no predilection for negative partial charges
was observed at the same level. No suggestions were made
around the western aromatic moiety due to the limited number
of different rings in this area present in the training set.
According to these results, further modifications seem to be
reasonable to potentiate the inhibitory potency, such as
introduction of bulky substituents onto the meta- or para-
positions of the distal pyridine as well as hydrophobic groups of
suitable size onto the methylene bridge. However, one should
have in mind that these modifications would increase
lipophilicity of the compounds with eventual adverse effects
on pharmacokinetic properties.
a
Mean value of at least three experiments. The deviations were <25%.
b
Hamster fibroblasts expressing human CYP11B1 or CYP11B2;
c
substrate 11-deoxycorticosterone, 100 nM. SF: IC50 CYP11B2
IC50 CYP11B1
sources.
/
d
. See ref 6a, IC50 values differ due to different enzyme
Selectivities over CYP17 and CYP19. Because of the
crucial roles of CYP17 and CYP19 in the biosynthesis of
androgens and estrogens, the inhibitors of CYP1711 and
CYP1912 have been applied in clinic for the treatment of
prostate and breast cancer, respectively. However, as for
CYP11B1 inhibitors, the inhibition of these enzymes would
lead to severe side effects due to the same reason. The
selectivities over CYP17 and CYP19 are therefore important
safety criteria, and inhibition of these enzymes by the selected
compounds 15, 39, and 44 was determined (Table 4). As the
tested inhibitors exhibited IC50 values below 35 nM toward
human CYP11B1, a more than 50-fold higher concentration (2
μM) was regarded as sufficient for the determination of CYP17
and CYP19 inhibition. Compound 15 showed only 13%
inhibition of CYP17 at a concentration of 2 μM, whereas
compounds 39 and 44 were considered as inactive as they
exhibited no significant inhibition (≤5% at 2 μM). Because
these compounds are very potent against CYP11B1 (IC50 < 35
nM), they were regarded to be selective over CYP17. As for
CYP19, none of the compounds showed inhibitory effects at a
concentration of 2 μM. These selectivity profiles guarantee no
interference with the production of androgens and estrogens
under therapeutic doses.
than Ref 1. More important is that the selectivity was also
enhanced to 15. 5-Ph substitution at the pyridyl led to the most
potent compound in this study (compound 52, IC50 = 1.3 nM),
albeit with a reduced SF of 0.4. These results are probably
caused by the bulky phenyl group occupying an additional
hydrophobic pocket near the heme, leading to the elevated
inhibition of both CYP11B1 and CYP11B2. As expected, a 5-
OMe substituent (48) improved the inhibition to 5 nM,
whereas the SF was reduced to 5.1. By contrast, compound 49,
hydroxylated in the 5-position, exhibited a slightly reduced
inhibition of CYP11B1 (IC50 = 51 nM) compared to the parent
compound but achieved the highest SF in this study of 16.4.
The optimization process yielded many compounds that are
more potent than the lead compound Ref 1. Some compounds
(18, 22, 41, 43, 44, 48, and 52) are more potent than the
clinically used drug metyrapone (IC50 = 15 nM, SF = 4.8).
More importantly, among them, compound 44 is also more
selective than metyrapone (SF = 15).
3D-QSAR Study. The CYP11B1 crystal structure is not
resolved yet, however, a homology model was reported before.9
We decided to employ a ligand based approach to further
illustrate the structure−activity relationship and built a 3D-
E
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Figure 1. Predicted vs determined pIC50 values for both training set (black) and test set compounds (purple).
Table 4. Inhibition of CYP17 and CYP19 by Compounds 15,
39, and 44.
b
inhibition (%)
a,
c
a,d
compd
ref 1
CYP17
CYP19
2
13
4
0
1
0
1
0
15
39
44
5
metyrapone
3
a
Mean value of at least three experiments. The deviations were <25%.
b
Compounds concentration 2 μM. Inhibition values ≤5% are not
c
significant. E. coli expressing human CYP17; substrate progesterone,
25 μM. Human placental CYP19; substrate androstenedione, 500
d
nM.
Inhibition of Rat CYP11B1. As Ref 1 showed a poor
inhibition of the rat enzyme (IC50 > 10000 nM), compound 44
was evaluated for rat CYP11B1 inhibition to identify a new
candidate for in vivo experiments in rats. Indeed, compound 44
not only elevated the inhibition of human CYP11B1 but also
enhanced the inhibition of the rat enzyme to an IC50 of 2440
nM (mean value of more than three repeats). As we have
observed with aldosterone synthase (CYP11B2) inhibitors, in
vitro activities in this range are sufficient to elicit biological
effects in rats.5a
In Vitro Metabolic Stability. Compound 44 showed a
moderate metabolic stability toward rat liver S9 fraction with a
half−life (t1/2) of 16 min (mean value of more than three
repeats) as well as an extraordinary metabolic stability toward
human liver S9 fraction (t1/2 > 150 min) and human and rat
plasma (t1/2 > 150 min). These properties make compound 44
an appropriate candidate for further proof of concept in rats.
Cytotoxicity. The cytotoxicity of compound 44 was
evaluated in HEK293 cells with doxorubicin as positive control
and rifampicin as negative control. After 24 h of incubation, the
compounds showed LC50 values of 61.4, 4.3, and >100 μM
(mean value of more than three repeats), respectively. Thus,
the cytotoxic effect of compound 44 was only weak and should
Figure 2. Illustration of the PLS pseudocoefficient contour maps of
the 3D-QSAR model with the superposed compounds from both
training and test sets. (A) Steric effects. Green (level: 0.015) indicates
bulky groups are favored, whereas yellow (level: −0.015) indicates
bulky moieties are prohibited. (B) Electrostatic potential. Cyan (level:
0.005) indicates groups with positive partial charges are favored.
Selectivities over Hepatic CYP2A6 and CYP3A4.
Because CYP2A6 and CYP3A4 play important roles in the
metabolism of xenobiotics and drugs, compound 44 was tested
for inhibition of these enzymes. 44 showed IC50 values of 106
μM for CYP2A6 and 1.1 μM for CYP3A4, respectively. In spite
of the moderate CYP3A4 inhibition, the safety margin should
be sufficient due to the high potency toward CYP11B1 (IC50
=
2 nM, factor of 550).
F
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incubated with mammalian plasma (pooled, heparinized) of the
indicated species at 37 °C for 0, 10, 30, 60, and 150 min. The
incubation was stopped by precipitation of plasma proteins with 5
volumes of cold acetonitrile containing an internal standard, and the
remaining compound concentration was analyzed by LC-MS/MS.
