Replacement of imidazolyl by pyridyl in biphenylmethylenes results in selective CYP17 and dual CYP17/CYP11B1 inhibitors for the treatment of prostate cancer

Article abstract of DOI:10.1021/jm100317b

Androgens are well-known to stimulate prostate cancer (PC) growth. Thus, blockade of androgen production in testes and adrenals by CYP17 inhibition is a promising strategy for the treatment of PC. Moreover, many PC patients suffer from glucocorticoid overproduction, and importantly mutated androgen receptors can be stimulated by glucocorticoids. In this study, the first dual inhibitor of CYP17 and CYP11B1 (the enzyme responsible for the last step in glucocorticoid biosynthesis) is described. A series of biphenylmethylene pyridines has been designed, synthesized, and tested as CYP17 and CYP11B1 inhibitors. The most active compounds were also tested for selectivity against CYP11B2 (aldosterone synthase), CYP19 (aromatase), and hepatic CYP3A4. In detail, compound 6 was identified as a dual inhibitor of CYP17/CYP11B1 (IC₅₀ values of 226 and 287 nM) showing little inhibition of the other enzymes as well as compound 9 as a selective, highly potent CYP17 inhibitor (IC₅₀ = 52 nM) exceeding abiraterone in terms of activity and selectivity.

Full text of DOI:10.1021/jm100317b

J. Med. Chem. 2010, 53, 5749–5758 5749
DOI: 10.1021/jm100317b
Replacement of Imidazolyl by Pyridyl in Biphenylmethylenes Results in Selective CYP17 and Dual
CYP17/CYP11B1 Inhibitors for the Treatment of Prostate Cancer
†
Qingzhong Hu, Carsten Jagusch, Ulrike E. Hille, Jorg Haupenthal, and Rolf W. Hartmann*
€
Pharmaceutical and Medicinal Chemistry and Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarland University,
Campus C23, D-66123 Saarbru€cken, Germany. †Present address: Dottikon Exclusive Synthesis AG, 5605 Dottikon, Switzerland.
Received March 10, 2010
Androgens are well-known to stimulate prostate cancer (PC) growth. Thus, blockade of androgen
production in testes and adrenals by CYP17 inhibition is a promising strategy for the treatment of PC.
Moreover, many PC patients suffer from glucocorticoid overproduction, and importantly mutated
androgen receptors can be stimulated by glucocorticoids. In this study, the first dual inhibitor of CYP17
and CYP11B1 (the enzyme responsible for the last step in glucocorticoid biosynthesis) is described. A
series of biphenylmethylene pyridines has been designed, synthesized, and tested as CYP17 and
CYP11B1 inhibitors. The most active compounds were also tested for selectivity against CYP11B2
(aldosterone synthase), CYP19 (aromatase), and hepatic CYP3A4. In detail, compound 6 was identified
as a dual inhibitor of CYP17/CYP11B1 (IC50 values of 226 and 287 nM) showing little inhibition of the
other enzymes as well as compound 9 as a selective, highly potent CYP17 inhibitor (IC50 = 52 nM)
exceeding abiraterone in terms of activity and selectivity.
Introduction
(CYP17). CYP17 is the crucial enzyme catalyzing the conver-
sion of pregnenolone and progesterone to dehydroepiandro-
sterone (DHEA) and androstenedione in gonadal and adrenal
glands6 (Chart 1). Subsequently, these two weak androgens
are converted to testosterone. In the prostate cells, the latter
steroid is transformed into the most potent androgen dihy-
drotestosterone (DHT) by 5R-reductase (5R-R). Because in-
hibitors of 5R-R7 reduce the intracellular prostatic DHT
stimulation, they are exploited in the treatment of benign pro-
static hyperplasia. For PC treatment, however, total blockade
of androgen production is necessary.
However, a major portion of patients becomes “castration
resistant”, which might be caused by androgen receptor muta-
tions, as mentioned above. Androgen receptor activation by
glucocorticoids, especially cortisol, stimulates cancer cell pro-
liferation.5 Moreover, small cell anaplastic prostate carcinoma,
which originates from neuroendocrine cells, is found to be
capable of ectopic ACTH production.8a The elevated ACTH
levels promote adrenals to synthesize and release high con-
centrations of cortisol leading to Cushing’s syndrome,8b,c,d
diabetes mellitus, osteoporosis, hypertension, and obesity. High
cortisol levels have also been considered as a sign of PC cell
growth.8b Importantly, some patients die of severe infections
largely due to the immunosuppression caused by the gluco-
corticoid.8c,d Hence, for these patients the control of cortisol
concentration is urgently needed. As the key step in the bio-
synthesis of this hormon is catalyzed by CYP11B1 (Chart 1),
additionalinhibitionofthisenzyme couldbea substantialway
to improve curative effects, relieve symptoms, and increase
survival of prostate cancer patients.
As the growth of up to 80% of prostate carcinoma is
androgen dependent,1 blockage of androgen production or
action will effectively prevent cancer cells from proliferation.
Currently, the standard therapy for prostate tumor is the so-
called “combined androgen blockade” (CABa), which means
orchidectomy or treatment with gonadotropin-releasing hor-
mone (GnRH) analogues (chemical castration)2 combined
with androgen receptor antagonists.3 In CAB therapy, orch-
idectomy or GnRH treatment is employed to annihilate
testicular androgen production and consequently to reduce
plasma androgen concentration. However, although about
90% of androgens are no longer produced, the minor amount
of androgens from the adrenals is still sufficient to prompt
cancer growth. To solve this problem, androgen receptor anta-
gonists are additionally applied to prevent androgen stimula-
tion. Nonetheless, mutations in androgen receptor are the
reason for the emerging resistance to this therapy. The mutated
receptor can be activated by recognizing antiandrogens4 and
glucocorticoids5 as agonists.
A promising alternative to CAB is the total blockage of
androgen biosynthesis in both testes and adrenals. An elegant
way to achieve this is to inhibit 17R-hydroxylase-17,20-lyase
*To whom correspondence should be addressed. Address: Pharma-
ceutical and Medicinal Chemistry, Saarland University, P.O. Box 151150,
€
D-66123 Saarbrucken, Germany. Phone: þ(49) 681 302 70302. Fax:
þ(49) 681 302 70308. E-mail: rwh@mx.uni-saarland.de. Home page:
http://www.PharmMedChem.de.
aAbbreviations: PC, prostate cancer; CYP, cytochrome P450; CAB,
combined androgen blockade; GnRH, gonadotropin-releasing hor-
mone; CYP17, 17R-hydroxylase-17,20-lyase; CYP11B1, 11β- hydroxy-
lase; CPY19, aromatase, estrogen synthase; CYP11B2, aldosterone
synthase; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone;
DOC, 11-deoxycorticosterone; 5R-R, 5R-reductase, TBAB, tetrabuty-
lammonium bromide; DCM, dichloromethane.
