Identification of 4-(4-nitro-2-phenethoxyphenyl)pyridine as a promising new lead for discovering inhibitors of both human and rat 11β-Hydroxylase

Article abstract of DOI:10.1016/j.ejmech.2015.04.013

The inhibition of 11β-hydroxylase is a promising strategy for the treatment of Cushing's syndrome, in particular for the recurrent and subclinical cases. To achieve proof of concept in rats, efforts were paid to identify novel lead compounds inhibiting both human and rat CYP11B1. Modifications on a potent promiscuous inhibitor of hCYP11B1, hCYP11B2 and hCYP19 (compound IV) that exhibited moderate rCYP11B1 inhibition led to compound 8 as a new promising lead compound. Significant improvements compared to starting point IV were achieved regarding inhibitory potency against both human and rat CYP11B1 (IC₅₀ values of 2 and 163 nM, respectively) as well as selectivity over hCYP19 (IC₅₀ Combining double low line 1900 nM). Accordingly, compound 8 was around 7-and 28-fold more potent than metyrapone regarding the inhibition of human and rat CYP11B1 and exhibited a comparable selectivity over hCYP11B2 (SF of 3.5 vs 4.9). With further optimizations on this new lead compound 8, drug candidates with satisfying profiles are expected to be discovered.

Full text of DOI:10.1016/j.ejmech.2015.04.013

European Journal of Medicinal Chemistry 96 (2015) 139e150
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Identification of 4-(4-nitro-2-phenethoxyphenyl)pyridine as a
promising new lead for discovering inhibitors of both human and rat
11b-Hydroxylase
,
, b, *
Qingzhong Hu a b, Jessica Kunde a, Nina Hanke c, Rolf W. Hartmann a
a Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, D-66123 Saarbrücken, Germany
b Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus C2.3, D-66123 Saarbrücken, Germany
c Elexopharm GmbH, Campus A1, 66123 Saarbrücken, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The inhibition of 11b-hydroxylase is a promising strategy for the treatment of Cushing's syndrome, in
Received 28 January 2015
Received in revised form
3 April 2015
Accepted 6 April 2015
Available online 8 April 2015
particular for the recurrent and subclinical cases. To achieve proof of concept in rats, efforts were paid to
identify novel lead compounds inhibiting both human and rat CYP11B1. Modifications on a potent
promiscuous inhibitor of hCYP11B1, hCYP11B2 and hCYP19 (compound IV) that exhibited moderate
rCYP11B1 inhibition led to compound 8 as a new promising lead compound. Significant improvements
compared to starting point IV were achieved regarding inhibitory potency against both human and rat
CYP11B1 (IC50 values of 2 and 163 nM, respectively) as well as selectivity over hCYP19 (IC50 ¼ 1900 nM).
Accordingly, compound 8 was around 7- and 28-fold more potent than metyrapone regarding the in-
hibition of human and rat CYP11B1 and exhibited a comparable selectivity over hCYP11B2 (SF of 3.5 vs
4.9). With further optimizations on this new lead compound 8, drug candidates with satisfying profiles
are expected to be discovered.
Keywords:
11b-hydroxylase
Human and rat CYP11B1
Inhibitors
Cushing's syndrome
Selectivity
© 2015 Elsevier Masson SAS. All rights reserved.
Aromatase
1. Introduction
this syndrome is considered to exacerbate concomitant diseases.
More severe is that subclinical Cushing's syndrome is deemed to be
Although endogenous Cushing's syndrome is a rare disease with
an annual incidence as low as 2e3 cases per million of the popu-
lation [1], it leads to a high mortality via co-morbidities including
cardio- and cerebrovascular diseases [1,2]. As for the majority of
this syndrome that are caused by pituitary adenomas and termed
Cushing's disease, the 5 years' survival is only about 50% if without
effective treatments [3]. Despite of the fact that the surgical
removal of tumors leads to cure or remission in 65e85% of patients,
recurrences are observed in up to 20% of cases [1]. Furthermore,
investigations indicate a high prevalence of undiagnosed subclini-
cal Cushing's syndrome in particular in patients with type II dia-
betes and osteoporosis [4,5]. Although no evident symptoms other
than high levels of cortisol in plasma are observed in these patients,
associated with metabolic dementia and to promote the progres-
sion of Alzheimer disease [6]. Apparently, for these recurrent and
subclinical cases, pharmacotherapy via the inhibition of steroid
11b-hydroxylase (hCYP11B1), which catalyzes the hydroxylation of
11-deoxycortisol to cortisol, to reduce the circulating cortisol levels
is a superior approach. Metyrapone (IC50 ¼ 15 nM, Chart 1) as an
inhibitor of adrenal steroidogenesis has been employed to relieve
patients' symptoms before surgery [7]; and its long-term applica-
tions were also reported to successfully control cortisol levels and
psychiatric manifestations [8,9]. In a phase I clinical study, an
experimental hCYP11B1 inhibitor LCI699 (IC50 ¼ 2.9 nM, Chart 1)
normalized urinary free cortisol levels or reduced its concentra-
tions by more than half from baseline in patients with moderate-to-
severe Cushing's disease [10]. However, simultaneously, the plasma
aldosterone levels were significantly decreased by around 3-fold
from 4.2 to 1.3 ng/dL because of the potent inhibition of aldoste-
rone synthase (hCYP11B2) by LCI699 (IC50 ¼ 0.2 nM). Although this
inhibition contributed to the reduction of both systolic and diastolic
blood pressures (10.0 and 6.0 mmHg, respectively), it, together with
the twofold elevated adrenocorticotropic hormone in plasma that
Abbreviations: CYP, cytochrome P450; hCYP11B1, steroid 11
b-hydroxylase;
hCYP11B2, aldosterone synthase; hCYP17, 17 -hydroxylase-17,20-lyase; hCYP19,
a
aromatase; SF, selectivity factor ¼ IC50 hCYP11B2/IC50
.
hCYP11B1
* Corresponding author. Pharmaceutical and Medicinal Chemistry, Saarland
University, Campus C2 3, D-66123 Saarbrücken, Germany.
E-mail address: rolf.hartmann@helmholtz-hzi.de (R.W. Hartmann).
http://dx.doi.org/10.1016/j.ejmech.2015.04.013
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

140
Q. Hu et al. / European Journal of Medicinal Chemistry 96 (2015) 139e150
Chart 1. The structures of typical hCYP11B1 inhibitors (Metyrapone, LCI699, compounds I and II) as well as lead compounds III, IV and designed compounds 1e25.
is the response to the drop of cortisol levels, boosted the concen-
trations of its precursor 11-deoxycorticosterone by 42-fold from 3.5
to 147.5 ng/dL. The binding of these over-produced 11-
deoxycorticosterone to mineralocorticoid receptors induced
around 8% reduction of the plasma potassium levels and conse-
quently hypokalemia in one-third (4 out of 12) of patients. As
evidenced by the decreased plasma renin levels (13e8 ng/L), which
remained suppressed two weeks after the treatment discontinua-
tion, the renineangiotensinealdosterone system was impaired.
Therefore, the selectivity over hCYP11B2 is considered to be a
crucial criterion for safety when treating Cushing's syndrome via
hCYP11B1 inhibition. Besides this indication, topical application of
hCYP11B1 inhibitors has also been proposed as a novel strategy to
promote the healing of chronic wounds [11] based on the obser-
vation that cutaneously over-expressed cortisol impairs fibroblast
proliferation and collagen synthesis, and thus induces chronic
wounds and ulcers [12]. Cortisol also stimulates the proliferation of
prostate cancer cells via certain mutated androgen receptors [13],
therefore the combinatory application of hCYP11B1 inhibitors [14]
of compounds with novel structures inhibiting both human and rat
CYP11B1 is necessary and urgent. In this study, we describe our
efforts to identify new lead compounds exploiting previously ac-
quired knowledge on developing selective inhibitors of steroido-
genic CYP enzymes.
2. Results
2.1. Design concept
In our previous investigation of pyridyl substituted 3,4-
dihydroquinolin-2(1H)-ones as potent inhibitors of hCYP11B2, an
interesting SAR was observed that the bulkiness augment of the
alkoxy substituent at the 7-position largely increased the hCYP11B1
inhibition [42]. The benzyloxy compound IV exhibited a 56-fold
stronger hCYP11B1 inhibition than the corresponding methoxy
analog III (IC50 ¼ 2487 nM) with an IC50 value of 44 nM (Chart 1).
Although the inhibition of hCYP11B2 was accordingly enhanced to
22 nM thus only showing an inadequate selectivity factor of 0.5 for
hCYP11B1, a trend of reverse preference between hCYP11B1 and
hCYP11B2 inhibition was anticipated when comparing it to that of
compound III (SF ¼ 0.1). More important is that compound IV
showed a moderate inhibition of rCYP11B1 (IC50 ¼ 1500 nM), which
is apparently superior to the clinical used drug metyrapone
(IC50 ¼ 4607 nM) and the previously identified potent hCYP11B1
inhibitors I (IC50 > 10,000 nM) and II (IC50 ¼ 2440 nM) (Chart 1 &
Table 1). Due to the difficulty of identifying inhibitors of both hu-
man and rat CYP11B1 to facilitate the proof of concept in rats,
compound IV was considered as a promising starting point for
further modifications. However, as compound IV actually origi-
nated from a series of dual inhibitors of hCYP11B2 and hCYP19
[42,43], it showed significant inhibition of hCYP19 as well
(IC50 ¼ 35 nM). Therefore, besides elevating the potencies against
human and rat CYP11B1, improvement of the selectivity over both
hCYP11B2 and hCYP19 is another challenge encountered. To solve
these problems, modifications were performed as shown in Chart 1.
Since the 3,4-dihydroquinolin-2(1H)-one core was regarded as a
privileged structure for hCYP11B2 inhibition [44], the lactam ring
was opened and simplified into a phenyl substituted by various
groups. As the insertion of a nitrogen atom into the core was
demonstrated to be beneficial for the selectivity over hCYP11B2
[45], a pyridyl was also attempted replacing the benzene core.