Cytotoxicity Assay. HEK 293 cells (2 × 105 cells per well) were
seeded in 24-well flat-bottom plates. Culturing of cells, incubations,
and OD measurements were performed as described previously16 with
minor modifications. Twenty-four h after seeding the cells, the
incubation was started by the addition of compounds in a final DMSO
concentration of 1%. The living cell mass was determined after 24 h,
followed by the calculation of LC50 values.
not give rise to problems in vivo due to the strong in vitro
CYP11B1 inhibitory activity of 44 and the low therapeutic
doses necessary.
CONCLUSION
■
For a better management of Cushing’s syndrome, CYP11B1
inhibitors that are more potent and selective than the clinically
used drug metyrapone are urgently needed. Optimizations have
been performed with the previously identified CYP11B1
inhibitor Ref 1 regarding the potencies toward the human
enzyme as well as the selectivity over CYP11B2 (SF = 13).
Modifications such as the introduction of various substituents
onto the methylene bridge and the heme Fe-complexing pyridyl
ring, replacement of the central pyridine moiety and the
imidazolyl ring by other heterocycles and the isosteric exchange
of the methylene bridge with NH or O have been performed.
These efforts resulted in potent CYP11B1 inhibitors with IC50
values lower than 10 nM (compounds 41, 43, 44, 48, and 52).
Among them, compound 44 turned out to be the best one, with
an IC50 value of 2 nM toward CYP11B1 (50-fold more potent
than Ref 1). This compound also showed selectivities over
CYP11B2 (SF = 15), CYP17, and CYP19 as well as hepatic
CYP2A6 and CYP3A4. To facilitate proof of concept (in vivo)
studies with the best compound 44, inhibition of rat CYP11B1
was investigated and a moderate activity was found (IC50 of
2440 nM, Ref 1 > 10000). Satisfying metabolic stabilities in
both plasma and liver S9 fractions from human and rat origin as
well as a negligible cytotoxicity were observed. Because these
profiles are clearly superior to that of metyrapone and Ref 1,
compound 44 can be considered as a candidate for further in
vivo studies in rats.
Chemistry. General Experimental. 1H NMR and 13C spectra were
recorded on a Bruker DRX-500 instrument. Chemical shifts are given
in parts per million (ppm), and spectra are obtained from DMSO-d6
or CDCl3 solutions (reported in ppm), using chloroform as the
reference standard (7.26 ppm) or DMSO-d6 (2.50 ppm). The
following abbreviations are used to denote signal multiplicities: s =
singlet, d = doublet, t = triplet, and m = multiplet. All coupling
constants (J) are given in hertz (Hz). Mass spectra (LC/MS) were
measured on an MSQ electro spray mass spectrometer (Thermo-
Fisher, Dreieich, Germany). An RP-C18 NUCLEODUR 100-5 (125
mm × 3 mm) column (Macherey-Nagel GmbH, Duren, Germany)
̈
was used as stationary phase. All solvents were HPLC grade. The
system was operated by the standard software Xcalibur. In a gradient
run, the percentage of acetonitrile (containing 0.1% trifluoroacetic
acid) was increased from an initial concentration of 0% at 0 min to
100% at 15 min and kept at 100% for 5 min. The injection volume was
10 μL, and the flow rate was set to 800 μL/min. MS analysis was
carried out at a spray voltage of 3800 V, a capillary temperature of 350
°C, and a source CID of 10 V. Spectra were acquired in positive mode
from 100 to 1000 m/z and at 254 nm for the UV trace. The relative
peak area in the UV chromatogram was used to determine the purity
of the compounds. The purity of all compounds was ≥95%. Reagents
and solvents were used as obtained from commercial suppliers without
further purification or drying. Yields refer to purified products and are
not optimized. Flash chromatography was performed on silica gel 40
(35/40−63/70 μM) with petroleum ether/ethyl acetate mixtures as
eluents, and the reaction progress was determined by thin-layer
chromatography analyses on Alugram SIL G/UV254 (Macherey
Nagel). Visualization was accomplished with UV light.
EXPERIMENTAL SECTION
■
Biological Test Procedures. Inhibition of CYP11B1 and
CYP11B2. V79MZh cells expressing human or rat CYP11B1 or
CYP11B2 were incubated with [1,2-3H]-11-deoxycorticosterone (100
nM) as the substrate and the inhibitor at different concentrations. The
assay was performed as previously described.13
Method A: Suzuki Coupling. The corresponding brominated
aromatic compound (1 equiv) and the boronic acid (1.5 equiv)
were dissolved in toluene (10 mL), ethanol (10 mL), and aq Na2CO3
(2.0 M, 2.5 mL). The mixture was degassed under reduced pressure
and flushed with N2 for three times before Pd(PPh3)4 (5 mol %) was
added. The resulting suspension was then heated under reflux for 4 h.
After cooling down, the phases were separated, and the aqueous phase
was extracted two times with EtOAc. The combined organic extracts
were dried over MgSO4 and concentrated under reduced pressure to
give the crude product, which was purified with flash chromatography
on silica gel.
Method B: Grignard Reaction. To a solution of the Grignard
reagent (2 equiv) in dry diethyl ether (10 mL), the corresponding
carbonyl compound (1 equiv) in dry diethyl ether (5 mL) was added
dropwise. The reaction mixture was heated to reflux for 2 h. Afterward,
ice was added followed by the addition of HCl (1 M) until the
resulting precipitate disappeared. The phases were separated, and the
aqueous phase was extracted twice with diethyl ether. The combined
organic layers were washed with saturated sodium hydrogen carbonate
solution and brine. After drying over MgSO4 and concentration under
vacuum, the crude product was purified by flash chromatography on
silica gel.
CYP17 Preparation and Assay. Human CYP17 was expressed in E.
coli (coexpressing human CYP17 and rat NADPH-P450 reductase),
and the assay was performed using the method previously described
with progesterone (25 μM) as the substrate and NADPH as the
cofactor.14
CYP19 Preparation and Assay. Human CYP19 was obtained from
microsomal preparations of human placenta, and the assay was
performed using the 3H2O-method as previously described with
[1β-3H]androstenedione (500 nM) as the substrate.15
CYP2A6 and CYP3A4 Assay. The inhibition of CYP2A6 was
determined using the CYP2A6/coumarin inhibitor screening kit (BD
Gentest) with coumarin (3 μM) as the substrate. Similarly, the
inhibition of CYP3A4 was determined using the CYP3A4/BFC
inhibitor screening kit (BD Gentest) with 7-benzyloxy-trifluorome-
thylcoumarin (50 μM) as the substrate. Both assays were performed
according to the manufacturer’s instruction.
Metabolic Stability Tests in Human and Rat Liver S9 Fraction.
For the evaluation of phase I and II metabolic stability, the compound
(1 μM) was incubated with 1 mg/mL pooled mammalian liver S9
fraction (BD Gentest), 2 mM NADPH regenerating system, 1 mM
UDPGA, and 0.1 mM PAPS at 37 °C for 0, 5, 15, and 60 min. The
incubation was stopped by precipitation of S9 enzymes with 2 volumes
of cold acetonitrile containing internal standard. Concentration of the
remaining test compound at the different time points was analyzed by
LC-MS/MS and used to determine half−life (t1/2) and intrinsic
clearance (CLint).