Two decades ago, the first attempt to cure prostate cancer
via CYP17 inhibition was described: the antimycotic ketoco-
nazole (Chart 2) was exploited as CYP17 inhibitor for the
treatment of prostate cancer clinically.9a Although curative
r
2010 American Chemical Society
Published on Web 07/15/2010
pubs.acs.org/jmc

5750 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Chart 1. Androgen Biosynthesis
Hu et al.
Chart 2. Inhibitor Design Conception
not be neglected which are caused by the affinity of steroidal
scaffolds toward steroid receptors. Therefore, nonsteroidal
CYP17 inhibitors, such as heterocycle substituted tetrahydro-
naphthalenes,12a adamantine carboxylates,12b and biphenyls,13
attract more and more attention.
Our group has reported about several series of biphenyl
methylene imidazoles13 as potent CYP17 inhibitors. All these
compounds were designed on the basis of the mechanism that
the sp2 hybrid nitrogen can coordinate with the heme iron,
which was first14a identified for aromatase (CYP19, estrogen
synthase) inhibitors14 and later was also proven to be valid for
aldosterone synthase (CYP11B2)15 and CYP17 inhibitors.10,12,13
Although potent inhibitors, like 1K13e (Chart 2, IC50
=
131 nM; for comparison, abiraterone IC50 = 72 nM), were
obtained after systematic modification on the biphenyl core
and methylene bridge, activity and selectivity could possibly
be further improved. Furthermore, compounds with addi-
tional CYP11B1 inhibition seemed feasible. After abirater-
one, 1K, and metyrapone (Chart 2) were superimposed using
their heterocyclic N as a common atom, it is apparent that all
of three compounds are composed of three structural features:
the hydrophobic core, an alkyl linker in between, and a
heterocycle containing N. Since metyrapone is a CYP11B1
inhibitor, which has been applied in the clinic to treat Cush-
ing’s syndrome for nearly half a century,16 and 1K also exhi-
bits weak CYP11B1 inhibition, this common structural pat-
tern might be the basis of dual inhibition while the inhibitory
potency toward each enzyme depends on the choice of the
structural features. It is obvious that the steroidal scaffold
of abiraterone and the biphenyl core of 1K mate sterically.
Nevertheless, the steroidal scaffold was avoided when choos-
ing the hydrophobic core because of the potential affinities of
steroidal structures for various steroidal receptors. As for
metyrapone, the nicotinoyl moiety occupies the same space as
the steroidal C-ring. Since pyridyl as C-ring leads to a total
loss of CYP17 inhibition,13d the nicotinoyl moiety was re-
placed by a biphenyl moiety furnished with polar substituents.
Moreover, a substituted methylene bridge, observed for these
three inhibitors, was sustained, as we regard it to be crucial for
inhibition of the two enzymes. Furthermore, inspired by the
response was observed,9b ketoconazole was withdrawn be-
cause of side effects associated with its poor selectivity against
other steroidogenic and hepatic CYP enzymes. Later, several
steroidal inhibitors were designed on the basis of endogenous
substrates.10 Recently, abiraterone11a (Chart 2) entered into
phase II and phase III clinical trials. Castration resistant
patients showed good response to abiraterone,11b which con-
firms the superiority of prostate carcinoma treatment via
CYP17 inhibition. However, the potential side effects might

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5751
Table 2. Inhibition of CYP17 by Compounds 5-20
Table 1. Inhibition of CYP17 by Compounds 1-4
compd
R1
NH2
R2
IC50 (nM)b
compd
R1
R2
heterocycles
IC50 (nM)b
5
H
408
226
6
H
NH2
NH2
H
ref 1
1
Et
Et
Et
H
OMe
OMe
OMe
OH
1-Im
3-Py
4-Py
1-Im
3-Py
4-Py
>5000
>5000
1610
7
NH2
OH
337
248
4
2
ref 2c
3
>5000
4040
248
8
H
OH
OH
OH
F
97
52
9
H
OH
OH
10
11
12
13
14
15
16
17
18
19
20
MYPa
KTZa
ABTa
OH
AcNH
H
186
>5000
876
4
H
MYPa
KTZa
ABTa
>10000
2780
72
H
AcNH
H
NH2CO
F
1790
386
H
a MYP: metyrapone. KTZ: ketoconazole. ABT: abiraterone. b Con-
centration of inhibitors required to give 50% inhibition. The given
values are mean values of at least three experiments. The deviations
were within (10%. The assay was run with human CYP17 expressed in
E.coli using progesterone as substrate (25 μM). c ref 2 was taken from ref
13a, where the inhibitory potency was tested with human testicular micro-
somes as 0.31 μM.
F
F
598
OMe
BocNH
BocNH
BocNH
OMe
F
3340
1370
>10000
>10000
>10000
>10000
2780
72
H
F
BocNH
BocNH
facts that pyridyl has already been successfully employed in
other types of CYP17 inhibitors,12a,b leading to compounds
being more potent than the corresponding imidazoles,12a and
importantly that the pyridyl group is a common feature of
CYP17 and CYP11B1 inhibitors, we replaced imidazole by
pyridine. Accordingly, by use of this segmentation and hy-
bridization procedure, biphenylmethylene pyridines were de-
signed. Depending on the substituents at the core, these new
compounds could be dual inhibitors of CYP17 and CYP11B2
orselectiveCYP17 inhibitors. This is becausemaximizationof
CYP17 inhibition was pursued as the first priority during the
segment selection by choosing biphenyl instead of nicotinoyl.
High inhibitory potency toward this enzyme should be the
result, whereas inhibition toward CYP11B1 will largely de-
pend on the substituents at the core.
a MYP: metyrapone. KTZ: ketoconazole. ABT: abiraterone. b Con-
centration of inhibitors required to give 50% inhibition. The given
values are mean values of at least three experiments. The deviations
were within (10%. The assay was run with human CYP17 expressed in
E.coli using progesterone as substrate (25 μM).
Table 3. Inhibition of CYP17 by Compounds 21-25
As the first step, the effects of this design strategy on CYP17
inhibition were substantiated by comparing the imidazole and
3- and 4-pyridine analogues, i.e., ref 1,13f ref 2,13a and com-
pounds 1-4 (Table 1). Encouraged by the positive results,
more modifications on the A-ring were performed by introdu-
cing groups with different electrostatic potential, H-bond
forming properties, and various steric demand to improve
inhibition toward CYP17 and CYP11B1 (5-25, Tables 2 and
3). After determination of CYP17 inhibition, the most potent
compounds were further tested for CYP11B1 inhibition and for
selectivity against CYP11B2, CYP19, and hepatic CYP3A4.
compd
heterocycle
R
IC50 (nM)b
21
22
2-thiophene
3-thiophene
5-indole
577
647
23
24
760
OMe
OH
2000
438
25
MYPa
KTZa
ABTa
>10000
2780
72
a MYP: metyrapone. KTZ: ketoconazole. ABT: abiraterone. b Con-
centration of inhibitors required to give 50% inhibition. The given
values are mean values of at least three experiments. The deviations
were within (10%. The assay was run with human CYP17 expressed in
E.coli using progesterone as substrate (25 μM).