Based on the comparison between compounds III and IV, the
or dual inhibitors of 17a-hydroxylase-17,20-lyase (hCYP17) and
hCYP11B1 [15] could be superior approaches to improve the sur-
vival of prostate cancer patients. Since several other steroidogenic
cytochrome P450 (CYP) enzymes including hCYP11B2, hCYP17 and
aromatase (hCYP19) catalyze the biosynthesis of important steroi-
dal hormones (mineralocorticoids, androgens and estrogens,
respectively), drug candidates inhibiting hCYP11B1 ought to show
adequate selectivity over these enzymes, in particular hCYP11B2 (as
indicated by the results of the clinical trial of LCI699). The selec-
tivity between hCYP11B1 and hCYP11B2 is especially challenging to
achieve because of the high homology (>93%) between these
two enzymes. Despite of this difficulty, potent and selective in-
hibitors of hCYP11B2 [16e21] were discovered recently. Aided by
our broad experience in developing selective inhibitors of ste-
roidogenic CYP enzymes, including hCYP11B2 [22e26], hCYP17
[27e33] and hCYP19 [34e38], several classes of potent hCYP11B1
inhibitors were identified [39e41]. Examples like compounds I
(IC50 ¼ 107 nM) [45] and II (IC50 ¼ 2 nM) [41] exhibited both potent
inhibition of hCYP11B1 (Chart 1 & Table 1) and promising selec-
tivity over other CYP enzymes (for hCYP11B2, selectivity factors
(SF ¼ IC50 hCYP11B2/IC50 hCYP11B1) around 15 were observed). How-
ever, these compounds showed no (I, IC50 > 10,000 nM) to low (II,
IC50 ¼ 2440 nM) inhibition toward rat CYP11B1 (Table 1), which
makes it difficult to prove the concept in rats. Therefore, discovery

Q. Hu et al. / European Journal of Medicinal Chemistry 96 (2015) 139e150
141
Table 1
Inhibition of hCYP11B1, hCYP11B2, hCYP19 and rCYP11B1 by compounds 1e25 as well as references IeIV and metyrapone.
Compd.
Structure
R
IC50 (nM)a
SFd
IC50 (nM)a
Py
n
hCYP11B1b
hCYP11B2b
hCYP19c
rCYP11B1b
I
II
III
IV
Mete
1
2
3
4
5
107
2
2487
44
1423
33
268
22
13.3
15.1
0.1
0.5
4.9
2.3
0.6
0.5
0.7
4.1
2.5
1.7
3.5
2.4
0.9
0.6
1.6
1.2
1.3
1.5
1.2
0.9
1.6
1.9
0.5
0.7
0.9
0.9
1.3
2.7
>5000
>5000
447
35
>5000
>10,000
2440
1500
4607
15
72
AcNH
4-F PhCONH
NH2
NO2
NO2
NO2
NO2
NO2
NO2
3
3
3
3
3
3
4
4
4
3
4
3
4
3
4
3
4
3
4
3
4
3
4
3
4
0
0
0
0
1
2
0
1
2
279
1207
540
23
13
6
50
2
8
508
1201
51
60
183
46
812
26
38
643
756
260
17
53
15
87
7
18
447
714
84
74
230
70
945
24
63
146
75
137
2425
1894
4993
1493
1307
2083
648
163
370
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
F
F
1
1
1
1
1
1
1
1
1
1
OMe
OMe
CF3
CF3
CN
CN
CONMe2
CONMe2
2090
165
1546
543
441
12
23
594
1150
1665
2665
182
6
299
816
1453
2441
246
16
128
157
706
2849
a
Mean value of at least three experiments, standard deviation less than 25%.
b
Hamster fibroblasts expressing hCYP11B1, hCYP11B2 or rCYP11B1, respectively; substrate deoxycorticosterone, 100 nM for hCYP11B1 and hCYP11B2 and 500 nM for
rCYP11B1.
c
human placental microsomes, substrate androstenedione, 500 nM.
d
e
SF ¼ IC50 hCYP11B2/IC50 hCYP11B1
.
Met: Metyrapone.
alkoxyl moiety was speculated to be a key for both potency and
selectivity. The influences of the chain length and the substituting
position of the alkoxyl group were therefore investigated. Since
such kind of inhibitors competitively inhibit CYP enzymes via the
coordination between their sp2 hybrid N and the heme iron, the
convenience to form these interactions, which largely depend on
the relative position of the N with regard to the hydrophobic core,
would exhibit significant impacts on both potency and selectivity.
This factor was therefore examined by introducing either 3- or 4-
pyridyl moieties as the heme-coordinating heterocycles as well as
by inserting a methylene moiety between the pyridyl ring and the
core, which have been found to alter the inhibitory pattern among
steroidogenic CYP enzymes [11,39,43]. These efforts led to com-
pounds 1e25 that were subsequently evaluated for their inhibition
of both human and rat CYP11B1 as well as the selectivity over
hCYP11B2, hCYP17 and hCYP19.
straightforward general route was employed (Scheme 1). The
starting bromo phenols that were substituted with various groups,
such as NO2, F, OMe, CF3, CN and CONMe2, were either commer-
cially available or obtained from cleavage of the corresponding
methoxy precursors. They were firstly alkylated using the corre-
sponding bromines to afford ether intermediates 4ae6a, 10a, 12a,
14a, 16a, 18a, 20a and 22a before the 3- or 4-pyridyl moieties were
introduced via Suzuki coupling reaction resulting in final com-
pounds 4e23. This reaction sequence was established after having
observed low yields of alkylation with pyridyl substituted phenols.
In most cases, the ordinary Suzuki coupling condition, i.e. boiling
the corresponding reactants with Pd(PPh3)4 and Na2CO3 in a
mixture of ethanol, toluene and water, gave satisfying yields; while,
for compounds 10e13, 18, 19 and 23, PdCl2(dppf) had to be
employed as an alternative. The nitro group in compound 4 was
reduced to amino (3) using hydrazine hydrate and Pd/C, which was
further acylated with acetyl chloride or 4-fluorobenzoyl chloride to
obtain final compounds 1 and 2, respectively. To achieve the
introduction of a methylene moiety between the pyridyl and the
benzene core, the bromo group in 5a was first converted into borate
(24a) using bis(pinacolato)diboron under the catalysis of
2.2. Chemistry
The syntheses of the designed compounds are shown in
Schemes 1e3. For the majority of desired compounds,
a

142
Q. Hu et al. / European Journal of Medicinal Chemistry 96 (2015) 139e150
Scheme 1. Syntheses of compounds 4e23. Reagents and conditions. (i) Method A: BBr3, DCM, ꢀ20 to RT, overnight. (ii) for 22b: HBr, 140 ꢁC, overnight. (iii) Method B: corresponding
brimide, K2CO3, KI, ethanol, reflux, overnight. (iv) for 10a: (2-bromoethyl)benzene, tetrabutylammonium iodide, DMF, Cs2CO3, 140 ꢁC, 3 h. (v) for 4e9, 14e17 and 20e22: Method C:
Pd(PPh3)4, corresponding pyridylboronic acid, Na2CO3, ethanol, toluene, H2O, reflux, 3e5 h. (vi) for 10e13, 18, 19 and 23: Method D: PdCl2(dppf), corresponding pyridylboronic acid,
Na2CO3, DME, H2O, reflux, overnight.
Scheme 2. Syntheses of compounds 1e3. Reagents and conditions. (i) hydrazine hydrate, Pd/C, EtOH, reflux, 1 h. (ii) acetyl chloride or 4-fluorobenzoyl chloride, triethylamine, N,N-
dimethylamionpyridine, dichloromethane, RT, overnight.
PdCl2(PPh3)2, which subsequently coupled with 3- or 4-(bromo-
methyl)pyridine to yield compounds 24 and 25, respectively.
after removing the astriction of the lactam ring. The replacement of
amido groups by an amino moiety (3) only led to a moderate in-
hibition of hCYP11B1 (IC50 ¼ 540 nM). Surprisingly, the nitro analog
4 showed potent hCYP11B1 inhibition with an IC50 value of 23 nM,
which is twice as potent as the lead IV (IC50 ¼ 44 nM). However,
compound 4 failed to exhibit any superiority to lead IV regarding
selectivity over hCYP11B2 (SF ¼ 0.7). With the nitro group sus-
tained, the influence of the chain length of the alkoxyl moiety was
subsequently investigated. As the augmentation of methylene
units, the inhibition of hCYP11B1 was elevated from 23 nM (4,
n ¼ 0, benzoxy) to 13 nM (5, n ¼ 1) and to 6 nM (6, n ¼ 2). Despite of
the fact that all compounds also showed potent inhibition of
hCYP11B2, improvements of selectivity compared to lead com-
pound IV (SF ¼ 0.5) was achieved, in particular for compound 5,
which exhibited a SF of 4.1. The displacement of 3-pyridyl by 4-
pyridyl did not influence much the inhibitory potency of
hCYP11B1. The corresponding benzoxy compound 7 was also a
potent inhibitor with an IC50 value of 50 nM. With the insertion of
more methylene units, a similar increase of inhibitory potency was
observed. Phenethoxy analog 8 (n ¼ 1, IC50 ¼ 2 nM) was 25-fold
more potent than the benzoxy compound 7; while an IC50 of
8 nM was observed for the phenylpropoxy compound 9. Being the
most potent hCYP11B1 inhibitor in this study, compound 8 was 22-
and 7-fold more potent than the lead compound IV and metyr-
apone, respectively, and also exhibited a similar selectivity over
2.3. Biological results and discussions
2.3.1. Inhibition of hCYP11B1 and hCYP11B2
The synthesized compounds 1e25 were tested for their inhibi-
tion of hCYP11B1 and hCYP11B2 in V79MZ cells expressing the
corresponding enzymes, respectively, with [3H]-11-deoxyc-
orticosterone as substrate [46]. Results are presented in Table 1
with metyrapone and compound IeIV as references.
The 3,4-dihydroquinolin-2(1H)-one core in the lead compound
IV was opened as the first step of modification because this struc-
ture was regarded as a privileged structure for hCYP11B2 inhibition
[44]. In contrast, the 3-pyridyl was left untouched to ensure direct
comparisons of inhibitory potencies. The lactam ring was simplified
into an acetamido group substituting the benzene core to maintain
potential hydrogen bonds formed by the amide group. As expected,
the resulting compound 1 exhibited preference for hCYP11B1 in-
hibition with a SF of 2.3, which is around 5-fold better than that of
the lead compound IV. However, its hCYP11B1 inhibition was
simultaneously reduced to 279 nM. A bulky 4-fluorobenzamido
group (2) further decreased the inhibitory potency by 4-
folde1207 nM. This observation is probably a result of steric
clashes caused by increased bulkiness and/or rotation freedom
Scheme 3. Syntheses of compounds 24 and 25. Reagents and conditions. (i) bis(pinacolato)diboron, PdCl2(PPh3)2, KOAc, dioxane, reflux, 4 h. (ii) Method D: PdCl2(dppf), corre-
sponding pyridylboronic acid, Na2CO3, DME, H2O, reflux, 4 h.