Method C: CDI Reaction. To a solution of the corresponding
alcohol (1 equiv) in NMP, CDI (5 equiv) was added. Then the
solution was heated to reflux for 16 h. After cooling to room
temperature, the reaction mixture was diluted with EtOAc and washed
with water and brine. The organic phase was dried over MgSO4 and
concentrated under vacuum. The crude product was purified by flash
chromatography on silica gel.
Metabolic Stability Tests in Human and Rat Plasma. For the
evaluation of mammalian plasma stability, the compound (1 μM) was
G
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Method D: Wohl−Ziegler Bromination. The methyl heteroar-
omatic compound was dissolved in 20 mL of dry carbon tetrachloride.
To this solution, N-bromsuccinimide (NBS) (1.1 equiv) and benzoyl
peroxide (5 mol %) were added and the mixture was refluxed
overnight. After cooling, the succinimide was removed by filtration and
the filtrate was concentrated under vacuum. The crude product was
purified by flash chromatography on silica gel.
Method E: SN Reaction. K2CO3 (5 equiv), imidazole (4 equiv), and
the corresponding methyl heteroaromatic bromide were suspended in
DMF (1 mL/mmol) or acetonitrile (1 mL/mmol). The resulting
mixture was heated to 120 °C for 2 h. After cooling, water (10 mL)
was added and the aqueous layer was extracted with EtOAc (3 × 10
mL). The combined organic layers were washed with brine (10 mL),
dried over MgSO4, and evaporated in vacuo. The crude product was
purified by column chromatography using SiO2.
Method F: Suzuki Coupling Using Microwave Irradiation. A
mixture of brominated aromatic compound (1 equiv), corresponding
boronic acid or boronic acid pinacolester (1.2 equiv), Cs2CO3 (3
equiv), and PdCl2(dppf) (5 mol %) were dissolved in DME/H2O/
EtOH (1 mL/1 mL/1 mL). The reaction mixture was stirred for 20
min at 150 °C, 150 W, and 18 bar in the microwave oven. After
addition of H2O (10 mL) and extraction with ethyl acetate (3 × 15
mL), the combined organic phases were dried over MgSO4 and
concentrated under reduced pressure. The purification was performed
by flash chromatography using SiO2. After flash chromatography, the
product was dissolved in ethyl acetate and a few drops of conc HCl
and water were added. After stirring for 30 min the phases were
separated and aqueous phase was neutralized with aqueous Na2CO3
solution (2M). After extraction with ethyl acetate and drying over
MgSO4, the solvent was removed under vacuum.
1.4 Hz, 1H), 7.11−7.15 (m, 1H), 7.40−7.51 (m, 4H), 7.64 (s, 1H),
7.71 (dd, J = 8.2, 0.6 Hz, 1H), 7.95−8.00 (m, 2H), 8.58 (dd, J = 1.6,
0.6 Hz, 1H). MS (ESI): m/z = 250.26 [M + H]+.
5-(1-(1H-Imidazol-1-yl)propyl)-2-phenylpyridine (16). Synthesized
using compound 6 (275 mg, 1.29 mmol), CDI (1.05 g, 6.45 mmol),
and NMP (10 mL) according to method E. Crude product was
purified by flash chromatography on silica gel using a mixture of ethyl
acetate/methanol (9:1) as eluent. Beige solid. Yield: 114 mg, 34%.
1
Melting point: 103−104 °C (ethyl acetate). H NMR (CDCl3, 500
MHz): δH (ppm) = 0.76 (t, J = 7.3 Hz, 3H), 1.98−2.13 (m, 2H), 4.87
(t, J = 7.7 Hz, 1H), 6.75 (t, J = 1.1 Hz, 1H), 6.89 (s, 1H), 7.16−7.33
(m, 4H), 7.41 (s, 1H), 7.47 (d, J = 8.2 Hz, 1H), 7.71−7.79 (m, 2H),
8.37 (d, J = 2.2 Hz, 1H). MS (ESI): m/z = 264.37 [M + H]+.
5-(Cyclopropyl(1H-imidazol-1-yl)methyl)-2-phenylpyridine (17).
Synthesized using compound 7 (200 mg, 0.89 mmol), CDI (720
mg, 4.44 mmol), and acetonitrile (12 mL) according to method C.
Crude product was purified by flash chromatography on silica gel using
a mixture of hexane/ethyl acetate (1:3) as eluent. White solid. Yield:
1
45 mg, 18%. Melting point: 122−124 °C (ethyl acetate). H NMR
(CDCl3, 500 MHz): δH (ppm) = 0.50−0.61 (m, 2H), 0.81−0.95 (m,
2H), 1.58 (m, 1H), 4.49 (d, J = 9.5 Hz, 1H), 6.98 (t, J = 1.3 Hz, 1H),
7.13 (t, J = 0.9 Hz, 1H), 7.40−7.51 (m, 4H), 7.72 (dd, J = 9.9, 1.4 Hz,
2H), 7.97−8.02 (m, 2H), 8.63 (d, J = 2.2 Hz, 1H). MS (ESI): m/z =
276.28 [M + H]+.
5-(1-(1H-Imidazol-1-yl)-2-methylpropyl)-2-phenylpyridine (18).
Synthesized using compound 8 (231 mg, 1.02 mmol), CDI (825
mg, 5.09 mmol), and NMP (10 mL) according to method C. Crude
product was purified by flash chromatography on silica gel using a
mixture of ethyl acetate/methanol (9:1) as eluent. Beige solid. Yield:
1
71 mg, 25%. Melting point: 127−129 °C (ethyl acetate). H NMR
6-Bromonicotinaldehyde (1). To a suspension of 2,5-dibromopyr-
idine (2.00 g, 8.44 mmol) in dry diethyl ether (25 mL) was added n-
BuLi (3.55 mL, 8.87 mmol, 2.5 M solution in hexane) at −80 °C
under a nitrogen atmosphere. After stirring for 1 h at −80 °C, dry
DMF (0.68 mg, 9.28 mmol) was added. Reaction mixture was stirred
for an additional hour at −80 °C, warmed slowly to 0 °C, and HCl
(18.0 mL, 1 M) was added. After stirring for 15 min, the phases were
separated and aqueous layer was extracted twice with diethyl ether.
The combined organic layers were washed with water (50 mL) and
brine (50 mL) and dried over MgSO4. The organic phase was
concentrated under reduced pressure, and the crude product was
purified by flash chromatography on silica gel using a mixture of
hexane/ethyl acetate (8:1) as eluent. White solid. Yield: 1.03 g, 66%.