Results
Chemistry. The synthesis of compounds 1-25 is shown in
Schemes 1 and 2. A general strategy was employed starting
from the corresponding (4-bromophenyl)pyridylmethanones,
which are commercially available but were easily prepared
from bromobenzene and the corresponding nicotinic or iso-
nicotinic acid via Friedel-Crafts acylation. The starting ma-
terial and the corresponding boronic acids were first reacted
via Suzuki coupling to form the biaryl moieties, and then the
ketone group was transferred by Wolff-Kishner reduction to
afford the final methylene products. Subsequently, a more
efficient strategy was used by first synthesizing the 4-bromo-
benzylpyridine 5a as a common building block, followed by
introduction of the aryl ring via Suzuki coupling. For
compounds 1 and 2 substituted with an ethyl on the methyl-
ene bridge, the ketone intermediates were reacted with
ethylmagnesium bromide to give the corresponding alcohols
1b and 2b. After H2O elimination in formic acid using
Pd(OAc)2 as catalyst, hydrogenation was performed to yield
the final compounds. Moreover, the OH analogues 8, 9, 10,

5752 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Hu et al.
Scheme 1a
a Reagents and conditions. (i) Method A: Pd(OAc)2, 4-methoxyphenylboronic acid, Na2CO3, TBAB, toluene, H2O, ethanol, reflux, 6 h. (ii) Method
C: EtMgCl, THF. (iii) Method E: Pd(OAc)2, HCOOH, reflux, 16 h. (iv) Method F: Pd/C, H2. (v) Method B: N2H4, KOH, ethylene glycol, reflux, 4 h.
(vi) Method D: BBr3, DCM.
Scheme 2a
a Reagents and conditions. (i) Method A: Pd(OAc)2, corresponding boronic acid, Na2CO3, TBAB, toluene, H2O, ethanol, reflux, 6 h. (ii) Method B:
N2H4, KOH, ethylene glycol, reflux, 4 h.
and 25 were obtained by ether cleavage of the corresponding
methoxy compounds 8a, 9a, 16, and 24, respectively, using
boron tribromide.
hydroxy at the 4-position of the A-ring increase the
activity.13d-g Taking these SARs into consideration, refer-
ence compounds ref 113f and ref 213a were chosen, each
possessing one advantageous structural feature, ethyl or
hydroxy, but showing low activity. Accordingly, the 3- and
4-pyridyl analogues were synthesized (Table 1). It becomes
apparent that no matter which substituent was furnished, the
3-pyridine analogues 1 and 3 showed similar inhibitory pot-
encies compared to the reference compounds (IC50 g 5000
nM). However, the 4-pyridine analogue with an ethyl sub-
stituent on the methylene bridge exhibited an IC50 value of
1610 nM, thus being more potent than the corresponding
imidazole analogue ref 1 (IC50 > 5000 nM). The most active
compound was the 4-pyridine analogue 4 furnished with
hydroxy at the 4-position of the A-ring, showing an IC50
value of 248 nM.
CYP17 Inhibition. Inhibitory activities of the synthesized
compounds for CYP17 were determined using the 50 000
sediment after homogenation of E.coli expressing human
CYP17 as well as cytochrome P450 reductase.17a The assay
was run with progesterone as substrate at high concentra-
tions of 25 μM and NADPH as cofactor.13c Separation of
substrate and product was accomplished by HPLC using UV
detection. IC50 values are presented in comparison to keto-
conazole and abiraterone in Tables 1-3.
To validate the possible benefits achieved after re-
placement of imidazolyl by pyridyl, the inhibitory potencies
of imidazole, 3-pyridine, and 4-pyridine analogues were
compared. The SARs obtained from our previous work on
biphenylmethylene imidazoles confirm that (a) an ethyl
group substituted on the methylene bridge elevates the
inhibitory potency and (b) H-bond forming groups such as
Encouraged by these results, more compounds were de-
signed on the basis of the biphenylmethylene 4-pyridine
scaffold (Table 2). It can be seen that the substituents at

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5753
the A-ring showed a significant influence on the inhibitory
potencies. The 4-amino substituted compound 5 showed an
IC50 value of 408 nM, whereas its 3-amino analogue 6 was
even more potent (IC50 = 226 nM). Interestingly, the 3,4-
diamino compound 7 exhibited potent activity with an IC50
value in between (337 nM). Moreover, the hydroxy analo-
gues were more potent than the amino derivatives. The 3-OH
compound 8 was 3-fold more potent (IC50 = 97 nM) com-
pared to its corresponding NH2 analogue and to the 4-OH
compound 4 (IC50 = 248 nM). The 3,4-di-OH compound 9
turned out to be the most potent one in this series with an
IC50 value of 52 nM, being even more potent than abira-
terone (IC50 = 72 nM). The introduction of fluorine in the
3-position of compound 4, resulting in compound 10, in-
creased activity to 186 nM. The phenomenon that 3-sub-
stituted analogues are more potent than the corresponding
4-substituted compounds was also observed with the 4-acet-
amido compound 11 and its 3-analogue 12 (IC50 = 876 nM).
The retroamide 13 (IC50 = 1790 nM) showed a slightly in-
creased potency compared to compound 11. As for fluorine
derivatives, introduction of additional F resulted in loss of
inhibitory potency (15), which is in accordance with our
previous findings,13g while the compound with a single
fluorine in the 4-position of the A-ring (14) exhibited strong
inhibition (IC50 = 386 nM). Another interesting observation
was the little activity of compounds 17-20, indicating that
the Boc-amido group is not appropriate for highly act-
ive compounds, no matter what is introduced at the 3- or
4-position.