Q. Hu et al. / European Journal of Medicinal Chemistry 96 (2015) 139e150
143
hCYP11B2 as metyrapone. The fact that both 3- and 4-pyridyl
compounds showed similar potency is an indication of a relative
large pocket, in which the benzene cycle could drift a bit to provide
the suitable angle to form the NeFe coordination. The high flexi-
bility of the alkoxyl side chain could enable the compounds to
adapt to the angle change. In contrast, the position of the alkoxyl
group was obviously more decisive for hCYP11B1 inhibition. The
shift from the ortho-(with regard to the pyridyl) to the meta-
position resulted in deceases of potency by 85- and 600-fold for
the 3- and 4-pyridyl analogs (10, IC50 ¼ 508 nM; and 11,
IC50 ¼ 1201 nM), respectively. Since phenethoxy analogs showed
better selectivity (SF of 4.1 for compound 5 and 3.5 for compound
8), this chain length was sustained in the subsequent modifications.
In contrast, both 3- and 4-pyridyl analogs were synthesized to
avoid missing any potential strong inhibitors. Although nitro
groups present in a considerable number of drugs in clinical use,
such as nifedipine, they have the potential of being reduced to ar-
omatic nitrosos, which can covalently binds to DNA and proteins,
and thus are carcinogenic [47]. Wariness was therefore put on the
nitro groups and the replacement of nitro by other substituents was
attempted. When relatively small groups were introduced into the
molecules (F in 12 & 13; OMe in 15; CF3 in 17; and CN in 18 & 19),
potent hCYP11B1 inhibition was observed with IC50 values less than
60 nM. CN in combination with 4-pyridyl led to the most potent
compound in this small set with an IC50 value of 12 nM. An
exception is OMe in the 3-pyridyl compound 14, which showed a
moderate hCYP11B1 inhibition (IC50 ¼ 183 nM); whereas CF3 in
combination with 3-pyridyl surprisingly only resulted in a weak
inhibitory potency (IC50 ¼ 812 nM). Accordingly, the bulkier
CONMe2 substituents led to weak inhibition with IC50 values of 594
and 1150 nM for 3-Py (20) and 4-Py (21) compounds, respectively,
indicating potential steric hindrances. In contrast to bulkiness,
other properties, such as electrostatic potential and the ability to
form hydrogen bonds, seem to play only minor roles. When the
substituent is as small as F, no significant difference in potency was
found between 3- and 4-pyridyl compounds 12 and 13 (IC50 values
of 51 and 60 nM, respectively); while for other groups with larger
size (OMe, CF3 and CN in compounds 14e19), the 4-pyridyl analogs
were significantly more potent than the corresponding 3-pyridyl
compounds. Although more potent hCYP11B1 inhibitors were
identified via these replacements, no improvement of selectivity
over hCYP11B2 was observed (SFs < 2). As the replacement of the
benzene core by a pyridyl moiety in the biaryl methylene
substituted heterocycle class of hCYP11B1 inhibitors increased the
selectivity over hCYP11B2 [45], a nitrogen atom was inserted into
the benzene core leading to compounds 22 and 23. However, this
modification strongly reduced the hCYP11B1 inhibition
(IC50 > 1400 nM). Moreover, the introduction of a methylene
moiety between the benzene core and the 3-pyridyl group (24)
decreased the potency by around 10-fold compared to the corre-
sponding analog 5; whereas the same modification on 4-pyridyl
compound 25 led to a potent hCYP11B1 inhibitor with similar po-
tency (IC50 ¼ 6 nM) as the parent compound 8.
very weak to no inhibition toward hCYP19 with IC50 values ranging
from 1500 to 5000 nM. The only exception was compound 25
(IC50 ¼ 157 nM) probably due to the methylene moiety between the
heme-coordinating pyridyl and the benzene core. With an
improvement of more than 40-fold on hCYP19 selectivity compared
to lead IV, these 4-pyridyl compounds are not likely to reduce es-
trogen biosynthesis under the treatment doses when taking into
consideration their very strong inhibitory potency against the
target enzyme hCYP11B1.
Since hCYP17 is of crucial importance for the production of
androgens, the selectivity over this steroidogenic enzyme was
employed as another criterion for safety. The above-mentioned
compounds were further investigated for their hCYP17 inhibition
using the 50,000 g sediment of an Escherichia coli homogenate
recombinantly expressing hCYP17 with progesterone (25
mM) as a
substrate [49]. No inhibition (IC50 > 10 M) was observed for all
m
compounds tested indicating safety with regard to this aspect.
2.3.3. Inhibition of rat CYP11B1
As one of the major tasks of this study, the inhibition of
rCYP11B1 was determined for the most potent inhibitors of the
human enzyme (same as mentioned in the last section) in V79MZ
cells expressing rCYP11B2 [50]. Interestingly, for nitro compounds,
all 4-pyridyl analogs 7e9 (IC50 < 650 nM) were much stronger than
3-pyridyl compounds 4e6 (IC50 ranging from 1300 to 2000 nM).
Compound 8 as the most potent one exhibited an IC50 value of
163 nM, which was near 10- and 28-fold stronger than lead IV and
metyrapone, respectively. In contrast, when substituted with CN,
no significant difference in potency between 3- and 4-pyridyl
compounds 18 and 19 was observed (IC50 around 500 nM);
whereas, in the case of methylene compounds, the 4-pyridyl de-
rivative 25 showed an about 4-fold weaker inhibition compared to
the corresponding 3-pyridyl compound 24 (IC50 of 2850 vs
706 nM), which is in contrast to the SAR observed with the nitro
compounds.
2.3.4. Modeling studies
To further investigate the interactions between the synthesized
inhibitors and the targeting enzyme, a homology model of human
CYP11B1 was established using the reported crystal structure of
hCYP11B2 as the template. The sequences of both hCYP11B1
(UniProt code: P15538) and hCYP11B2 (UniProt code: P19099)
comprise 503 amino acids, among which 473 are identical
(Figure S1). In the crystal of hCYP11B2 in complex with deoxy-
corticosterone (PDB ID: 4DVQ, chain A), amino acids 34-502 pre-
sent. Since amino acids 1e24 as a transit peptide is less relevant to
biological functions and configuration, amino acids 1e33 in the
hCYP11B1 sequence was omitted similar as in the template crystal
when establishing the model. A library of ten models was initially
generated and the best one was selected according to electrostatic
solvation energy and root mean square deviation of each inter-
mediate model to the average position of all models. This pre-
liminary model was subsequently further refined via molecular
dynamic simulation to yield the final one. The quality of the
resulting homology model was evaluated regarding the stereo-
chemical measurements of each amino acid including bond
2.3.2. Inhibition of human CYP17 and CYP19
Since this series of compounds were originated from the lead
compound IV, which showed a potent inhibition of hCYP19
(IC50 ¼ 35 nM), the improvement of the corresponding selectivity
was a tough task throughout the modifications. The most potent
hCYP11B1 inhibitors 4e9,17e9, 24 and 25 were therefore evaluated
for their inhibition of hCYP19 with human placental microsomes
lengths, angles, dihedrals and 4e
j torsions. As shown in the
Ramachandran scatter plot (Figure S2), 451 out of 470 amino acid
(96%) clustered in favorable regions, whereas only one amino acid
(Ser288) was considered as an outlier (0.2%). This hCYP11B1 ho-
mology model presented characteristic cytochrome P450 folds and
nicely superposed to the template structure (Fig. 1A). Although 30
amino acids differ in the sequences of these two enzymes, they
locate outside the active sites. It is therefore not surprising that the
configuration of their active sites are very similar. However, a
using [1b
-3H]androstenedione (500 nM) as a substrate [48].
Apparently, all 3-pyridyl compounds (4e6, 18 and 24) exhibited
potent inhibition of hCYP19 with IC50 values below 170 nM
(Table 1). In contrast, 4-pyridyl analogs (7e9, 17 and 29) showed

144
Q. Hu et al. / European Journal of Medicinal Chemistry 96 (2015) 139e150
scrutiny on the active sites still revealed minor differences
(Fig. 1B), which could be the basis and keys for inhibitors to
selectively inhibit an individual enzyme. In the hCYP11B1 ho-
mology model, the middle part of the I-helix extruded slightly
model to investigate the interactions between inhibitors and tar-
geting enzyme. The molecule was anchored by the coordination
between the sp2 hybrid N in pyridyl and the heme iron and leaned
on the b
2-sheet with an angle of approximate 60ꢁ with respect to
toward the opposite
b
2-sheet compared to that in the hCYP11B2
the heme plane (Fig. 2A). The phenethoxyl group stretched out
pointing to the BeB0 loop and fitted into the hydrophobic
pocket mentioned above, which was delimitated by Arg110e
Met111eSer112eLeu113eLeu131eGlu383 (Fig. 2B). The phenyl
side chains of Phe130 and Phe381 served as two gates of this
crystal thus narrowing the binding pocket; while the F-helix and
the heme moiety as the roof and the floor of the active site,
respectively, subsided by around 2 Å synchronously. Although
drifts were also observed for the
b
2-sheet, the BeB0 loop and the
(b
4-1)-(
b
4-2) loop, alterations were identified in the B0-C loop that
pocket and formed pep interactions with the phenethoxyl group
are subtle nevertheless of greater importance in changing the
binding site (Fig. 1C). The side chain of Leu131 jostled toward the
in parallel and perpendicular manners, respectively. An are-
neecation interaction was also observed between the phenethoxyl
substituent and the guanidyl of Arg110. Since the side chains of
Glu383 and Met111 were accessible, the introduction of positive-
charged groups and/or hydrogen bond donors onto the phene-
thoxyl moiety to form salt bridge and/or hydrogen bond would
elevate inhibitory potency. As this pocket in hCYP11B1 homology
model exhibited a more convenient entrance (Phe130) but a
reduced volume (Leu131) compared to that in the hCYP11B2
crystal structure, this feature could be exploited to achieve more
b2-sheet thus shrinking the hydrophobic pocket in between. In
contrast, the phenyl group of Phe130 turned downward to heme
moiety leading to more convenience for inhibitors to enter
and exploit this pocket. The most promising compound (8) in this
study was subsequently docked into the hCYP11B1 homology
selectivity. Furthermore, Phe130 and Phe381 formed
pep in-
teractions with the pyridyl moiety (parallel) and the phenyl core
(perpendicular) and thus promoted affinity and stabilized the
binding pose. An additional pep interaction between Phe487 and
the phenyl core (parallel) as well as hydrogen bonds formed by the
nitro group with Tyr485 and Leu380 also contributed significantly
with this regard. The drift of I-helix toward the
b2-sheet nar-
rowing the active site possibly facilitated the formation of these
hydrogen bonds, but also increased the risks of steric clashes for
bulkier substituents, which is in accordance with the SAR dis-
cussed above.
Fig. 1. (A) Superposition of the hCYP11B1 homology model (green) onto the hCYP11B2
crystal structure (magenta, PDB ID: 4DVQ-A). (B) Comparison of the active sites. (C)
Configurational changes of F130 and L131 strongly influenced the hydrophobic pocket
Fig. 2. Docking of compound 8 (magenta) in the hCYP11B1 homology model. In-
teractions between the inhibitor and the enzyme are depicted in gold dashed lines.