1H NMR (CDCl3, 500 MHz): δH (ppm) = 7.67−7.71 ppm (m, 1H),
8.02 (dd, J = 8.2, 2.5 Hz, 1H), 8.84 (dd, J = 2.5, 0.6 Hz, 1H), 10.10 (s,
1H). MS (ESI): m/z = 187.19 [M + H]+.
(CDCl3, 500 MHz): δH (ppm) = 0.93−1.02 (m, 6H), 2.59−2.66 (m,
1H), 4.72 (d, J = 10.4 Hz, 1H), 7.02−7.07 (m, 1H), 7.09 (s, 1H),
7.40−7.51 (m, 3H), 7.62−7.70 (m, 2H), 7.70−7.76 (m, 1H), 7.95−
8.02 (m, 2H), 8.67 (d, J = 2.2 Hz, 1H). MS (ESI): m/z = 278.41 [M +
H]+.
5-(Furan-2-yl(1H-imidazol-1-yl)methyl)-2-phenylpyridine (19).
Synthesized using compound 9 (631 mg, 2.51 mmol), CDI (2.04 g,
12.56 mmol), and NMP (4 mL) according to method C. Crude
product was purified by flash chromatography on silica gel using ethyl
1
acetate/methanol (9:1) as eluent. Brown oil. Yield: 276 mg, 37%. H
NMR (CDCl3, 500 MHz): δH (ppm) = 6.25−6.29 (m, 1H), 6.42 (dd,
J = 3.5, 1.9 Hz, 1H), 6.55 (s, 1H), 6.95 (t, J = 1.3 Hz, 1H), 7.14 (t, J =
1.1 Hz, 1H), 7.42−7.57 (m, 6H), 7.75 (dd, J = 8.5, 0.6 Hz, 1H), 7.97−
8.04 (m, 2H), 8.54 (dt, J = 1.6, 0.8 Hz, 1H). MS (ESI): m/z = 301.96
[M + H]+.
5-((1H-Imidazol-1-yl)(phenyl)methyl)-2-phenylpyridine (20). Syn-
thesized using compound 10 (299 mg, 1.14 mmol), CDI (928 mg,
5.72 mmol), and NMP (8 mL) according to method C. Crude product
was purified by flash chromatography on silica gel using ethyl acetate
as eluent. Beige solid. Yield: 75 mg, 21%. Melting point: 124−126 °C
6-Phenylnicotinaldehyde (2). Synthesized using compound 1 (1.20
g, 6.44 mmol) and phenylboronic acid (1.18 g, 9.65 mmol) according
to method A. Crude product was purified by flash chromatography on
silica gel using a mixture of hexane/ethyl acetate (10:1) as eluent.
1
1
Light-yellow solid. Yield: 1.10 g, 94%. H NMR (CDCl3, 500 MHz):
(ethyl acetate). H NMR (CDCl3, 500 MHz): δH (ppm) = 6.58 (s,
δH (ppm) = 7.44−7.52 ppm (m, 3H), 7.87 (d, J = 8.2 Hz, 1H), 8.04−
8.08 (m, 2H), 8.20 (dd, J = 8.2, 2.2 Hz, 1H), 9.10 (dd, J = 2.2, 0.6 Hz,
1H), 10.11 (s, 1H). MS (ESI): m/z = 184.31 [M + H]+.
1H), 6.87−6.91 (m, 1H), 7.11−7.19 (m, 3H), 7.34−7.54 (m, 8H),
7.73 (d, J = 8.2 Hz, 1H), 7.98−8.04 (m, 2H), 8.49 (d, J = 2.5 Hz, 1H).
MS (ESI): m/z = 312.01 [M + H]+.
1-(6-Phenylpyridin-3-yl)ethanol (5). Synthesized using compound
2 (210 mg, 1.15 mmol) and methylmagnesium bromide (2.29 mL,
2.29 mmol, 1 M in THF) according to method B. Crude product was
purified by flash chromatography on silica gel using a mixture of
hexane/ethyl acetate (2:1) as eluent. Light-yellow solid. Yield: 201 mg,
5-(1-(1H-Imidazol-1-yl)ethyl)-2-(thiophen-3-yl)pyridine (21). Syn-
thesized using compound 11 (227 mg, 1.11 mmol), CDI (897.0 mg,
5.53 mmol), and NMP (8 mL) according to method C. Crude product
was purified by flash chromatography on silica gel using ethyl acetate/
1
methanol (9:1) as eluent. Brown oil. Yield: 78 mg, 28%. H NMR
1
88%. H NMR (CDCl3, 500 MHz): δH (ppm) = 1.51 (d, J = 6.6 Hz,
(CDCl3, 500 MHz): δH (ppm) = 1.75 (d, J = 6.9 Hz, 3H), 5.25 (q, J =
7.0 Hz, 1H), 6.79 (t, J = 1.3 Hz, 1H), 6.96 (t, J = 0.9 Hz, 1H), 7.21−
7.28 (m, 2H), 7.40−7.52 (m, 3H), 7.74 (dd, J = 2.8, 1.3 Hz, 1H), 8.34
(d, J = 2.5 Hz, 1H). MS (ESI): m/z = 256.08 [M + H]+.
5-(Cyclopropyl(1H-Imidazol-1-yl)methyl)-2-(thiophen-3-yl)-
pyridine (22). Synthesized using compound 12 (116 mg, 0.50 mmol),
CDI (407 mg, 2.51 mmol), and NMP (5 mL) according to method C.
Crude product was purified by flash chromatography on silica gel using
ethyl acetate as eluent. Brown oil. Yield: 70 mg, 50%. 1H NMR
(CDCl3, 500 MHz): δH (ppm) = 0.49−0.60 (m, 2H), 0.83−0.91 (m,
2H), 1.53−1.64 (m, 1H), 4.47 (d, J = 9.5 Hz, 1H), 6.97 (t, J = 1.3 Hz,
3H), 3.05 (br, s, 1H), 4.92 (q, J = 6.6 Hz, 1H), 7.38−7.50 (m, 3H),
7.65 (d, J = 7.9 Hz, 1H), 7.74 (dd, J = 8.2, 2.2 Hz, 1H), 7.91−7.97 (m,
2H), 8.58 (d, J = 2.2 Hz, 1H). MS (ESI): m/z = 200.32 [M + H]+.
5-(1-(1H-Imidazol-1-yl)ethyl)-2-phenylpyridine (15). Synthesized
using compound 5 (164 mg, 0.82 mmol), CDI (667 mg, 4.12 mmol),
and NMP (8 mL) according to method C. Crude product was purified
by flash chromatography on silica gel using a mixture of ethyl acetate/
methanol (9:1) as eluent. White solid. Yield: 20 mg, 10%. Melting
1
point: 64−67 °C (ethyl acetate). H NMR (CDCl3, 500 MHz): δH
(ppm) = 1.93 (d, J = 7.3 Hz, 3H), 5.45 (q, J = 6.9 Hz, 1H), 6.97 (t, J =
H
dx.doi.org/10.1021/jm400240r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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1H), 7.12 (t, J = 1.1 Hz, 1H), 7.39−7.46 (m, 2H), 7.59−7.63 (m, 1H),
7.64−7.68 (m, 1H), 7.72 (s, 1H), 7.91 (dd, J = 3.2, 1.3 Hz, 1H), 8.56
(dd, J = 1.6, 0.6 Hz, 1H). MS (ESI): m/z = 282.30 [M + H]+.