Table 4. Inhibition of CYP11B1, CYP11B2, CYP19, and CYP3A4 by
Selected Compounds
IC50 (nM)b
compd
CYP11B1c
CYP11B2c
CYP19d
CYP3A4e
4
251
522
287
902
342
1400
850
307
928
4742
422
415
469
627
15
341
406
921
367
261
948
210
567
515
273
331
796
231
1130
72
3070
24500
2830
>25000
663
3210
2450
1520
nda
5
6
7
8
538
7580
896
nda
9
10
2440
2693
nda
12
14
nda
668
nda
15
21
nda
nda
nda
22
23
nda
nda
nda
nda
25
nda
1140
nda
MYPa
KTZa
ABTa
>5000
>5000
>5000
127
1610
67
1750
57
2700
a MYP: metyrapone. KTZ: ketoconazole. ABT: abiraterone. nd: not
determined. b Standard deviations were within (5%. All the data are the
mean values of at least three tests. c Hamster fibroblasts expressing
human CYP11B1 or CYP11B2 are used with deoxycorticosterone as the
substrate at 100 nM. d Human placental CYP19 is used with androste-
nedione as the substrate at 500 nM. e Recombinantly expressed enzyme
from baculovirus-infected insect microsome is used with 7-benzyloxytri-
fluoromethyl coumarin as the substrate at 50 μM.
homology between these two enzymes of 93%. It can be seen
that although compounds 8, 10, and 15 exhibited strong
inhibition toward CYP11B2 (IC50 values of around 250 nM),
some compounds (4, 5, 7, 12, 14, and 21-23) showed modest
inhibition with IC50 values ranging from 300 to 800 nM,
whereas other compounds (6, 9, and 25) were very select-
ive with IC50 values around 1000 nM. Important is that
compound 6 as a dual inhibitor of CYP17 and CYP11B1,
exhibiting a selectivity factor of 3.2 between CYP11B1 and
CYP11B2.
Finally, the A-ring phenyl group was exchanged by het-
erocycles and substituted naphthalenes (Table 3). The thi-
ophene (21 and 22) and indole (23) compounds showed
modest activity with IC50 values around 600 nM. For the
naphthalene analogues, the great influence of the substituent
on the A-ring was again observed. The hydroxy derivative 25
showed rather potent inhibition (IC50 = 438 nM), while the
corresponding methoxy derivative 24 was less active.
CYP11B1 Inhibition. After it has been shown that the
replacement of imidazolyl by pyridyl significantly increased
the CYP17 inhibition, selected compounds (4-10, 12, 14, 15,
21-23, and 25) were examined for their CYP11B1 inhibition
(Table 4). The inhibitory activities were determined in V79
MZh cells expressing human CYP11B1.17b,c The V79 MZh
cells were incubated with [14C]deoxycorticosterone as sub-
strate and the inhibitor in different concentrations. Product
formation was monitored by HPTLC using a phosphoima-
ger. It becomes apparent that the substituents on the A-ring
also show profound influence on the inhibition of this
enzyme. Compounds 4 and 6 exhibited rather strong effects
with IC50 values of around 250 nM. Compounds 9 and 15
showed only weak or no inhibition (IC50 values of 1400 and
4742 nM, respectively), thus being rather selective inhibitors
of CYP17, whereas the rest of the compounds 5, 7, 8, 10, 12,
14, 15, 21-23, and 25 showed modest inhibition values
between 300 and 930 nM.
Selectivity. The inhibition of CYP11B2 by selected potent
compounds was determined as well using V79 MZh cells
expressing human CYP11B2 (Table 4). CYP11B2 is the
crucial enzyme responsible for the final steps in aldosterone
biosynthesis (Chart 1). Inhibition of CYP11B2 could cause
hyponatremia, hyperkalemia, and a series of recessive dis-
orders, such as adrenal hyperplasia and hypovolemic shock.18a
Selectivityover CYP11B2 is difficult toreach especially for the
compounds inhibiting CYP11B1 because of the very high
Furthermore, the selectivity of these potent compounds
toward CYP19 and hepatic CYP3A4 has also been tested as
a criterion for safety (Table 4). CYP19 is a unique enzyme
catalyzing the peripheral conversion of androgens to estro-
gens by hydroxylation and subsequent elimination of the
C19 methyl group, resulting in steroid A-ring aromatization.
Estrogen deficiency causes osteoporosis, increased fracture
risk,18b and memory loss.18c Under CYP17 inhibition, there
is a reduction of the estrogen plasma concentrations because
the androgens, as substrates for estrogen formation, are de-
creased. A further reduction of estrogen levels by CYP19
inhibition would be detrimental. As can be seen, all the tested
compounds except 8 showed no inhibition of CYP19 (IC50
values above 2000 nM).
The important role of CYP3A4 in drug metabolism and
drug-drug interaction has been addressed, and the com-
pounds have been tested for inhibition of this enzyme as well.
Most of them showed weak or no inhibition of CYP3A4
(IC50 > 1000 nM). Compounds 4 and 9 exhibited a better
selectivity than abiraterone (IC50 values of 3210 and 7580
nM vs 2700 nM).
Discussion and Conclusion
The design concept applied in the present paper using the
steroidal CYP17 inhibitor abiraterone and the CYP11B1
inhibitor metyrapone and our experience in nonsteroidal

5754 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Hu et al.
biphenylmethylene based CYP17 inhibitors (including 1K)
was successful. Exchange of the imidazolyl moiety by a 4-pyridyl
rest led (depending on the substitution pattern at the A-ring) on
the one hand to compound 9, exceeding abiraterone in activity
and selectivity, and on the other hand to compound 6, a dual
inhibitor of CYP17 and CYP11B1.
improved. Structure modifications are presently being per-
formed.
The use of selective multitarget-directed ligands has already
been proposed for the treatment of other diseases to enhance
efficacy and to improve safety, for example, agents inhibiting
angiotensin converting enzyme (ACE) and neutral endopep-
tidase (NEP) in the treatment of hypertension, multikinase
inhibitors (MKI) with combined inhibition of vascular en-
dothelial growth factor (VEGF) and platelet-derived growth
factor (PDGF) for cancer therapy, and dual binding site
acetylcholinesterase inhibitors (AChEI) for Alzheimer’s
disease.20 Compared to the traditional combinational appli-
cation of two or more drugs, multitarget-directed agents can
reduce the risk of drug-drug interactions and achieve better
compliance. The conception of selective dual inhibition of
CYP17 and CYP11B1 is a novel treatment for PC. There is
indeed a high medical need in prostate cancer patients to
combat elevated glucocorticoid levels and to prevent stimu-
lation of mutated androgen receptors by glucocorticoids.