(For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
delimitated by B0-C loop, BeB0 loop and
b2-sheet. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)

Q. Hu et al. / European Journal of Medicinal Chemistry 96 (2015) 139e150
145
3. Conclusions
combined organic layers were dried over Na2SO4. The solvent was
removed under reduced pressure and the crude product was pu-
rified by flash chromatography on silica gel.
As evidenced by the clinical application of metyrapone and
the phase I clinical study of LCI699, the inhibition of hCYP11B1 is
an elegant approach to treat Cushing's syndrome, in particular
for the recurrent and subclinical cases. To overcome the incon-
venience in achieving proof of concept in rats, which is caused by
the impotency of our previously discovered hCYP11B1 inhibitors
against the corresponding rat enzyme, efforts were paid to
identify novel lead compounds with different structures. Modi-
fications on a potent promiscuous inhibitor (IV) of hCYP11B1,
hCYP11B2 and hCYP19 (IC50 values of 44, 22 and 35 nM,
respectively) that exhibited moderate rCYP11B1 inhibition
(IC50 ¼ 1500 nM) led to compound 8 as a new promising lead
compound. Compared to the starting point compound IV, this
inhibitor showed not only 22- and 9-fold elevations on both
human and rat CYP11B1 inhibition with IC50 values of 2 and
163 nM, respectively, but also significant improvement of selec-
tivity over hCYP19 (54-fold, IC50 ¼ 1900 nM). Accordingly, com-
pound 8 was around 7- and 28-fold more potent than the drug
metyrapone regarding the inhibition of human and rat CYP11B1.
Although its selectivity over hCYP11B2 was also increased by a
factor of 7 (SF of 3.5 vs 0.5) compared to compound IV reaching a
similar level as metyrapone (SF ¼ 4.9), it was considered insuf-
ficient and remains to be improved. With further optimizations
on the new lead compound 8 that was successfully identified in
this study, drug candidates with satisfying profiles are expected
to be discovered for the consequent drug development and the
research of pathophysiology involving CYP11B1.
4.1.3. General procedure B: etherization
To the corresponding bromophenol in ethanol (5 mL/mmol) was
added K2CO3 (1.2 eq). The resulted mixture was stirred for 2 h
under reflux conditions (90 ꢁC) before the corresponding bromide
(1.3 eq) and KI (5 mol%) were added. Reflux was continued over-
night. After cooling down, the solvent was removed under reduced
pressure and the residue was washed with brine and extracted
three times with ethyl acetate. The combined organic layers were
dried over Na2SO4, the solvent was removed under reduced pres-
sure and the crude product was purified by flash chromatography
on silica gel.
4.1.4. General procedure C: Suzuki coupling catalyzed by Pd(PPh3)4
The corresponding bromide (1.0 eq) and pyridylboronic acid (1.5
eq) were dissolved in a mixture of ethanol (10 mL/100 mg), toluene
(10 mL/100 mg) and an aqueous 2.0 M Na2CO3 solution (2.5 mL/
100 mg). The mixture was deoxygenated and flushed with nitrogen
three times before Pd(PPh3)4 (5 mol%) was added. The resulting
suspension was heated under reflux for 3e5 h. After cooling down,
water and ethyl acetate were added and the phases were separated.
The water phase was extracted three times with ethyl acetate and
the combined organic layers were dried over Na2SO4. The solvent
was removed under reduced pressure and the crude product was
purified by flash chromatography on silica gel.
4.1.5. General procedure D: Suzuki coupling catalyzed by
PdCl2(dppf)
4. Experimental section
The corresponding bromide (1.0 eq) and pyridylboronic acid (1.5
eq) were dissolved in a mixture of DME (10 mL/100 mg) and an
aqueous 2.0 M Na2CO3 solution (2.5 mL/100 mg). The mixture was
deoxygenated and flushed with nitrogen three times before
PdCl2(dppf) (5 mol%) was added. The resulting suspension was
heated under reflux for 3e5 h or overnight. After cooling down,
water and ethyl acetate were added and the phases were separated.
The water phase was extracted three times with ethyl acetate and
the combined organic layers were dried over Na2SO4. The solvent
was removed under reduced pressure and the crude product was
purified by flash chromatography on silica gel.
4.1. Chemistry
4.1.1. Chemical and analytical methods
Melting points were measured on a Mettler FP1 melting point
apparatus and are uncorrected. 1H NMR and 13C NMR were
recorded on a Bruker AM500 spectrometer at 500 MHz and
125 MHz, respectively, at 300 K. Chemical shifts are reported in
parts per million (ppm), by reference to the hydrogenated resi-
dues of the deuterated solvent as internal standard. All coupling
constants (J) are given in Hertz (Hz). Mass spectra (LC/UV/MS: ESI)
were recorded on a SpectraSystem/MSQ Plus (ThermoFinnigan)
instrument with a RP18-100-5 column (MachereyeNagel). A wa-
ter/acetonitrile gradient was used as eluent system. All com-
pounds are >95% chemical pure as measured by LC/UV trace at
254 nm. Reagents were used as obtained from commercial sup-
pliers without further purification. Solvents were distilled before
use. If necessary, solvents were dried by distillation from appro-
priate drying reagents prior to use. Flash chromatography was
4.1.6. N-(3-(benzyloxy)-4-(pyridin-3-yl)phenyl)acetamide (1)
To the solution of 3-(benzyloxy)-4-(pyridin-3-yl)aniline
3
(100 mg, 0.36 mmol), triethylamine (0.10 mL, 0.72 mmol) and N,N-
dimethylpyridin-4-amine (2 mg) in dicholoromathane (20 mL) was
dropped in acetyl chloride (29.8 mg, 0.38 mmol) in an ice bath. The
resulting reaction mixture was subsequently stirred at room tem-
perature overnight before water (20 mL) was added and the phases
were separated. The water phase was extracted three times with
dicholoromathane (10 mL) and the combined organic layers were
dried over Na2SO4. The solvent was removed under reduced pres-
sure and the crude product was purified by flash chromatography
on silica gel using a mixture of dicholromethane/methanol (50:1)
as eluent. Yield: 109 mg (95%); yellow solid: mp 157e159 ꢁC;
Rf ¼ 0.29 (n-Hex/EtOAc 1:2); Anal. C20H18N2O2 (C, H, N, O). 1H NMR
(CDCl3, 500 MHz): dH ¼ 8.78 (d, J ¼ 1.7 Hz, 1H), 8.54 (dd, J ¼ 1.7,
4.8 Hz,1H), 7.87e7.89 (m, 1H), 7.47 (d, J ¼ 1.7 Hz,1H), 7.37 (s, br,1H),
7.31e7.34 (m, 1H), 7.24e7.27 (m, 3H), 7.15e7.18 (m, 1H), 7.10e7.12
performed on silica gel 40 (35/40e63/70
mM) with petroleum
ether/ethyl acetate or CH2Cl2/methanol mixtures as eluents. Re-
action progress was monitored by thin-layer chromatography
(TLC) on TLC Silica Gel 60 F254 (Merck KGaA). Visualization was
accomplished with UV light.
4.1.2. General procedure A: methoxyl cleavage with boron
tribromide
A solution of the corresponding methoxyl starting material in
DCM (4 mL/mmol) was cooled to ꢀ20 ꢁC before boron tribromide (3
eq, 1 M in DCM) was added in a nitrogen atmosphere at the same
temperature. The stirred solution was subsequently warmed to
ambient temperature overnight. The resulted reaction mixture was
diluted with water and the phases were separated. The aqueous
layer was extracted three times with ethyl acetate and the
(m, 2H), 6.97 (dd, J ¼ 1.9, 8.2 Hz, 1H), 4.79 (s, 2H), 2.20 (s, 3H); 13
C
NMR (CDCl3, 125 MHz): dC ¼ 156.4, 150.1, 147.7, 141.3, 139.3, 136.8,
136.7, 133.9, 130.6, 128.4, 125.9, 123.0, 122.8, 111.7, 104.3, 70.4, 20.5;
MS (ESI): m/z ¼ 319.14 [MþH]þ.

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Q. Hu et al. / European Journal of Medicinal Chemistry 96 (2015) 139e150
4.1.7. N-(3-(benzyloxy)-4-(pyridin-3-yl)phenyl)-4-
fluorobenzamide (2)
petroleum ether/ethyl acetate (2:1) as eluent. Yield: 137 mg (78%);
yellow solid: mp 125e126 ꢁC; Rf ¼ 0.39 (n-Hex/EtOAc 1:2); Anal.
To the solution of 3-(benzyloxy)-4-(pyridin-3-yl)aniline
3
C
19H16N2O3 (C, H, N, O). 1H NMR (CDCl3, 500 MHz): dH ¼ 8.77 (s,1H),
(100 mg, 0.36 mmol), triethylamine (0.10 mL, 0.72 mmol) and N,N-
dimethylpyridin-4-amine (2 mg) in dicholoromathane (20 mL) was
dropped in 4-fluorobenzoyl chloride (60.0 mg, 0.38 mmol) in an ice
bath. The resulting reaction mixture was subsequently stirred at
room temperature overnight before water (20 mL) was added and
the phases were separated. The water phase was extracted three
times with dicholoromathane (10 mL) and the combined organic
layers were dried over Na2SO4. The solvent was removed under
reduced pressure and the crude product was purified by flash
chromatography on silica gel using a mixture of dicholromethane/
methanol (50:1) as eluent. Yield: 130 mg (91%); yellow solid: mp
166e167 ꢁC; Rf ¼ 0.35 (n-Hex/EtOAc 1:2); Anal. C25H19FN2O2 (C, H,
N, O). 1H NMR (CDCl3, 500 MHz): dH ¼ 8.82 (d, J ¼ 1.7 Hz, 1H), 8.63
(dd, J ¼ 1.7, 4.8 Hz, 1H), 8.10 (dd, J ¼ 1.9, 8.2 Hz, 2H), 7.92e7.94 (m,
1H), 7.65 (d, J ¼ 1.7 Hz, 1H), 7.59 (s, br, 1H), 7.24e7.35 (m, 6H),
7.17e7.22 (m, 3H), 6.95 (m, 1H), 4.76 (s, 2H); 13C NMR (CDCl3,
125 MHz): dC ¼ 159.7 (d, JCF ¼ 245.7 Hz), 154.7, 147.3, 138.8, 137.9,
134.7, 126.9, 123.4, 119.8 (d, JCF ¼ 10.1 Hz), 119.7 (d, JCF ¼ 10.6 Hz),
119.2, 118.9, 118.7, 117.2, 117.1, 114.0, 111.1, 105.8 (d, JCF ¼ 26.6 Hz),
105.6 (d, JCF ¼ 26.1 Hz), 103.7, 102.0, 71.5; MS (ESI): m/z ¼ 399.14
[MþH]þ.