5-(1-(1H-Imidazol-1-yl)ethyl)-2-(naphthalen-1-yl)pyridine (23).
Synthesized using compound 13 (147 mg, 0.59 mmol), CDI (478
mg, 2.95 mmol), and NMP (6 mL) according to method C. Crude
product was purified by flash chromatography on silica gel using ethyl
acetate as eluent. Brown oil. Yield: 59 mg, 33%. 1H NMR (CDCl3, 500
MHz): δH (ppm) = 1.97 (d, J = 7.3 Hz, 3H), 5.50 (q, J = 6.9 Hz, 1H),
7.02−7.06 (m, 1H), 7.16 (s, 1H), 7.45−7.63 (m, 6H), 7.69 (s, 1H),
7.92 (td, J = 4.9, 2.5 Hz, 2H), 8.06 (dd, J = 8.2, 0.9 Hz, 1H), 8.68 (d, J
= 2.2 Hz, 1H). MS (ESI): m/z = 300.03 [M + H]+.
aqueous phase was neutralized with aqueous Na2CO3 solution (2M).
After extraction with ethyl acetate and drying over MgSO4, the solvent
was removed under vacuum. Beige solid. Yield: 140 mg, 40%. Melting
1
point: 72−75 °C (ethyl acetate). H NMR (CDCl3, 500 MHz): δH
(ppm) = 3.77 (s, 2H), 6.88−6.93 (m, 2H), 7.15−7.21 (m, 1H), 7.21−
7.31 (m, 3H), 7.45 (dd, J = 8.2, 0.9 Hz, 1H), 7.72−7.78 (m, 2H),
8.28−8.33 (m, 2H), 8.33−8.37 (m, 1H). MS (ESI): m/z = 246.85 [M
+ H]+.
4-((6-Phenylpyridin-3-yl)methyl)isoquinoline (41). Synthesized
using compound 38 (109 mg, 0.44 mmol) and 4-isoquinolineboronic
acid (114 mg, 0.66 mmol) according to method A. Crude product was
purified by flash chromatography on silica gel using a mixture of
hexane/ethyl acetate (3:1) as eluent. After flash chromatography, the
product was dissolved in ethyl acetate and a few drops of conc HCl
and water were added. After stirring for 30 min, the phases were
separated and aqueous phase was neutralized with aqueous Na2CO3
solution (2M). After extraction with ethyl acetate and drying over
MgSO4, the solvent was removed under vacuum. Beige solid. Yield: 20
mg, 19%. Melting point: 133−136 °C (ethyl acetate). 1H NMR
(CDCl3, 500 MHz): δH (ppm) = 4.43 (s, 2H), 7.37−7.54 (m, 4H),
7.59−7.65 (m, 2H), 7.65−7.75 (m, 1H), 7.89−8.07 (m, 4H), 8.48 (s,
1H), 8.65−8.70 (m, 1H), 9.23 (s, 1H). MS (ESI): m/z = 296.95 [M +
H]+.
5-((4-Methylpyridin-3-yl)methyl)-2-phenylpyridine (43). Synthe-
sized using compound 38 (100 mg, 0.40 mmol) and 4-methylpyridine-
3-boronic acid (83 mg, 0.61 mmol) according to method A. Crude
product was purified by flash chromatography on silica gel using ethyl
acetate as eluent. After flash chromatography, the product was
dissolved in ethyl acetate and a few drops of conc HCl and water were
added. After stirring for 30 min, the phases were separated and
aqueous phase was neutralized with aqueous Na2CO3 solution (2M).
After extraction with ethyl acetate and drying over MgSO4, the solvent
was removed under vacuum. Light-yellow solid. Yield: 54 mg, 52%.
Melting point: 82−84 °C (ethyl acetate). 1H NMR (CDCl3, 500
MHz): δH (ppm) = 2.22−2.29 (m, 3H), 4.02 (s, 2H), 7.11 (d, J = 5.0
Hz, 1H), 7.37−7.51 (m, 4H), 7.64 (dd, J = 8.2, 0.6 Hz, 1H), 7.95−
8.01 (m, 2H), 8.38−8.46 (m, 2H), 8.52−8.57 (m, 1H). MS (ESI): m/z
= 260.85 [M + H]+.
5-(Cyclopropyl(1H-imidazol-1-yl)methyl)-2-(naphthalen-1-yl)-
pyridine (24). Synthesized using compound 14 (210 mg, 0.76 mmol),
CDI (619 mg, 3.82 mmol), and NMP (8 mL) according to method C.
Crude product was purified by flash chromatography on silica gel using
a mixture of ethyl acetate/methanol (9:1) as eluent. Brown oil. Yield:
1
38 mg, 15%. H NMR (CDCl3, 500 MHz): δH (ppm) = 0.54−0.66
(m, 2H), 0.86−0.98 (m, 2H), 1.61−1.68 (m, 1H), 4.54 (d, J = 9.5 Hz,
1H), 7.06 (s, 1H), 7.17 (s, 1H), 7.46−7.64 (m, 6H), 7.78 (s, 1H),
7.89−7.97 (m, 2H), 8.06−8.12 (m, 1H), 8.75 (dd, J = 1.3, 0.6 Hz,
1H). MS (ESI): m/z = 325.96 [M + H]+.
4-((1H-Imidazol-1-yl)methyl)-1-phenylisoquinoline (36). Synthe-
sized using compound 32 (110 mg, 0.37 mmol), imidazole (101 mg,
1.48 mmol), and K2CO3 (256 mg, 1.85 mmol) in DMF according to
method E. Crude product was purified by flash chromatography on
silica gel using a mixture of ethyl acetate/methanol (9:1) as eluent.
White solid. Yield: 30 mg, 28%. Melting point: 138−140 °C (ethyl
1
acetate). H NMR (CDCl3, 500 MHz): δH (ppm) = 5.56 (s, 2 H),
6.96 (m, 1 H), 7.09 (m, 1 H), 7.49−7.60 (m, 4 H), 7.62 (s, 1 H),
7.66−7.75 (m, 3 H), 7.86 (d, J = 8.2 Hz, 1 H), 8.17 (d, J = 8.5 Hz, 1
H), 8.49 (s, 1 H). MS (ESI): m/z = 285.96 [M + H]+.