CYP11B1 catalyzing the last step in cortisol biosynthesis is
an ideal target to decrease corticosterone and cortisol produc-
tion. However, there are no highly selective inhibitors of this
enzyme described so far that could be used in a combination
therapy with CYP17 inhibitors. Metyrapone is a compound
not selective enough to inhibit other steroidogenic CYP
enzymes and therefore shows severe side effects. Accordingly
it is not an appropriate candidate for this purpose, although it
has been used in the treatment of Cushing’s syndrome for a
long time. Another unselective compound, the antimycotic
ketoconazole, has already been employed to treat prostate
cancer patients with ectopic adrenocorticotropic hormone
syndrome8d because it inhibits both androgen and corticos-
teroid (not only glucocorticoid but also mineralocorticoid)
biosynthesis. However, the response was not satisfactory
The elevated inhibition of CYP17 caused by the novel pyri-
dines compared to the corresponding imidazoles might be due
to the prolonged distance between the sp2 hybrid N and the
methylene C. This elongation of the molecule places the
H-bond forming groups at the A-ring closer to the amino acid
residues, facilitating better H-bond formation. Moreover, it
has been found that substituents on the A-ring showed pro-
found influence on the inhibitory potency of both CYP17 and
CYP11B1. Hydrogen bond forming groups, like OH and NH2,
significantly elevated CYP17 inhibition, probably because of
the interaction with Arg109, Lys231, His235, and Asp298,
similar to what we described recently.13f This finding indicates
that imidazoles and pyridines may adopt the same binding
mode in the enzyme pocket. Among them, OH analogues were
found to be more potent than the corresponding NH2 analo-
gues (e.g., compounds 4 and 5(IC50 values of 248 and 408 nM),
compounds 8 and 6 (IC50 values of 97 and 226 nM), and
compounds 9 and 7 (IC50 values of 52 and 337 nM)), probably
because the hydrogen bonds formed by O are stronger than the
ones formed by N.19 Interestingly, meta-substituted analogues
are more potent than the corresponding para-analogues. After
methylation of OH (compounds 16 and 20) or acylation of
NH2 (11, 12, 17-20), the inhibitory potency dropped along
with the augment of bulk, which is probably due to the steric
hindrance with His235, Arg109, and the proximal I-helix
residues as described previously.13f
Regarding the additional CYP11B1 inhibition we were
aiming at in this study, the exchange of the nicotinoyl
moiety from metyrapone (because it leads to a loss of
CYP17 inhibition) by a biphenyl moiety with small polar
substituents was successful and resulted in compounds
with dual, CYP17 and CYP11B1, inhibition. It was found
that compounds 4-6, 8, and 12 with one single H-bond
donor showed potent to modest inhibition (IC50 values
ranged from 250 to 350 nM, with compound 5 as an
exception showing IC50 value of 522 nM), whereas H-bond
acceptors or two donors resulted in rather weak inhibitors
of CYP11B1 (compounds 7, 9, and 10; IC50 values of
greater than 800 nM). Since these compounds with differ-
ing CYP11B1 inhibition are potent CYP17 inhibitors, they
are accordingly either dual inhibitors of CYP17/11B1 or
selective CYP17 inhibitors.
because of the weak potency of ketoconazole (CYP17 IC50
=
2780 nM). Compound 6should be a much better candidate than
ketoconazole showing a good dual inhibition of CYP17 and
CYP11B1 with IC50 values around 200 nM for both enzymes, a
3-fold selectivity of CYP11B1 over CYP11B2, and nearly no
inhibition of CYP19 and CYP3A4. Because selectivity for
CYP11B2 should be further enhanced, compound 6 could be
an ideal candidate for this optimization process.
Importantly, compound 9 showed excellent selectivity with
almost no inhibition compared with all other enzymes tested.
It is highly active showing an IC50 value of 52 nM. As in our
experimental setup with high substrate concentrations, this
value has to be ranked higher than similar IC50 values ob-
tained by other groups using much lower substrate concentra-
tions. The fact that compound 9 not only shows strong inhi-
bition of androgen formation (CYP17) but also does not
block androgen conversion to estrogens (CYP19) provides
maximally low androgen levels (a prerequisite for the ther-
apeutic success). After validation in vivo, this compound
could be a promising drug candidate for further development.
On the basis of the results of the present study, we propose a
novel strategy for the treatment of prostate cancer: CYP17
inhibitors with different selectivity profiles according to the
status of the patients should be applied. For normal patients,
selective CYP17 inhibitors that do not interfere with other
steroidogentic CYPs should be used to avoid side effects,
whereas for patients with mutated androgen receptors or ect-
opic adrenocorticotropic hormone syndrome, dual inhibitors
of CYP17 and CYP11B1 are the best choice for personalized
medicine.
The A-ring substituents also diversified the inhibitory
potency towardthe nontarget enzyme CYP11B2. Itwasfound
that compounds with strong CYP11B1 inhibition were also
associated with CYP11B2 inhibition. This is not surprising,
since homology between thesetwo enzymes is morethan 93%.
As inhibition of CYP11B2 has to be avoided, the most inter-
esting dual inhibitor in this study is compound 6 showing a
selectivity factor of 3.2 regarding CYP11B1 and CYP11B2.
For the application as a dual CYP17/CYP11B1 inhibitor, this
selectivity toward CYP11B2 is certainly not sufficient. How-
ever, compound 6 should be an interesting lead for further
optimization. Since highly active and selective CYP11B2
inhibitors with in vivo activity15a reaching selectivity factors
of 100015b have been identified by our group for the treatment
of congestive heart failure and myocardial fibrosis, we are
confident that the selectivity of compound 6 can be further

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5755
Experimental Section
20:1); δH (CDCl3, 500 MHz) 3.48 (s, br, 4H, NH2), 3.98 (s, 2H,
CH2), 6.76 (d, J = 8.2 Hz, 1H,), 6.92-6.96 (m, 2H), 7.13 (d, J =
6.0 Hz, 2H), 7.18 (d, J = 8.2 Hz, 2H), 7.47 (d, J = 8.2 Hz, 2H),
8.50 (dd, J = 1.6, 6.0 Hz, 2H); MS (ESI) m/z = 276 [Mþ þ H].
4-[(30,40-Dimethoxybiphenyl-4-yl)methyl]pyridine (9a). 9a was
synthesized according to method A using 5a (0.31 g, 1.25 mmol)
and 3,4-dimethoxyphenylboronic acid (0.34 g, 1.88 mmol).
Yield, 0.34 g, (90%). This compound was used directly in the
next step without further purification and characterization.
40-(Pyridin-4-ylmethyl)biphenyl-4-carboxamide (13). 13 was
synthesized according to method A using 5a (0.25 g, 1.01 mmol)
and 4-carbamoylphenylboronic acid (0.25 g, 1.51 mmol). Yield,
0.26 g (89%); white solid; mp 241-242 °C; Rf = 0.21 (DCM/
MeOH, 50:1); δH (CD3OD, 500 MHz) 4.09 (s, 2H,CH2),
7.09-7.11 (m, 4H), 7.64 (d, J = 7.9 Hz, 2H), 7.71 (d, J = 7.8
Hz, 2H), 7.94 (d, J = 8.1 Hz, 2H), 8.42 (d, J = 4.7 Hz, 2H, Py
3,5-H); MS (ESI) m/z = 289 [Mþ þ H].
CYP17 Preparation and Assay. Human CYP17 was expres-
sed in E. coli17a (coexpressing human CYP17 and NADPH-
P450 reductase), and the assay was performed as previously
described.13c
Inhibition of Hepatic CYP3A4. The recombinantly expressed
enzyme from baculovirus-infected insect microsomes (supersomes)
was used, and the manufacturer’s instructions (www.gentest.com)
were followed.