8.72 (d, J ¼ 4.8 Hz, 1H), 8.00 (d, J ¼ 8.3 Hz, 1H), 7.90 (s, 1H), 7.75 (d,
J ¼ 7.9 Hz, 1H), 7.52 (d, J ¼ 8.3 Hz, 1H), 7.31e7.41 (m, 4H), 7.23 (d,
J ¼ 7.4 Hz, 2H), 4.40 (t, J ¼ 6.5 Hz, 2H), 3.15 (t, J ¼ 6.5 Hz, 2H); 13C
NMR (CDCl3, 125 MHz): dC ¼ 156.3, 149.9, 149.2, 148.5, 137.6, 136.9,
133.8, 132.2, 130.9, 129.0, 128.5, 126.7, 122.9, 116.1, 107.0, 69.9, 35.4;
MS (ESI): m/z ¼ 321.12 [MþH]þ.
4.1.11. 3-(4-Nitro-2-(3-phenylpropoxy)phenyl)pyridine (6)
The title compound was synthesized from 1-bromo-4-nitro-2-
phenethoxybenzene 6a (125 mg, 0.37 mmol) according to
method C using 3-pyridylboronic acid. The crude product was pu-
rified by flash chromatography on silica gel using a mixture of
petroleum ether/ethyl acetate (2:1) as eluent. Yield: 89 mg (72%);
yellow solid: mp 117e119 ꢁC; Rf ¼ 0.37 (n-Hex/EtOAc 1:2); Anal.
C
20H18N2O3 (C, H, N, O). 1H NMR (CDCl3, 500 MHz): dH ¼ 8.79 (d,
J ¼ 41.5 Hz, 1H), 8.62 (dd, J ¼ 1.5, 4.8 Hz, 1H), 7.92 (dd, J ¼ 2.1, 8.4 Hz,
1H), 7.87 (dt, J ¼ 1.8, 7.9 Hz, 1H), 7.77 (d, J ¼ 2.1 Hz, 1H), 7.45 (d,
J ¼ 8.3 Hz, 1H), 7.36e7.39 (m, 1H), 7.23e7.25 (m, 2H), 7.15e7.18 (m,
2H), 7.08 (d, J ¼ 7.0 Hz, 2H), 4.07 (t, J ¼ 6.2 Hz, 2H), 2.68 (t, J ¼ 7.3 Hz,
2H), 2.06e2.09 (m, 2H); 13C NMR (CDCl3, 125 MHz): dC ¼ 156.6,
150.3,149.5,148.9,141.0, 137.0,134.1, 132.6,131.1, 128.8,128.6,126.4,
123.2, 116.4, 107.4, 68.4, 32.3, 30.7; MS (ESI): m/z ¼ 335.13 [MþH]þ.
4.1.8. 3-(Benzyloxy)-4-(pyridin-3-yl)aniline (3)
To the suspension of 3-(2-(benzyloxy)-4-nitrophenyl)pyridine 4
(25 mg, 0.08 mmol) and Pd/C (2.2 mg, 10% mmol) in ethanol (5 mL)
was added hydrazine hydrate (11.2 mg, 0.25 mmol) slowly in an ice
bath. The resulting reaction mixture was subsequently heated to
reflux for 1 h. After cooling down to room temperature, water
(10 mL) and ethyl acetate (10 mL) were added and the phases were
separated. The water phase was extracted three times with ethyl
acetate (10 mL) and the combined organic layers were dried over
Na2SO4. The solvent was removed under reduced pressure and the
crude product was purified by flash chromatography on silica gel
using a mixture of dicholromethane/methanol (50:1) as eluent.
Yield: 19 mg (86%); yellow solid: mp 172e174 ꢁC; Rf ¼ 0.21 (n-Hex/
EtOAc 1:2); Anal. C18H16N2O (C, H, N, O). 1H NMR (CDCl3, 500 MHz):
dH ¼ 8.67 (d, J ¼ 2.6 Hz, 1H), 8.47 (dd, J ¼ 1.7, 4.8 Hz, 1H), 7.65e7.67
(m,1H), 7.26e7.29 (m, 2H), 7.15e7.17 (m, 2H), 7.09 (d, J ¼ 8.1 Hz,1H),
6.35 (dd, J ¼ 2.1, 8.1 Hz, 1H), 6.30 (d, J ¼ 2.1 Hz,1H), 4.79 (s, 2H), 3.85
(s, br, 2H, NH2); 13C NMR (CDCl3, 125 MHz): dC ¼ 156.9, 150.0, 147.9,
146.9, 136.7, 134.4, 129.0, 128.4, 126.5, 122.6, 117.5, 107.6, 99.4, 70.4;
MS (ESI): m/z ¼ 277.13 [MþH]þ.
4.1.12. 4-(2-(Benzyloxy)-4-nitrophenyl)pyridine (7)
The title compound was synthesized from 2-(benzyloxy)-1-
bromo-4-nitrobenzene 4a (150 mg, 0.49 mmol) according to
method C using 4-pyridylboronic acid. The crude product was pu-
rified by flash chromatography on silica gel using a mixture of
petroleum ether/ethyl acetate (2:1) as eluent. Yield: 107 mg (72%);
yellow solid: mp 129e121 ꢁC; Rf ¼ 0.33 (n-Hex/EtOAc 1:2); Anal.
C
18H14N2O3 (C, H, N, O). 1H NMR (CDCl3, 500 MHz): dH ¼ 8.69 (s,
2H), 7.94e7.96 (m, 2H), 7.47e7.51 (m, 2H), 7.33e7.37 (m, 5H), 5.12
(s, 2H); 13C NMR (CDCl3, 125 MHz): dC ¼ 156.2, 150.1, 149.1, 144.3,
135.5, 134.9, 131.2, 129.0, 128.7, 127.4, 124.3, 115.7, 108.2, 71.4; MS
(ESI): m/z ¼ 307.10 [MþH]þ.
4.1.13. 4-(4-Nitro-2-phenethoxyphenyl)pyridine (8)
The title compound was synthesized from 1-bromo-4-nitro-2-
phenethoxybenzene 5a (150 mg, 0.47 mmol) according to
method C using 4-pyridylboronic acid. The crude product was pu-
rified by flash chromatography on silica gel using a mixture of
petroleum ether/ethyl acetate (2:1) as eluent. Yield: 104 mg (70%);
yellow solid: mp 112e114 ꢁC; Rf ¼ 0.32 (n-Hex/EtOAc 1:2); Anal.
4.1.9. 3-(2-(Benzyloxy)-4-nitrophenyl)pyridine (4)
C
19H16N2O3 (C, H, N, O). 1H NMR (CDCl3, 500 MHz): dH ¼ 8.60 (dd,
The title compound was synthesized from 2-(benzyloxy)-1-
bromo-4-nitrobenzene 4a (108 mg, 0.35 mmol) according to
method C using 3-pyridylboronic acid. The crude product was pu-
rified by flash chromatography on silica gel using a mixture of
petroleum ether/ethyl acetate (2:1) as eluent. Yield: 80 mg (75%);
lemon yellow solid: mp 121e123 ꢁC; Rf ¼ 0.37 (n-Hex/EtOAc 1:2);
Anal. C18H14N2O3 (C, H, N, O). 1H NMR (CDCl3, 500 MHz): dH ¼ 8.71
(d, J ¼ 1.9 Hz, 1H), 8.53 (dd, J ¼ 1.4, 4.8 Hz, 1H), 7.85e7.88 (m, 2H),
7.79e7.81 (m, 1H), 7.40 (d, J ¼ 8.2 Hz, 1H), 7.23e7.28 (m, 6H), 5.12 (s,
2H); 13C NMR (CDCl3, 125 MHz): dC ¼ 156.0, 150.0, 149.3, 148.6,
136.8,135.3,134.2,132.2,131.0,128.8,128.4,127.1,123.0,116.5,107.9,
71.1; MS (ESI): m/z ¼ 307.10 [MþH]þ.
J ¼ 1.6, 4.5 Hz, 2H), 7.87 (dd, J ¼ 2.2, 8.4 Hz, 1H), 7.79 (d, J ¼ 2.1 Hz,
1H), 7.40 (d, J ¼ 8.4 Hz, 1H), 7.25e7.29 (m, 2H), 7.22e7.24 (m, 3H),
7.12e7.14 (m, 2H), 4.31 (t, J ¼ 6.4 Hz, 2H), 3.04 (t, J ¼ 6.5 Hz, 2H); 13
C
NMR (CDCl3, 125 MHz): dC ¼ 156.5, 150.0, 149.1, 144.2, 137.8, 134.5,
131.0, 129.2, 128.8, 127.0, 124.3, 116.3, 107.3, 70.1, 35.6; MS (ESI): m/
z ¼ 321.12 [MþH]þ.
4.1.14. 4-(4-Nitro-2-(3-phenylpropoxy)phenyl)pyridine (9)
The title compound was synthesized from 1-bromo-4-nitro-2-
phenethoxybenzene 6a (150 mg, 0.45 mmol) according to
method C using 4-pyridylboronic acid. The crude product was pu-
rified by flash chromatography on silica gel using a mixture of
petroleum ether/ethyl acetate (2:1) as eluent. Yield: 113 mg (76%);
yellow solid: mp 127e199 ꢁC; Rf ¼ 0.32 (n-Hex/EtOAc 1:2); Anal.
4.1.10. 3-(4-Nitro-2-phenethoxyphenyl)pyridine (5)
The title compound was synthesized from 1-bromo-4-nitro-2-
phenethoxybenzene 5a (175 mg, 0.54 mmol) according to
method C using 3-pyridylboronic acid. The crude product was pu-
rified by flash chromatography on silica gel using a mixture of
C
20H18N2O3 (C, H, N, O). 1H NMR (CDCl3, 500 MHz): dH ¼ 8.71 (s, br,
2H), 7.91 (s, br, 1H), 7.79 (s, 1H), 7.45e7.47 (m, 3H), 7.23e7.27 (m,
2H), 7.15e7.19 (m, 1H), 7.10e7.12 (m, 2H), 4.09 (t, J ¼ 6.2 Hz, 2H),
2.71 (t, J ¼ 7.3 Hz, 2H), 2.06e2.10 (m, 2H); 13C NMR (CDCl3,

Q. Hu et al. / European Journal of Medicinal Chemistry 96 (2015) 139e150
147
125 MHz): dC ¼ 156.3, 149.8, 148.9, 144.2, 140.7, 134.3, 130.8, 128.6,
128.3, 126.2, 124.0, 116.0, 107.2, 68.2, 32.1, 30.5; MS (ESI): m/
z ¼ 335.13 [MþH]þ.