5-Methyl-2-phenylpyridine (37). Synthesized using 2-bromo-5-
methylpyridine (2.92 g, 16.95 mmol) and phenylboronic acid (3.09 g,
25.4 mmol) according to method A. Crude product was purified by
flash chromatography on silica gel using a mixture of hexane/ethyl
1
acetate (8:1) as eluent. White solid. Yield: 1.99 g, 70%. H NMR
(CDCl3, 500 MHz): δH (ppm) = 2.38 (s, 3H), 7.37−7.43 (m, 1H),
7.44−7.51 (m, 2H), 7.54−7.60 (m, 1H), 7.60−7.67 (m, 1H), 7.95−
8.00 (m, 2H), 8.51−8.55 (m, 1H). (ESI): m/z = 169.97 [M + H]+.
5-(Bromomethyl)-2-phenylpyridine (38). Synthesized using com-
pound 37 (760 mg, 4.49 mmol), NBS (878 mg, 4.93 mmol), and
DBPO (54 mg, 0.23 mmol) in carbon tetrachloride according to
method D. Crude product was purified by flash chromatography on
silica gel using hexane/ethyl acetate (8:1) as eluent. White solid. Yield:
297 mg, 73%. 1H NMR (CDCl3, 500 MHz): δH (ppm) = 4.54 (s, 2H),
7.42−7.53 (m, 3H), 7.71−7.77 (m, 1H), 7.80−7.85 (m, 1H), 7.98−
8.04 (m, 2H), 8.72−8.76 (m, 1H). (ESI): m/z = 249.66 [M + H]+.
2-Phenyl-5-(pyridin-3-ylmethyl)pyridine (39). Synthesized using
compound 38 (100 mg, 0.40 mmol) and 3-pyridineboronic acid (74
mg, 0.61 mmol) according to method A. Crude product was purified
by flash chromatography on silica gel using a mixture of hexane/ethyl
acetate (2:1) as eluent. After flash chromatography, the product was
dissolved in ethyl acetate, and a few drops of conc HCl and water were
added. After stirring for 30 min, the phases were separated and
aqueous phase was neutralized with aqueous Na2CO3 solution (2M).
After extraction with ethyl acetate and drying over MgSO4, the solvent
was removed under vacuum. Beige solid. Yield: 32 mg, 32%. Melting
5-((5-Methylpyridin-3-yl)methyl)-2-phenylpyridine (44). Synthe-
sized using compound 38 (70 mg, 0.28 mmol) and 5-methylpyridine-
3-boronic acid (58 mg, 0.42 mmol) according to method A. Crude
product was purified by flash chromatography on silica gel using ethyl
acetate as eluent. After flash chromatography, the product was
dissolved in ethyl acetate and a few drops of conc HCl and water were
added. After stirring for 30 min, the phases were separated and
aqueous phase was neutralized with aqueous Na2CO3 solution (2M).
After extraction with ethyl acetate and drying over MgSO4, the solvent
was removed under vacuum. Light-yellow solid. Yield: 56 mg, 77%.
Melting point: 79−81 °C (ethyl acetate). 1H NMR (CDCl3, 500
MHz): δH (ppm) = 2.30 (d, J = 0.6 Hz, 3H), 3.98 (s, 2H), 7.28−7.32
(m, 1H), 7.38−7.44 (m, 1H), 7.44−7.55 (m, 3H), 7.67 (dd, J = 8.0,
0.8 Hz, 1H), 7.96 − 8.01 (m, 2H), 8.36 (d, J = 1.6 Hz, 1H), 8.33 (d, J
= 1.6 Hz, 1H), 8.58 (dd, J = 1.6, 0.6 Hz, 1H). MS (ESI): m/z = 260.84
[M + H]+.
5-((6-Phenylpyridin-3-yl)methyl)nicotinamide (45). Synthesized
using compound 38 (57.0 mg, 0.23 mmol) and 5-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)nicotinonitrile (64.0 mg, 0.28
mmol), Cs2CO3 (225 mg, 0.7 mmol), and PdCl2(dppf) (8.40 mg,
0.01 mmol) according to method F. Crude product was purified by
flash chromatography on silica gel using a mixture of ethyl acetate/
methanol (10:1) as eluent. White solid. Yield: 18.0 mg, 27%. Meltiong
1
point: 86−88 °C (ethyl acetate). H NMR (CDCl3, 500 MHz): δH
(ppm) = 3.80 (s, 2H), 6.99−7.06 (m, 1H), 7.15−7.20 (m, 1H), 7.21−
7.35 (m, 4H), 7.45 (d, J = 8.2 Hz, 1H), 7.72−7.77 (m, 2H), 8.28 (dd, J
= 4.7, 1.3 Hz, 1H), 8.36 (d, J = 1.9 Hz, 1H), 8.33 (d, J = 1.9 Hz, 1H).
MS (ESI): m/z = 246.98 [M + H]+.
1
point: 170−173 °C (ethyl acetate). H NMR (DMSO-d6, 500 MHz):
δH (ppm) = 4.11 (s, 2 H), 7.36−7.53 (m, 3 H), 7.58 (s, 1 H), 7.76 (dd,
J = 8.2, 1.9 Hz, 1 H), 7.90 (d, J = 7.9 Hz, 1 H), 8.05 (d, J = 7.3 Hz, 2
H), 8.08−8.22 (m, 2 H), 8.61−8.68 (m, 1 H), 8.71 (d, J = 1.6 Hz, 1
H), 8.90 (d, J = 1.6 Hz, 1 H); MS (ESI): m/z = 290.04 [M + H]+.
5-((5-Fluoropyridin-3-yl)methyl)-2-phenylpyridine (46). Synthe-
sized using compound 38 (57.0 mg, 0.23 mmol) and 5-
fluoropyridine-3-boronic acid (39.0 mg, 0.28 mmol), Cs2CO3 (225
mg, 0.7 mmol), and PdCl2(dppf) (50 mg, 0.07 mmol) according to
2-Phenyl-5-(pyridin-4-ylmethyl)pyridine (40). Synthesized using
compound 38 (355 mg, 1.43 mmol) and 4-pyridineboronic acid (264
mg, 2.15 mmol) according to method A. Crude product was purified
by flash chromatography on silica gel using a mixture of hexane/ethyl
acetate (2:1) as eluent. After flash chromatography the product was
dissolved in ethyl acetate, and a few drops of conc HCl and water were
added. After stirring for 30 min, the phases were separated and
I
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Journal of Medicinal Chemistry
Article
method F. Crude product was purified by flash chromatography on
silica gel using a mixture of hexane/ethyl acetate (2:1) as eluent. White
solid. Yield: 20.0 mg, 33%. Melting point: 89−92 °C (ethyl acetate).