Inhibition of CYP11B1 and CYP11B2. V79MZh cells expres-
sing human CYP11B1 or CYP11B2 were incubated with [14C]11-
deoxycorticosterone as substrate. The assay was performed as
previously described.17b,c
Inhibition of CYP19. The inhibition of CYP19 was deter-
mined in vitro using human placental microsomes with [1β-3H]-
androstenedione as substrate.17d
Chemistry Section. General. Melting points were determined
on a Mettler FP1 melting point apparatus and are uncorrected.
IR spectra were recorded neat on a Bruker Vector 33FT infrared
spectrometer. 1H NMR spectra were measured on a Bruker
DRX-500 (500 MHz). Chemical shifts are given in parts per mil-
lion (ppm), and TMS was used as an internal standard for spec-
tra. All coupling constants (J) are given in Hz. ESI (electrospray
ionization) mass spectra were determined on a TSQ quantum
(Thermo Electron Corp.) instrument. The purities of the final
compounds were controlled by a Surveyor LC system. Purit-
ies were greater than 95%. Column chromatography was per-
formed using silica gel 60 (50-200 μm), and reaction progress
was determined by TLC analysis on Alugram SIL G/UV254
(Macherey-Nagel). Boronic acids and bromoaryls used as start-
ing materials were commercially obtained (CombiBlocks,
Chempur, Aldrich, Acros).
Method A: Suzuki Coupling. The corresponding brominated
aromatic compound (1 equiv) was dissolved in toluene (7 mL/
mmol), and an aqueous 2.0 M Na2CO3 solution (3.2 mL/mmol),
an ethanolic solution (3.2 mL/mmol) of the corresponding
boronic acid (1.5-2.0 equiv), and tetrabutylammonium bro-
mide (1 equiv) were added. The mixture was deoxygenated
under reduced pressure and flushed with nitrogen. After this
cycle was repeated several times, Pd(OAc)2 (5 mol %) was added
and the resulting suspension was heated under reflux for 2-6 h.
After the mixture was cooled, ethyl acetate (10 mL) and water
(10 mL) were added and the organic phase was separated. The
water phase was extracted with ethyl acetate (2 ꢀ 10 mL). The
combined organic phases were washed with brine, dried over
Na2SO4, filtered over a short plug of Celite, and evaporated
under reduced pressure. The compounds were purified by flash
chromatography on silica gel.
4-[(40-Fluorobiphenyl-4-yl)methyl]-pyridine (14). 14 was syn-
thesized according to method A using 5a (301 mg, 1.21 mmol)
and 4-fluorophenylboronic acid (254 mg, 1.82 mmol). Yield, 168
mg (53%); white solid; mp 114-115 °C; Rf = 0.49 (DCM/
MeOH, 95:5) δH (CDCl3, 500 MHz) 3.96 (s, 2H), 7.03-7.07 (m,
2H), 7.12 (d, J = 5.4 Hz, 2H), 7.17 (d, J = 8.5 Hz, 2H), 7.43 (d,
J = 8.2 Hz, 2H), 7.44-7.47 (m, 2H), 8.45 (d, J = 5.3 Hz, 2H);
MS (ESI) m/z = 264 [Mþ þ H]
4-[(30,40-Difluorobiphenyl-4-yl)methyl]pyridine (15). 15 was
synthesized according to method A using 5a (0.125 g, 0.5 mmol)
and 3,4-difluorophenylboronic acid (0.21 g, 0.75 mmol). Yield,
0.12 g (87%); colorless oil; Rf = 0.27 (hexane/EtOAc, 1:1); δH
(CDCl3, 500 MHz) 3.93 (s, 2H), 7.05 (d, J = 6.1 Hz, 2H), 7.10-
7.22 (m, 4H), 7.26-7.31 (m, 1H), 7.39 (d, J = 8.2 Hz, 2H), 8.44
(d, J = 6.1 Hz, 2H); MS (ESI) m/z = 282 [Mþ þ H].
tert-Butyl 40-(Pyridin-4-ylmethyl)biphenyl-4-ylcarbamate (17).
17 was synthesized according to method A using 5a (0.30 g,
1.21 mmol) and 4-(tert-butoxycarbonylamino)phenylboronic
acid (0.43 g, 1.81 mmol). Yield, 0.38 g (87%); white solid; mp
198-199 °C; Rf = 0.26 (DCM/MeOH, 50:1); δH (CD3OD,
500 MHz) 1.53 (s, 9H, Boc), 4.05 (s, 2H, CH2), 7.28-7.29 (m,
4H), 7.51-7.53 (m, 6H), 8.40 (d, J = 4.9 Hz, 2H); MS (ESI)
m/z = 361 [Mþ þ H].
4-[4-(Thiophen-2-yl)benzyl]pyridine (21). 21 was synthesized
according to method A using 5a (0.35 g, 1.41 mmol) and thio-
phen-2-ylboronic acid (0.27 g, 2.12 mmol). Yield, 0.32 g (91%);
white solid; mp 82-83 °C; Rf = 0.26 (DCM/MeOH, 50:1); δH
(CD3OD, 500 MHz) 4.00 (s, 2H, CH2), 7.05 (dd, J = 4.0, 4.6 Hz,
1H), 7.22 (d, J = 7.2 Hz, 2H), 7.26 (d, J = 4.6 Hz, 2H), 7.33 (d,
J = 4.7 Hz, 2H), 7.56 (d, J = 7.9 Hz, 2H), 8.40 (d, J = 3.9 Hz,
2H); MS (ESI) m/z = 252 [Mþ þ H].
4-[4-(Thiophen-3-yl)benzyl]pyridine (22). 22 was synthesized
according to method A using 5a (0.35 g, 1.41 mmol) and thio-
phen-3-ylboronic acid (0.27 g, 2.12 mmol). Yield, 0.31 g (90%);
white solid; mp 97-98 °C; Rf = 0.26 (DCM/MeOH, 50:1); δH
(CD3OD, 500 MHz) 4.03 (s, 1H, CH2), 7.24 (d, J = 8.2 Hz, 2H),
7.28 (d, J = 6.0 Hz, 2H), 7.43-7.45 (m, 2H), 7.60-7.63 (m, 3H),
8.40 (d, J = 6.45 Hz, 2H); MS (ESI) m/z = 252 [Mþ þ H].
5-[4-(Pyridin-4-ylmethyl)phenyl]-1H-indole (23). 23 was syn-
thesized according to method A using 5a (0.30 g, 1.21 mmol) and
1H-indol-5-ylboronic acid (0.27 g, 2.12 mmol). Yield, 0.30 g
(87%); white solid; mp 170-171 °C; Rf = 0.23 (DCM/MeOH,
20:1); δH (CD3OD, 500 MHz) 4.04 (s, 2H, CH2), 6.48 (d, J = 3.7
Hz, 1H), 7.25-7.27 (m, 3H), 7.30 (d, J = 5.9 Hz, 2H), 7.35-7.37
(m, 1H), 7.42 (d, J = 8.5 Hz, 2H), 7.58 (d, J = 8.2 Hz, 2H),
7.76-7.78 (m, 1H), 8.40 (d, J = 5.9 Hz, 2H); MS (ESI) m/z =
285 [Mþ þ H].