JCF ¼ 22.0 Hz), 123.6 (d, JCF ¼ 3.7 Hz), 124.4, 126.7, 128.5, 129.0, 131.3
(d, JCF ¼ 10.1 Hz), 137.8, 146.0, 148.8, 157.0 (d, JCF ¼ 9.2 Hz), 163.9 (d,
JCF ¼ 248.4 Hz); MS (ESI): m/z ¼ 293.91 [MþH]þ.
4.1.15. 3-(4-Nitro-3-phenethoxyphenyl)pyridine (10)
4.1.19. 3-(4-Methoxy-2-phenethoxyphenyl)pyridine (14)
The title compound was synthesized from 4-bromo-1-nitro-2-
phenethoxybenzene 10a (108 mg, 0.34 mmol) according to
method D using 3-pyridylboronic acid. The crude product was
purified by flash chromatography on silica gel using a mixture of
petroleum ether/ethyl acetate (2:1) as eluent. Yield: 106 mg, (97%);
orange solid: mp ¼ 91e92 ꢁC; Rf ¼ 0.36 (n-Hex/EtOAc 1:2); Anal.
The title compound was synthesized from 1-bromo-4-methoxy-
2-phenethoxybenzene 14a (100 mg, 0.33 mmol) according to
method C using 3-pyridylboronic acid. The crude product was pu-
rified by flash chromatography on silica gel using a mixture of
petroleum ether/ethyl acetate (2:1) as eluent. Yield: 78 mg (77%);
lemon yellow oil; Rf ¼ 0.55 (n-Hex/EtOAc 1:2); Anal. C20H19NO2 (C,
H, N, O). IR (ATR): n (cmꢀ1) ¼ 3028 (m), 2934 (m), 2836 (m), 1883
(w), 1608 (s), 1165 (vs), 699 (vs); 1H NMR (CDCl3, 500 MHz):
dH ¼ 8.72 (d, J ¼ 1.9 Hz, 1H), 8.55 (dd, J ¼ 1.6, 4.7 Hz, 1H), 7.71e7.75
(m, 1H), 7.24e7.35 (m, 5H), 7.19e7.22 (m, 2H), 6.62 (dd, J ¼ 2.2,
8.4 Hz, 1H), 6.58 (d, J ¼ 2.2 Hz, 1H), 4.23 (t, J ¼ 6.6 Hz, 2H), 3.87 (s,
3H), 3.07 (t, J ¼ 6.6 Hz, 2H); 13C NMR (CDCl3, 125 MHz): dC ¼ 161.0,
156.9,149.5,146.7,138.1,137.4,134.3,131.1,129.0,128.4,126.5,122.8,
119.8, 105.3, 99.8, 69.2, 55.4, 35.6; MS (ESI): m/z ¼ 305.85 [MþH]þ.
C
19H16N2O3 (C, H, N, O). IR (ATR): n (cmꢀ1) ¼ 3040 (m), 2940 (m),
1586 (s), 1505 (s), 1228 (s), 1022 (s), 734 (vs), 694 (vs); 1H NMR
(CDCl3, 500 MHz): dH ¼ 8.78 (s, 1H), 8.63 (d, J ¼ 4.7 Hz, 1H), 7.90 (d,
J ¼ 8.5 Hz, 1H), 7.88 (d, J ¼ 7.3 Hz, 1H), 7.40e7.46 (m, 1H), 7.26e7.30
(m, 4H), 7.18e7.23 (m, 1H), 7.14 (dd, J ¼ 1.4, 8.4 Hz, 1H), 7.11 (s, 1H),
4.32 (t, J ¼ 6.8 Hz, 2H), 3.14 (t, J ¼ 6.8 Hz, 2H); 13C NMR (CDCl3,
125 MHz); dC ¼ 152.9, 148.5, 147.0, 143.3, 139.5, 137.4, 135.8, 135.4,
129.2, 128.6, 126.8, 126.5, 124.2, 119.1, 113.2, 70.9, 35.7; MS (ESI): m/
z ¼ 320.88 [MþH]þ.
4.1.20. 4-(4-Methoxy-2-phenethoxyphenyl)pyridine (15)
4.1.16. 4-(4-Nitro-3-phenethoxyphenyl)pyridine (11)
The title compound was synthesized from 1-bromo-4-methoxy-
2-phenethoxybenzene 14a (106 mg, 0.35 mmol) according to
method C using 4-pyridylboronic acid. The crude product was pu-
rified by flash chromatography on silica gel using a mixture of
petroleum ether/ethyl acetate (1:1) as eluent. Yield: 76 mg (71%);
lemon yellow solid: mp 63e64 ꢁC; Rf ¼ 0.40 (n-Hex/EtOAc 1:2);
Anal. C20H19NO2 (C, H, N, O). IR (ATR): n (cmꢀ1) ¼ 3028 (m), 2955
(m), 2835 (m), 1941 (w), 1610 (s), 1207 (vs), 696 (vs), 783 (vs); 1H
NMR (CDCl3, 500 MHz): dH ¼ 8.47 (dd, J ¼ 1.6, 4.7 Hz, 2H), 7.17e7.28
(m, 6H), 7.13e7.17 (m, 2H), 6.53 (dd, J ¼ 2.5, 8.5 Hz, 1H), 6.49 (d,
J ¼ 2.5 Hz, 1H), 4.17 (t, J ¼ 6.5 Hz, 2H), 3.79 (s, 3H), 3.00 (t, J ¼ 6.5 Hz,
2H); 13C NMR (CDCl3,125 MHz): dC ¼ 161.5,157.0,148.8,146.5,138.1,
131.1, 129.0, 128.5, 126.6, 124.2, 120.4, 105.4, 99.8, 69.1, 55.4, 35.6;
MS (ESI): m/z ¼ 305.86 [MþH]þ.
The title compound was synthesized from 4-bromo-1-nitro-2-
phenethoxybenzene 11a (139 mg, 0.43 mmol) according to
method D using 4-pyridylboronic acid. The crude product was
purified by flash chromatography on silica gel using a mixture of
petroleum ether/ethyl acetate (1:1) as eluent. Yield: 129 mg (94%);
ochre solid: mp ¼ 98 ꢁC; Rf ¼ 0.27 (n-Hex/EtOAc 1:2); Anal.
C
19H16N2O3 (C, H, N, O). IR (ATR): n (cmꢀ1) ¼ 3032 (m), 2951 (m),
1586 (s), 1552 (s), 1509 (s), 1225 (s), 808 (vs), 755 (vs), 698 (vs); 1H
NMR (CDCl3, 500 MHz): dH ¼ 8.66 (d, J ¼ 5.4 Hz, 2H), 7.89 (d,
J ¼ 8.5 Hz, 1H), 7.44 (d, J ¼ 5.0 Hz, 2H), 7.25e7.30 (m, 4H), 7.17e7.22
(m, 2H), 7.15 (d, J ¼ 1.6 Hz, 1H), 4.32 (t, J ¼ 6.8 Hz, 2H), 3.14 (t,
J ¼ 6.8 Hz, 2H); 13C NMR (CDCl3, 125 MHz); dC ¼ 152.7, 149.7, 147.2,
143.8,140.0, 137.4, 129.2, 128.6, 126.9,126.4, 122.0, 119.0, 113.2, 70.9,
35.6; MS (ESI): m/z ¼ 320.96 [MþH]þ.
4.1.21. 3-(2-Phenethoxy-4-(trifluoromethyl)phenyl)pyridine (16)
The title compound was synthesized from 1-bromo-2-
phenethoxy-4-(trifluoromethyl)benzene 16a (106 mg, 0.31 mmol)
according to method C using 3-pyridylboronic acid. The crude
product was purified by flash chromatography on silica gel using a
mixture of hexane/ethyl acetate (2:1) as eluent. Yield: 75 mg (70%);
light yellow solid: mp ¼ 43e44 ꢁC; Rf ¼ 0.68 (n-Hex/EtOAc 1:2);
Anal. C20H16F3NO (C, H, N, O). IR (ATR): n (cmꢀ1) ¼ 3031 (m), 2949
(m),1614 (m),1406 (s), 1328 (vs),1105 (vs) 698 (vs); 1H NMR (CDCl3,
500 MHz): dH ¼ 8.68 (dd, J ¼ 0.8, 2.4 Hz,1H), 8.60 (dd, J ¼ 1.6, 4.7 Hz,
1H), 7.66e7.69 (m, 1H), 7.37e7.40 (m, 1H), 7.20e7.31 (m, 5H),
7.15e7.17 (m, 1H), 7.12e7.15 (m, 2H), 4.24 (t, J ¼ 6.6 Hz, 2H), 3.03 (t,
J ¼ 6.6 Hz, 2H); 13C NMR (CDCl3, 125 MHz): dC ¼ 156.0, 149.8, 148.4,
137.8, 137.2, 133.0, 131.5 (q, JCF ¼ 32.5 Hz), 130.9, 130.6, 129.0, 128.5,
126.6, 122.9, 123.8 (q, JCF ¼ 272.2 Hz), 117.8 (q, JCF ¼ 3.7 Hz), 108.8 (q,
JCF ¼ 3.7 Hz), 69.5, 35.5; MS (ESI): m/z ¼ 343.81 [MþH]þ.
4.1.17. 3-(4-Fluoro-2-phenethoxyphenyl)pyridine (12)
The title compound was synthesized from 1-bromo-4-fluoro-2-
phenethoxybenzene 12a (118 mg, 0.40 mmol) according to method
D using 3-pyridylboronic acid. The crude product was purified by
flash chromatography on silica gel using a mixture of hexane/ethyl
acetate (5:1) as eluent. Yield: 86 mg (73%); brown oil; Rf ¼ 0.66 (n-
Hex/EtOAc 1:2); Anal.
C19H16FNO (C, H, N, O). IR (ATR): n
(cmꢀ1) ¼ 3029 (m), 2935 (m), 1599 (s), 1279 (s), 1160 (vs), 699 (vs);
1H NMR (CDCl3, 500 MHz): dH ¼ 8.73 (d, J ¼ 1.9 Hz, 2H), 7.69 (d,
J ¼ 1.4 Hz, 1H), 7.21e7.34 (m, 5H), 7.15 (d, J ¼ 7.2 Hz, 2H), 6.70e6.76
(m, 2H), 4.19 (t, J ¼ 6.5 Hz), 3.03 (t, J ¼ 6.5 Hz); 13C NMR (CDCl3,
125 MHz): dC ¼ 162.9 (d, JCF ¼ 244.7 Hz), 156.7, 149.5, 147.8, 138.2,
136.6, 132.8, 131.4 (d, JCF ¼ 10.1 Hz), 136.9, 123.0, 117.5, 107.2 (d,
JCF ¼ 21.1 Hz), 100.7 (d, JCF ¼ 26.6 Hz), 69.1, 34.6; MS (ESI): m/
z ¼ 293.87 [MþH]þ.