1H NMR (CDCl3, 500 MHz): δH (ppm) = 4.05 (s, 2 H), 7.19−7.23
(m, 1 H), 7.39−7.44 (m, 1 H), 7.45−7.50 (m, 2 H), 7.53 (dd, J = 8.2,
2.5 Hz, 1 H), 7.70 (dd, J = 8.2, 0.6 Hz, 1 H), 7.96−8.00 (m, 2 H),
8.36−8.40 (m, 2 H), 8.57−8.60 (m, 1 H). MS (ESI): m/z = 265.00
[M + H]+.
7.58−7.62 (m, 1 H), 8.39 (dd, J = 2.5, 0.6 Hz, 1 H). (ESI): m/z =
251.62 [M + H]+.
2-Bromo-5-((5-bromopyridin-3-yl)methyl)pyridine (51). Synthe-
sized using compound 50 (283 mg, 1.13 mmol) and 5-bromopyr-
idine-3-boronic acid (228 mg, 1.13 mmol) according to method A.
Crude product was purified by flash chromatography on silica gel using
a mixture of hexane/ethyl acetate (5:1) as eluent. White solid. Yield:
1
70.0 mg, 19%. H NMR (CDCl3, 500 MHz): δH (ppm) = 3.94 (s, 2
2-Phenyl-5-((5-(trifluoromethyl)pyridin-3-yl)methyl)pyridine (47).
To a solution of 3-bromo-5-(trifluoromethyl)pyridine (502 mg, 2.22
mmol) in THF (20 mL) was added trimethylborate (240 mg, 26.0 mL,
2.31 mmol) under nitrogen atmosphere. After the mixture was cooled
to −78 °C, n-BuLi (1.6 M in hexane, 1.45 mL, 2.32 mmol) was added
dropwise over 10 min, keeping the temperature below 65 °C. Then the
reaction mixture was slowly warmed to room temperature and stirred
for 1 h. Afterward, HCl (1M, 10 mL) was added and the mixture was
stirred for 15 min. The phases were separated, and the organic phase
was washed with HCl (1M, 20 mL). The aqueous layers are combined
and washed with ethyl acetate (2 × 20 mL). Afterward, the aqueous
phases were neutralized with sodium hydroxide solution (2M) and
extracted with ethyl acetate (2 × 20 mL). The solvent was evaporated
in vacuum, and 5-(trifluoromethyl)pyridine-3-boronic acid (113 mg,
yellow solid) was obtained. The boronic acid was directly used in the
next step without further characterization and purification. A mixture
of compound 38 (171 mg, 0.69 mmol), 5-(trifluoromethyl)pyridine-3-
boronic acid (113 mg, 0.59 mmol), Cs2CO3 (684 mg, 2.10 mmol), and
PdCl2(dppf) (26 mg, 0.04 mmol) was used for synthesis according to
method F. Crude product was purified by flash chromatography on
silica gel using a mixture of hexane/ethyl acetate (5:1) as eluent. White
solid. Yield: 22 mg, 10%. Melting point: 95−98 °C (ethyl acetate). 1H
NMR (CDCl3, 500 MHz): δH (ppm) = 4.11 (s, 2 H), 7.40−7.56 (m, 4
H), 7.69−7.76 (m, 2 H), 7.97−8.02 (m, 2 H), 8.58−8.61 (m, 1 H),
8.73 (s, 1 H), 8.80 (s, 1 H). MS (ESI): m/z = 314.87 [M + H]+.
5-((5-Methoxypyridin-3-yl)methyl)-2-phenylpyridine (48). Synthe-
sized using compound 38 (338 mg, 1.36 mmol), 3-methoxy-5-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (413 mg, 1.76 mmol),
Cs2CO3 (1.33 g, 4.10 mmol), and PdCl2(dppf) (50 mg, 0.07 mmol)
according to method F. Crude product was purified by flash
chromatography on silica gel using a mixture of hexane/ethyl acetate
(1:1→1:2) as eluent. Light-yellow solid. Yield: 145 mg, 39%. Melting
H), 7.33 (dd, J = 8.2, 2.5 Hz, 1 H), 7.45 (d, J = 8.2 Hz, 1 H), 7.61 (s, 1
H), 8.27 (d, J = 2.2 Hz, 1 H), 8.42 (s, 1 H), 8.58 (s, 1 H). (ESI): m/z
= 328.64 [M + H]+.
2-Phenyl-5-((5-phenylpyridin-3-yl)methyl)pyridine (52). Synthe-
sized using compound 51 (70.0 mg, 0.21 mmol) and phenylboronic
acid (96.0 mg, 0.64 mmol) according to method A. Crude product was
purified by flash chromatography on silica gel using a mixture of
hexane/ethyl acetate (5:1) as eluent. After flash chromatography, the
product was dissolved in ethyl acetate and a few drops of conc HCl
and water were added. After stirring for 30 min, the phases were
separated and aqueous phase was neutralized with aqueous Na2CO3
solution (2M). After extraction with ethyl acetate and drying over
MgSO4, the solvent was removed under vacuum. White solid. Yield:
1
49.0 mg, 72%. Melting point: 115−117 °C (ethyl acetate). H NMR
(CDCl3, 500 MHz): δH (ppm) = 4.09 (s, 2 H), 7.37−7.44 (m, 2 H),
7.44−7.50 (m, 4 H), 7.53−7.59 (m, 3 H), 7.67−7.70 (m, 2 H), 7.97−
8.02 (m, 2 H), 8.53 (d, J = 2.2 Hz, 1 H), 8.63−8.66 (m, 1 H), 8.75 (d,
J = 2.2 Hz, 1 H). MS (ESI): m/z = 322.95 [M + H]+.
3D-QSAR Study. Ligands were built and energy minimized in the
MMFF94s force field with MOE before being aligned using the flexible
alignment module. Among the top three solutions, which showed
similar average strain energy (dU < 0.03), the one with the highest
similarity (dF = 0.0000) was selected as the final alignment. The
aligned compounds were subsequently imported into Open3DQSAR,
where a grid box around the molecules with a 0.5 Å step size and a 5.0
Å margin was set up. Molecular interaction fields regarding steric
factors and electrostatic potential were then calculated. After importing
the corresponding activity data, compounds 17, 35, 41, 44, and 46
were assigned to the test set, while the rest of the compounds were
employed as the training set. The MIF parameters were pretreated
with zeroing (level = 0.05), max/min cutoff (level = 30), standard
deviation cutoff, N-level variable elimination, and block unscaled
weighting before regression with pIC50 data using partial least-squares
analysis (PLS). The model was further improved with variable
selection procedures using smart region definition (SRD) and
fractional factorial design (FFD) methods. It was subsequently
cross-validated using the leave-many-out paradigm and challenged
with the test set compounds. The PLS pseudocoefficient contour maps
were finally illustrated with MOE.