40-(Pyridin-4-ylmethyl)biphenyl-4-amine (5). 5 was synthe-
sized according to method A using 5a (0.35 g, 1.41 mmol) and
4-aminophenylboronic acid (0.29 g, 2.12 mmol). Yield, 0.32 g
(87%); white solid; mp 217-218 °C; Rf = 0.19 (DCM/MeOH,
20:1); δH (CD3OD, 500 MHz) 4.04 (s, 2H), 6.77 (d, J = 8.5 Hz,
2H), 7.23 (d, J = 8.1 Hz, 2H), 7.29 (d, J = 5.9 Hz, 2H, Py 2,6-H),
7.36 (d, J = 8.5 Hz, 2H), 7.48 (d, J = 8.2 Hz, 2H), 8.40 (d, J =
6.1 Hz, 2H, Py 3,5-H); MS (ESI) m/z = 261 [Mþ þ H].
40-(Pyridin-4-ylmethyl)biphenyl-3-amine (6). 6 was synthe-
sized according to method A using 5a (0.30 g, 1.21 mmol) and
3-aminophenylboronic acid (0.25 g, 1.81 mmol). Yield, 0.26 g
(83%); white solid; mp 79-80 °C; Rf = 0.45 (DCM/MeOH,
95:5); δH (CDCl3, 500 MHz) 4.00 (s, 2H), 5.13 (s, 2H), 6.54 (dd,
J = 2.2, 7.9 Hz, 1H), 6.74 (dd, J = 1.6, 7.6 Hz, 1H), 6.81 (t, J =
1.9 Hz, 1H), 7.07 (t, J = 7.9 Hz, 1H), 7.27 (d, J = 5.9 Hz, 2H),
7.30 (d, J = 8.2 Hz, 2H), 7.49 (d, J = 7.9 Hz, 2H), 8.46 (d, J =
5.9 Hz, 2H); MS (ESI) m/z = 261 [Mþ þ H].
Method B: Wolff-Kishner Reduction. To an ice-cooled solu-
tion of the appropriate ketone (10 mmol) in ethylene glycol
(100 mL) were added hydrazine hydrate (70 mmol) and potas-
sium hydroxide (10 mmol). Then the resulting mixture was
heated to 150 °C for 1 h. After the mixture was cooled, a further
batch of potassium hydroxide (50 mmol) was added, and the
40-(Pyridin-4-ylmethyl)biphenyl-3,4-diamine (7). 7 was synthe-
sized according to method A using 5a (0.35 g, 1.41 mmol) and
3,4-diaminophenylboronic acid (0.32 g, 2.11 mmol). Yield, 0.32
g (83%); white solid; mp 180-181 °C; Rf = 0.15 (DCM/MeOH,

5756 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Hu et al.
mixture was heated to 210 °C for 2 h. After cooling to ambient
temperature, it was diluted with water (300 mL), and the re-
sulting mixture was extracted with ethyl acetate (3 ꢀ 50 mL).
The combined organic phases were washed with brine, dried
over MgSO4, and evaporated under reduced pressure. Then the
desired product was purified by chromatography on silica gel.
4-(4-Bromobenzyl)pyridine (5a). 5a was synthesized accord-
ing to method B using (4-bromophenyl)pyridin-4-ylmethanone
(0.39 g, 1.5 mmol). Yield, 0.21 g (52%); amber oil; Rf = 0.35
(PE/EtOAc, 10:1); δH (CDCl3, 500 MHz) 3.89 (s, 2H, CH2), 7.02
(d, J = 8.3 Hz, 2H), 7.05 (d, J = 5.9 Hz, 2H), 7.41 (d, J = 8.3 Hz,
2H), 8.48 (d, J = 5.9 Hz, 2H); δC (CDCl3, 125 MHz) 40.5 (CH2),
120.5, 124.0, 130.6, 131.7, 137.7, 149.8; MS (ESI) m/z = 249
[Mþ þ H].
8.2 Hz, 1H), 6.99 (t, J = 2.1 Hz, 1H), 7.04 (d, J = 7.9 Hz, 1H),
7.23 (t, J = 7.9 Hz 1H), 7.28 (d, J = 5.7 Hz, 2H), 7.31 (d, J = 8.2
Hz, 2H), 7.53 (d, J = 8.2 Hz, 2H), 8.47 (d, J = 5.7 Hz, 2H), 9.50
(s, 1H); MS (ESI) m/z = 262 [Mþ þ H].
40-(Pyridin-4-ylmethyl)biphenyl-3,4-diol (9). 9 was synthesized
according to method D using 9a (0.26 g, 0.85 mmol) and 1 M
BBr3 solution in DCM (4.20 mL, 4.20 mmol). Yield, 0.22 g
(93%); white solid; Rf = 0.55 (DCM/MeOH, 10:1); δH (DMSO-
d6, 500 MHz) 3.97 (s, 2H), 6.79 (d, J = 8.2 Hz, 1H), 6.90 (dd, J =
2.2, 8.2 Hz, 1H), 7.00 (d, J = 2.2 Hz, 1H), 7.22-7.29 (m, 4H),
7.45 (d, J = 8.2 Hz, 2H), 8.46 (d, J = 6.0 Hz, 2H), 8.98 (s, 1H),
9.02 (s, 1H); MS (ESI) m/z = 278 [Mþ þ H].
3-Fluoro-40-(pyridin-4-ylmethyl)biphenyl-4-ol (10). 10 was
synthesized according to method D using 16 (0.18 g, 0.64 mmol)
and 1 M BBr3 solution in DCM (3.20 mL, 3.20 mmol). Yield,
0.12 g (67%); white solid; mp 229-230 °C; Rf = 0.16 (DCM/
MeOH, 19:1); δH (DMSO-d6, 500 MHz) 3.98 (s, 2H), 7.00 (t,
J = 8.8 Hz, 1H), 7.25-7.31 (m, 5H), 7.44 (dd, J = 2.2 Hz,
2JHF = 12.9 Hz, 1H), 7.55 (d, J = 8.5 Hz, 2H), 8.46 (d, J =
5.9 Hz, 2H), 9.95 (s, 1H); MS (ESI) m/z = 280 [Mþ þ H].
6-[4-(Pyridin-4-ylmethyl)phenyl]naphthalen-2-ol (25). 25 was
synthesized according to method D using 24 (0.33 g, 1.00 mmol)
and 1 M BBr3 solution in DCM (3.00 mL, 3.00 mmol). Yield,
0.25 g (80%); white solid; Rf = 0.49 (DCM/MeOH, 10:1); δH
(DMSO-d6, 500 MHz) 4.00 (s, 2H, CH2), 7.11 (dd, J = 2.2, 8.8
Hz, 1H), 7.14 (d, J = 2.2 Hz, 1H), 7.27 (d, J = 6.0 Hz, 2H), 7.34
(d, J = 8.2 Hz, 2H), 7.68-7.70 (m, 3H), 7.74 (d, J = 8.8 Hz, 1H),
7.82 (d, J = 8.8 Hz, 1H), 8.04 (s, 1H), 8.46 (d, J = 6.0 Hz, 2H),
9.80 (s, 1H); MS (ESI) m/z = 312 [Mþ þ H].