4.1.18. 4-(4-Fluoro-2-phenethoxyphenyl)pyridine (13)
4.1.22. 4-(2-Phenethoxy-4-(trifluoromethyl)phenyl)pyridine (17)
The title compound was synthesized from 1-bromo-2-
phenethoxy-4-(trifluoromethyl)benzene 16a (100 mg, 0.29) ac-
cording to method C using 4-pyridylboronic acid. The crude prod-
uct was purified by flash chromatography on silica gel using a
mixture of petroleum ether/ethyl acetate (4:1) as eluent. Yield:
73 mg (73%); light fawn solid: mp ¼ 58e59 ꢁC; Rf ¼ 0.55 (n-Hex/
EtOAc 1:2); Anal. C20H16F3NO (C, H, N, O). IR (ATR): n (cmꢀ1) ¼ 3033
(m), 2946 (m), 1591 (s), 1329 (vs), 1109 (vs), 817 (vs); 1H NMR
(CDCl3, 500 MHz): dH ¼ 8.57 (dd, J ¼ 1.6, 4.4 Hz, 2H), 7.37 (dd, J ¼ 7.9,
0.6 Hz, 1H), 7.24e7.29 (m, 5H), 7.20e7.24 (m, 1H), 7.12e7.17 (m, 3H),
4.25 (t, J ¼ 6.3 Hz, 2H), 3.02 (t, J ¼ 6.3 Hz, 2H); 13C NMR (CDCl3,
The title compound was synthesized from 1-bromo-4-fluoro-2-
phenethoxybenzene 12a (128 mg, 0.43 mmol) according to method
D using 4-pyridylboronic acid. The crude product was purified by
flash chromatography on silica gel using a mixture of hexane/ethyl
acetate (5:1) as eluent. Yield: 89 mg (71%); fawn oil; Rf ¼ 0.54 (n-
Hex/EtOAc 1:2); Anal.
C19H16FNO (C, H, N, O). IR (ATR): n
(cmꢀ1) ¼ 3029 (m), 2936 (m), 1599 (vs), 1407 (s), 1280 (vs), 1161
(vs), 832 (vs), 699 (vs); 1H NMR (CDCl3, 500 MHz): dH ¼ 8.54 (dd,
J ¼ 4.7, 1.6 Hz, 2H), 7.21e7.31 (m, 6H), 7.14e7.18 (m, 2H), 6.67e6.75
(m, 2H), 4.20 (t, J ¼ 6.5 Hz, 2H), 3.03 (t, J ¼ 6.5 Hz, 2H); 13C NMR
(CDCl3, 125 MHz) dC ¼ 35.4, 69.4, 100.4 (d, JCF ¼ 25.7 Hz), 107.7 (d,

148
Q. Hu et al. / European Journal of Medicinal Chemistry 96 (2015) 139e150
125 MHz): dC ¼ 155.9, 149.2, 145.2, 137.8, 132.1 (q, JCF ¼ 33.0 Hz),
131.1, 130.8, 129.0, 128.5, 126.7, 124.3, 123.7 (q, JCF ¼ 272.2 Hz), 117.8
(q, JCF ¼ 3.7 Hz), 108.9 (q, JCF ¼ 3.7 Hz), 69.5, 35.5; MS (ESI): m/
z ¼ 343.82 [MþH]þ.
4.5 Hz, 2H), 7.25e7.30 (m, 2H), 7.19e7.24 (m, 3H), 7.15 (d, J ¼ 1.6 Hz,
1H), 7.05 (dd, J ¼ 1.6, 7.6 Hz, 1H), 4.30 (t, J ¼ 6.5 Hz, 2H), 2.85e3.05
(m, 8H); 13C NMR (DMSO-d6, 125 MHz): dC ¼ 169.4, 155.3, 149.3,
144.8, 138.5, 138.4, 130.2, 129.0, 128.2, 127.4, 126.3, 124.0, 119.4,
111.3, 68.9, 34.8; MS (ESI): m/z ¼ 346.80 [MþH]þ.
4.1.23. 3-Phenethoxy-4-(pyridin-3-yl)benzonitrile (18)
The title compound was synthesized from 4-bromo-3-
phenethoxybenzonitrile 18a (105 mg, 0.35 mmol) according to
method D using 3-pyridylboronic acid. The crude product was
purified by flash chromatography on silica gel using a mixture of
petroleum ether/ethyl acetate (1.5:1) as eluent. Yield: 82 mg, (78%);
brown oil; Rf ¼ 0.56 (n-Hex/EtOAc 1:2); Anal. C20H16N2O (C, H, N,
O). IR (ATR): n (cmꢀ1) ¼ 3029 (m), 2936 (m), 2228 (s), 1506 (s), 1397
(s), 1277 (vs), 1021 (s), 700 (vs); 1H NMR (CDCl3, 500 MHz): dH ¼ 8.7
(br, s, 1H) 8.6 (br, s, 1H) 7.7 (d, J ¼ 7.6 Hz, 1H) 7.3e7.4 (m, 3H) 7.2e7.3
(m, 4H) 7.1e7.2 (m, 2H) 4.3 (t, J ¼ 6.5 Hz, 2H) 3.1 (t, J ¼ 6.5 Hz, 2H);
13C NMR (CDCl3, 125 MHz): dC ¼ 156.0, 149.2, 148.2, 137.5, 134.4,
132.8, 131.8, 131.2, 128.9, 128.5, 126.7, 125.0, 123.1, 118.4, 115.1, 113.0,
69.6, 35.4; MS (ESI): m/z ¼ 300.96 [MþH]þ.
4.1.27. 3-Phenethoxy-2,30-bipyridine (22)
The title compound was synthesized from 2-bromo-3-
phenethoxypyridine 22a (95 mg, 0.342 mmol) according to
method C using 3-pyridylboronic acid. The crude product was pu-
rified by flash chromatography on silica gel using ethyl acetate as
eluent. Yield: 92 mg, (97.5%); light yellow solid: mp ¼ 126e127 ꢁC;
Rf ¼ 0.14 (n-Hex/EtOAc 1:2); Anal. C18H16N2O (C, H, N, O). IR (ATR): n
(cmꢀ1) ¼ 3510 (br, m), 2990 (m), 1579 (m), 1452 (s), 1321 (vs), 696
(s); 1H NMR (CDCl3, 500 MHz): dH ¼ 9.2 (dd, J ¼ 0.6, 2.2 Hz, 1H), 8.6
(dd, J ¼ 1.6, 5.0 Hz, 1H), 8.3 (dd, J ¼ 1.4, 4.6 Hz, 1H), 8.2 (dt, J ¼ 2.0,
8.0 Hz,1H), 7.4 (ddd, J ¼ 0.8, 4.8, 8.0 Hz,1H), 7.3e7.3 (m, 3H), 7.2e7.3
(m, 4H), 4.3 (t, J ¼ 6.6 Hz, 2H), 3.1 (t, J ¼ 6.6 Hz, 2H); 13C NMR (CDCl3,
125 MHz): dC ¼ 153.2, 149.8, 148.2, 144.7, 141.8, 137.6, 137.4, 133.7,
129.0, 128.6, 126.8, 123.8, 123.0, 119.5, 69.4, 35.6; MS (ESI): m/
z ¼ 276.95 [MþH]þ.
4.1.24. 3-Phenethoxy-4-(pyridin-4-yl)benzonitrile (19)
The title compound was synthesized from 4-bromo-3-
phenethoxybenzonitrile 18a (110 mg, 0.36 mmol) according to
method D using 4-pyridylboronic acid. The crude product was
purified by flash chromatography on silica gel using a mixture of
petroleum ether/ethyl acetate (1.5:1) as eluent. Yield: 92 mg, (85%);
white solid: mp ¼ 112e113 ꢁC; Rf ¼ 0.44 (n-Hex/EtOAc 1:2); Anal.
4.1.28. 3-Phenethoxy-2,40-bipyridine (23)
The title compound was synthesized from 2-bromo-3-
phenethoxypyridine 22a (50 mg, 0.18 mmol) according to method
D using 4-pyridylboronic acid. The crude product was purified by
flash chromatography on silica gel using ethyl acetate as eluent.
Yield: 35 mg, (70.4%); light brown solid: mp ¼ 39e40 ꢁC; Rf ¼ 0.16
(n-Hex/EtOAc 1:2); Anal. C18H16N2O (C, H, N, O). IR (ATR): n
(cmꢀ1) ¼ 3454 (br, m),1642 (m),1437 (s), 1322 (vs),1272 (s), 814 (s),
744 (s), 699 (s); 1H NMR (CDCl3, 500 MHz): dH ¼ 8.6 (d, J ¼ 4.7 Hz,
2H), 8.3 (dd, J ¼ 1.6, 4.4 Hz, 1H), 7.7 (d, J ¼ 5.4 Hz, 2H), 7.2e7.3 (m,
7H), 4.3 (t, J ¼ 6.6 Hz, 2H), 3.1 (t, J ¼ 6.6 Hz, 2H); 13C NMR (CDCl3,
125 MHz): dC ¼ 153.7, 149.6, 145.3, 145.1, 141.9, 137.9, 129.2, 128.8,
127.0,124.6,124.0,119.9, 69.5, 35.8; MS (ESI): m/z ¼ 276.97 [MþH]þ.
C
20H16N2O (C, H, N, O). IR (ATR): n (cmꢀ1) ¼ 3031 (m), 2928 (m),
2228 (s), 1595 (s), 1277 (s), 1024 (s), 808 (vs), 752 (vs), 705 (vs); 1H
NMR (CDCl3, 500 MHz): dH ¼ 8.6 (d, J ¼ 6.0 Hz, 2H), 7.4 (d, J ¼ 7.9 Hz,
1H), 7.3 (dd, J ¼ 1.6, 7.6 Hz, 1H), 7.2e7.3 (m, 5H), 7.2 (d, J ¼ 1.6 Hz,
1H) 7.1e7.2 (m, 2H), 4.3 (t, J ¼ 6.5 Hz, 2H), 3.1 (t, J ¼ 6.5 Hz, 2H); 13
C
NMR (CDCl3, 125 MHz): dC ¼ 155.9, 149.4, 144.5, 137.6, 132.6, 131.1,
128.9, 128.5, 126.8, 124.9, 124.1, 118.4, 115.2, 113.4, 69.6, 35.4; MS
(ESI): m/z ¼ 300.96 [MþH]þ.