1
point: 102−105 °C (ethyl acetate). H NMR (CDCl3, 500 MHz): δH
(ppm) = 3.82 (s, 3 H), 4.01 (s, 2 H), 6.98−7.01 (m, 1 H), 7.39−7.44
(m, 1 H), 7.45−7.50 (m, 2 H), 7.53 (dd, J = 8.2, 2.2 Hz, 1 H), 7.68
(dd, J = 8.2, 0.9 Hz, 1 H), 7.96−7.99 (m, 2 H), 8.17 (d, J = 1.6 Hz, 1
H), 8.21 (d, J = 2.8 Hz, 1 H), 8.59 (dd, J = 1.6, 0.6 Hz, 1 H). MS
(ESI): m/z = 276.95 [M + H]+.
5-((6-Phenylpyridin-3-yl)methyl)pyridin-3-ol (49). Compound 48
(145 mg, 0.53 mmol) was suspended in HBr (48% in water, 5 mL),
and the mixture was stirred at 130 °C overnight. After cooling down to
room temperature, saturated Na2CO3 solution was added to neutralize
the mixture. After extraction with ethyl acetate (3 × 10 mL) and
drying over MgSO4, the solvent was removed under vacuum. The
crude product was dissolved in ethyl acetate, and a few drops of conc
HCl and water were added. After stirring for 30 min, the phases were
separated and aqueous phase was neutralized with aqueous Na2CO3
solution (2M). After extraction with ethyl acetate and drying over
MgSO4, the solvent was removed under vacuum. Light-yellow solid.
ASSOCIATED CONTENT
■
S
* Supporting Information
The biotest procedures, synthetic procedures, and character-
izations of the final compounds 33−35, 42, 55, and 56 and
intermediates 3, 4, 6−14, 25−32, 53, and 54 as well as all the
13C NMR data. The predicted and determined values (pIC50)
of all compounds in both training and test sets as well as the
superposition of training set compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
1
Yield: 100 mg, 72%. Melting point: 181−183 °C (ethyl acetate). H
NMR (DMSO-d6, 500 MHz): δH (ppm) = 3.97 (s, 2 H), 7.02 (dd, J =
2.8, 1.9 Hz, 1 H), 7.39−7.43 (m, 1 H), 7.45−7.50 (m, 2 H), 7.72 (dd, J
= 8.2, 2.2 Hz, 1 H), 7.89 (dd, J = 8.2, 0.6 Hz, 1 H), 7.99 (d, J = 2.5 Hz,
1 H), 8.01−8.07 (m, 3 H), 8.59−8.62 (m, 1 H), 9.83 (s, 1 H). MS
(ESI): m/z = 263.00 [M + H]+.
AUTHOR INFORMATION
■
Corresponding Author
2-Bromo-5-(bromomethyl)pyridine (50). Synthesized using 2-
bromo-5-methylpyridine (2.30 g, 13.4 mmol), NBS (2.62 g, 14.7
mmol), and DBPO (162 mg, 0.67 mmol) in carbon tetrachloride
according to method D. Crude product was purified by flash
chromatography on silica gel using a mixture of hexane/ethyl acetate
(15:1→8:1) as eluent. Yellow solid. Yield: 1.50 g, 45%. 1H NMR
(CDCl3, 500 MHz): 4.42 (s, 2 H), 7.49 (dd, J = 8.2, 0.9 Hz, 1 H),
*Phone: +(49) 681 302 70300. Fax: +(49) 681 302 70308. E-
mail: rolf.hartmann@helmholtz-hzi.de. Homepage: http://
www.helmholtz-hzi.de/?id=3897.
Notes
The authors declare no competing financial interest.
J
dx.doi.org/10.1021/jm400240r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(7) Schenkman, J. B.; Sligar, S. G.; Cinti, D. L. Substrate interaction
with cytochrome P-450. Pharmacol. Ther. 1981, 12, 43−71.
(8) Yin, L.; Hu, Q.; Hartmann, R. W. Tetrahydropyrroloquinolinone
type dual inhibitors of aromatase/aldosterone synthase as a novel
strategy for breast cancer patients with elevated cardiovascular risks. J.
Med. Chem. 2013, 56, 460−470.
ACKNOWLEDGMENTS
■
We thank Drs. Christina Zimmer and Jorg Haupenthal as well
̈
as Victoria Kraemer, Martina Jankowski, Ahmed Saad, Marco
Gargano, and Jeannine Jung for their help in performing the in
vitro tests. Thanks are also due to Dr. Jan Hoyer for reading the
manuscript.
(9) Roumen, L.; Sanders, M. P.; Pieterse, K.; Hilbers, P. A.; Plate, R.;
Custers, E.; de Gooyer, M.; Smits, J. F.; Beugels, I.; Emmen, J.;
Ottenheijm, H. C.; Leysen, D.; Hermans, J. J. Construction of 3D
models of the CYP11B family as a tool to predict ligand binding
characteristics. J. Comput.-Aided Mol. Des. 2007, 21, 455−471.
(10) Tosco, P.; Balle, T. Open3DQSAR: a new open-source software
aimed at high-throughput chemometric analysis of molecular
interaction fields. J. Mol. Model. 2011, 17, 201−208.
ABBREVIATIONS USED
■
11β-HSD1, 11β-hydroxysteroid dehydrogenase type 1;
CYP11B1, 11β-hydroxylase; CYP11B2, aldosterone synthase;
CYP17, 17α-hydroxylase-17,20-lyase; CYP19, aromatase;
ACTH, adrenocorticotropic hormone; SF, selectivity factor =
IC50 CYP11B2/IC50 CYP11B1; t1/2, half-life; CDI, 1,1′-carbonyldii-
midazole; MIF, molecular interaction fields; PLS, partial least-
squares analysis; SRD, smart region definition; FFD, fractional
factorial design; ref, reference; BINAP, 2,2′-bis-
(diphenylphosphino)-1,1′-binaphthyl; SDEC, standard devia-
tion of error of calculation; SDEP, standard deviation of error
of prediction; CLint, intrinsic clearance
(11) (a) Hu, Q.; Negri, M.; Jahn-Hoffmann, K.; Zhuang, Y.; Olgen,
S.; Bartels, M.; Muller-Vieira, U.; Lauterbach, T.; Hartmann, R. W.
̈
Synthesis, biological evaluation, and molecular modeling studies of
methylene imidazole substituted biaryls as inhibitors of human 17α-
hydroxylase-17,20-lyase (CYP17). Part II: Core rigidification and
influence of substituents at the methylene bridge. Bioorg. Med. Chem.
2008, 16, 7715−7727. (b) Hille, U. E.; Hu, Q.; Vock, C.; Negri, M.;
Bartels, M.; Muller-Vieira, U.; Lauterbach, T.; Hartmann, R. W. Novel
̈
CYP17 inhibitors: Synthesis, biological evaluation, structure−activity
relationships and modeling of methoxy- and hydroxy-substituted
methyleneimidazolyl biphenyls. Eur. J. Med. Chem. 2009, 44, 2765−
2775.
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