N-(40-Isonicotinoylbiphenyl-3-yl)acetamide (12). 12 was syn-
thesized according to method B using 12a (0.32 g, 1.0 mmol).
Yield, 0.19 g (62%); yellowish solid; mp 123-124 °C; Rf = 0.27
(DCM/MeOH, 95:5); δH (DMSO-d6, 500 MHz) 2.06 (s, 3H,
CH3), 4.00 (s, 2H, CH2), 7.27 (d, J = 5.9 Hz, 2H), 7.29 (t, J = 1.9
Hz, 1H), 7.32-7.38 (m, 3H), 7.52-7.57 (m, 3H), 7.88 (t, J = 1.9
Hz, 1H), 8.47 (d, J = 5.9 Hz, 2H), 10.01 (s, 1H); MS (ESI) m/z =
303 [Mþ þ H].
4-[(30-Fluoro-40-methoxybiphenyl-4-yl)methyl]pyridine (16). 16 was
synthesized according to method B using 16a (0.43 g, 1.38 mmol).
Yield, 0.37 g (91%); white solid; mp 98-99 °C; Rf = 0.46 (hexane/
EtOAc, 1:1); δH (CDCl3, 500 MHz) 3.84 (s, 3H), 3.91 (s, 2H), 6.93
(t, J = 8.5 Hz, 1H), 7.05 (d, J = 6.3 Hz, 2H), 7.14 (d, J = 7.9 Hz,
2H), 7.18-7.26 (m, 1H), 7.21 (s, 1H), 7.39 (d, J = 7.9 Hz, 2H), 8.43
(d, J = 6.3 Hz, 2H); MS (ESI) m/z = 294 [Mþ þ H].
4-[4-(6-Methoxynaphthalen-2-yl)benzyl]pyridine (24). 24 was
synthesized according to method B using 24a (0.40 g, 1.18 mmol).
Yield, 0.34 g (89%); white solid; Rf = 0.49 (DCM/MeOH, 95:5);
δH (CDCl3, 500 MHz) 3.89 (s, 3H), 4.02 (s, 2H), 7.18 (dd, J = 2.5,
8.8 Hz, 1H), 7.29 (d, J = 6.0 Hz, 2H), 7.34 (d, J = 2.5 Hz, 1H),
7.37 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 8.2 Hz, 2H), 7.77 (dd, J =
1.9, 8.5Hz, 1H), 7.88 (d, J= 8.8Hz, 2H), 8.11 (d, J = 1.6 Hz, 1H),
8.47 (d, J = 6.0 Hz, 2H); MS (ESI) m/z = 326 [Mþ þ H].
Method C: Grignard Reaction. Under exclusion of air and
moisture a 1.0 M RMgBr (1.2 equiv) solution in THF was added
dropwise to a solution of the aldehyde or ketone (1 equiv) in
THF (12 mL/mmol). The mixture was stirred overnight at room
temperature. Then ethyl acetate (10 mL) and saturated ammo-
nium chloride solution (10 mL) were added and the organic
phase was separated. The organic phase was extracted with
water and brine, dried over Na2SO4, and evaporated under re-
duced pressure. The crude products were purified by flash
chromatography on silica gel.
Method E: Dehydroxylation with HCOOH. Under exclusion
of air and moisture the appropriate alcohol (1 mmol) was dis-
solved in formic acid (10 mL per mmol). Pd(OAc)2 (1 mol %)
was added, and the mixture was heated to reflux for 16 h. After
the reaction mixture was cooled, formic acid was distilled off
and the mixture was neutralized using saturated sodium bicar-
bonate solution. Then the mixture was extracted with ethyl
acetate (3 ꢀ 10 mL), and the combined organic phases were
washed with brine, dried over Na2SO4, evaporated to dryness,
and purified using column chromatography.
Method F: Hydrogenation. A solution of the corresponding
alkene (1 mmol) in dry THF (15 mL/mmol) was treated with
10% Pd on charcoal (15 mg). The reaction vessel was repeatedly
evacuated and flushed with hydrogen gas and left to stir at room
temperature for 3 h, pressurized with 1 bar of H2. The resulting
reaction mixture was filtered through a short plug of Celite, which
was washed with THF (40 mL). The filtrates were concentrated
with an oil pump and purified using column chromatography.
Method D: Ether Cleavage with BBr3. To a solution of the
corresponding ether (1 equiv) in DCM (5 mL/mmol) at -78 °C
was added 1 M boron tribromide in DCM (5 equiv). The re-
sulting mixture was stirred at room temperature for 16 h. Then
water (25 mL) was added and the emulsion was stirred for
further 30 min. The resulting mixture was extracted with ethyl
acetate (3 ꢀ 25 mL). The combined organic phases were washed
with brine, dried over Na2SO4, and evaporated under reduced
pressure. Then the desired product was purified by chromato-
graphy on silica gel.
Acknowledgment. The authors thank Professor J. Hermans,
Cardiovascular Research Institute, University of Maastricht,
The Netherlands, for providing us with V79MZh11B1 cells
expressing human CYP11B1 and Professor R. Bernhardt, Saar-
land University, Germany, for making the V79MZh11B2 cells
expressing human CYP11B2 available to us.
Supporting Information Available: Synthesis procedures and
characterization of compounds 1-3, 11, 18-20 and other
intermediates as well as HPLC purities and 13C NMR and IR
spectra of other final compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
4-[(40-Hydroxybiphenyl-4-yl)methyl]pyridine (4). 4 was syn-
thesized according to method D using 4a (0.38 g, 1.37 mmol) and
1 M BBr3 solution in DCM (6.85 mL, 6.85 mmol). Yield, 0.39 g
(10%); white solid; mp 224-225 °C; Rf = 0.24 (DCM/MeOH,
95:5); δH (DMSO-d6, 500 MHz) 3.97 (s, 2H), 6.83 (d, J = 8.5 Hz,
2H), 7.25-7.30 (m, 4H), 7.45 (d, J = 8.5 Hz, 2H), 7.50 (d, J =
8.2 Hz, 2H), 8.46 (d, J = 5.9 Hz, 2H), 9.52 (s, 1H); MS (ESI)
m/z = 262 [Mþ þ H].
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