4.1.25. N,N-dimethyl-3-phenethoxy-4-(pyridin-3-yl)benzamide
(20)
4.1.29. 3-(4-Nitro-2-phenethoxybenzyl)pyridine (24)
The title compound was synthesized from 4,4,5,5-tetramethyl-
2-(4-nitro-2-phenethoxyphenyl)-1,3,2-dioxaborolane 24a (97 mg,
0.26 mmol) according to method D using 3-pyridylboronic acid.
The crude product was purified by flash chromatography on silica
gel using a mixture of hexane/ethyl acetate (2:1) as eluent. Yield:
55 mg (63%); light brown solid: mp ¼ 82e83 ꢁC; Rf ¼ 0.48 (n-Hex/
EtOAc 1:2); Anal. C20H18N2O3 (C, H, N, O). IR (ATR): n (cmꢀ1) ¼ 3061
(m), 2928 (m), 2879 (m), 1585 (m), 1511 (vs), 1337 (vs), 1248 (vs),
1026 (s), 744 (vs), 704 (vs); 1H NMR (CDCl3, 500 MHz): dH ¼ 8.48
(br, s, 2H), 7.78 (dd, J ¼ 8.2, 2.2 Hz, 1H), 7.70 (d, J ¼ 2.2 Hz, 1H), 7.38
(d, J ¼ 7.9 Hz, 1H), 7.30e7.35 (m, 2H), 7.24e7.28 (m, 3H), 7.22 (d,
J ¼ 8.2 Hz, 1H), 7.18 (dd, J ¼ 7.4, 4.9 Hz, 1H), 4.30 (t, J ¼ 6.6 Hz, 2H),
3.95 (s, 2H), 3.13 (t, J ¼ 6.6 Hz, 2H); 13C NMR (CDCl3, 125 MHz)
dC ¼ 156.6, 149.8, 147.8, 147.4, 137.6, 136.5, 136.0, 134.9, 130.3, 128.8,
128.6, 126.7, 123.5, 115.9, 106.2, 69.3, 35.5, 33.4; MS (ESI): m/
z ¼ 335.03 [MþH]þ.
The title compound was synthesized from 4-bromo-N,N-
dimethyl-3-phenethoxybenzamide 20a (100 mg, 0.29 mmol) ac-
cording to method C using 3-pyridylboronic acid. The crude prod-
uct was purified by flash chromatography on silica gel using ethyl
acetate as eluent. Yield: 95 mg (95%); fawn solid: mp ¼ 161e162 ꢁC;
Rf ¼ 0.05 (n-Hex/EtOAc 1:2); Anal. C22H22N2O2 (C, H, N, O). IR (ATR):
n (cmꢀ1) ¼ 3451 (br, m), 2927 (m), 1711 (m), 1609 (s), 1406 (s), 1329
(vs), 703 (s); 1H NMR ((CD3)2CO, 500 MHz): dH ¼ 8.69 (dd, J ¼ 1.0,
2.2 Hz, 1H), 8.54 (dd, J ¼ 1.6, 4.7 Hz, 1H), 7.78 (ddd, J ¼ 1.9, 3.5,
7.9 Hz, 1H), 7.40 (d, J ¼ 7.9 Hz, 1H), 7.36 (ddd, J ¼ 0.8, 4.9, 7.9 Hz, 1H),
7.16 (d, J ¼ 1.6 Hz,1H), 7.09 (dd, J ¼ 1.4, 7.7 Hz,1H), 4.33 (t, J ¼ 6.5 Hz,
2H), 3.02e3.07 (m, 8H); 13C NMR ((CD3)2CO, 125 MHz): dC ¼ 171.1,
156.8, 150.9, 149.1, 139.6, 138.7, 137.7, 134.4, 131.1, 130.1, 129.2, 129.0,
127.2, 123.8, 120.6, 112.4, 70.2, 60.6, 36.3; MS (ESI): m/z ¼ 346.81
[MþH]þ.
4.1.26. N,N-dimethyl-3-phenethoxy-4-(pyridin-4-yl)benzamide
(21)
4.1.30. 4-(4-Nitro-2-phenethoxybenzyl)pyridine (25)
The title compound was synthesized from 4,4,5,5-tetramethyl-
2-(4-nitro-2-phenethoxyphenyl)-1,3,2-dioxaborolane 24a (80 mg,
0.22 mmol) according to method D using 4-pyridylboronic acid.
The crude product was purified by flash chromatography on silica
gel using a mixture of hexane/ethyl acetate (2:1) as eluent. Yield:
51 mg (69%); brown oil; Anal. C20H18N2O3 (C, H, N, O). Rf ¼ 0.35
(n-Hex/EtOAc 1:2); IR (ATR): n (cmꢀ1) ¼ 3028 (m), 2923 (m), 2851
(m), 1598 (m), 1517 (vs), 1341 (vs), 1247 (vs), 801 (s), 738 (vs), 700
(vs); 1H NMR (CDCl3, 500 MHz): dH ¼ 8.45 (d, J ¼ 6.0 Hz, 2H), 7.82
(dd, J ¼ 8.2, 2.2 Hz, 1H), 7.74 (d, J ¼ 2.2 Hz, 1H), 7.25e7.33 (m, 5H),
The title compound was synthesized from 4-bromo-N,N-
dimethyl-3-phenethoxybenzamide 20a (96 mg, 0.28 mmol) ac-
cording to method C using 4-pyridylboronic acid. The crude prod-
uct was purified by flash chromatography on silica gel using ethyl
acetate as eluent. Yield: 76 mg (78%); white solid:
mp ¼ 262e263 ꢁC; Rf ¼ 0.05 (n-Hex/EtOAc 1:2); Anal. C22H22N2O2
(C, H, N, O). IR (ATR): n (cmꢀ1) ¼ 3438 (br, m), 2933 (m), 1609 (s),
1414 (s), 1315 (vs), 700 (s); 1H NMR (DMSO-d6, 500 MHz): dH ¼ 8.53
(dd, J ¼ 1.6, 4.5 Hz, 2H), 7.41 (d, J ¼ 7.6 Hz, 1H), 7.35 (dd, J ¼ 1.6,

Q. Hu et al. / European Journal of Medicinal Chemistry 96 (2015) 139e150
149
7.21 (dd, J ¼ 7.7,1.7 Hz, 2H), 7.10 (d, J ¼ 6.3 Hz, 2H), 4.32 (t, J ¼ 6.5 Hz,
2H), 4.02 (s, 2H), 3.10 (t, J ¼ 6.5 Hz, 2H); 13C NMR (CDCl3, 125 MHz):
dC ¼ 156.7, 152.5, 148.2, 147.0, 140.7, 137.5, 134.1, 130.8, 128.7, 126.9,
124.7, 116.1, 106.4, 69.2, 35.9, 35.4; MS (ESI): m/z ¼ 334.99 [MþH]þ.
as the ligand was built and energy-minimized in the MMFF94s
force field with MOE. After importing both the protein model and
the ligand into GOLD, the heme iron was appointed as the origin of
active-site with a radius 19 Å, although the function of automatic
active-site detection was also switched on. A distance constraint of
1.5e4.5 Å between the heme iron and the coordinating N was set.
Ligand was docked in 50 independent genetic algorithm iterations
for each run with default parameters. The resulting binding poses
were evaluated using the goldscore function with p450_pdb pa-
rameters and subsequently ranked according to fitness. The diverse
binding modes were further investigated and illustrated with the
LigX module in MOE.
4.2. Biology
4.2.1. Inhibition of hCYP11B1, hCYP11B2 and rCYP11B1
V79MZhCYP11B1, V79MZhCYP11B2 or V79MZrCYP11B1 cells
were pre-incubated with inhibitor for 1 h at 37 ꢁC. The reaction was
started by addition of 100 nM (hCYP11B) or 500 nM (rCYP11B1)
[3H]-11-deoxycorticosterone as substrate. After incubation for
25 min (hCYP11B1), 45 min (hCYP11B2) or 7 h (rCYP11B1), the
enzyme reactions were stopped by extracting the supernatant with
ethyl acetate. Samples were centrifuged, and the ethyl acetate was
separated [46,50]. The steroids were separated by HPLC and
analyzed with radio flow detection.
Acknowledgments
We thank Jeannine Jung for performing the in vitro tests and are
grateful to Prof. J. J. Rob Hermans, University of Maastricht, The
Netherlands, for providing the V79MZhCYP11B1 cells.
4.2.2. Inhibition of hCYP19
The inhibition of hCYP19 was determined in vitro using human
placental microsomes with [1
[48].
Appendix A. Supplementary data
b
-3H]androstenedione as substrate
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.04.013.
4.2.3. Inhibition of hCYP17
Human CYP17 was expressed in E. coli (coexpressing human
CYP17 and NADPH-P450 reductase) and the assay was performed
as previously described [49].
References
[1] C. Steffensen, A.M. Bak, K.Z. Rubeck, J.O. Jørgensen, Epidemiology of Cushing's
syndrome, Neuroendocrinology 92 (Suppl. 1) (2010) 1e5.
[2] R.N. Clayton, Mortality in Cushing's disease, Neuroendocrinology 92 (Suppl. 1)
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4.3. Modeling studies
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The primary sequence of CYP11B1 Homo sapiens (UniProt code:
P15538) was obtained from the Universal Protein Resource (Uni-
Prot, http://www.uniprot.org/), which was subsequently subject to
similarity search in the UniProtKB database using the Basic Local
Alignment Search Tool (BLAST) sever in UniProt. Among the hits,
hCYP11B2 (UniProt code: P19099) with a crystal in complex with
deoxycorticosterone (PDB ID: 4DVQ, chain A) that showed 93.2%
identity and an E-value of 0.0 was selected as the template. The
target and template proteins subsequently underwent a tree-based
initial sequence alignment and then structural realignment taking
into consideration the coordinates of alpha carbons in the
hCYP11B2 crystal using MOE. After setting the force field to
Amber99 with Solvation R Field, a library of ten initial models was
generated employing the Generalized Born/Volume Integral (GB/
VI) scoring function, from which the best one was selected ac-
cording to electrostatic solvation energy and root mean square
deviation of each intermediate model to the average position of all
models.
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model, and the model was partially charged and energy-minimized
to RMS Gradient of 0.1 before the initiation of molecular dynamic
simulation using MOE. Conformations were generated in the sta-
tistical ensemble with thermodynamic variables of volume, tem-
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ꢀ
ꢀ
method of the NoseePoincareeAnderson equations of motion was
employed to create ensemble trajectory. The system was heated
from 0 K to 300 K in 60 ps and, after a run time of 1 ns, it was cooled
down to 0 K in 60 ps.
4.3.3. Molecular docking
The homology model was prepared by adding hydrogens and
partial charges using the Protonate3D module in MOE. Compound 8
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