Isopropylidene substitution increases activity and selectivity of biphenylmethylene 4-pyridine type CYP17 inhibitors

Article abstract of DOI:10.1021/jm100400a

CYP17 inhibition is a promising therapy for prostate cancer (PC) because proliferation of 80% of PC depends on androgen stimulation. Introduction of isopropylidene substituents onto the linker of biphenylmethylene 4-pyridines resulted in several strong CYP17 inhibitors, which were more potent and selective, regarding CYP 11B1, 11B2, 19 and 3A4, than the drug candidate abiraterone.

Full text of DOI:10.1021/jm100400a

J. Med. Chem. 2010, 53, 5049–5053 5049
DOI: 10.1021/jm100400a
Isopropylidene Substitution Increases Activity and Selectivity of Biphenylmethylene 4-Pyridine Type
CYP17 Inhibitors
Qingzhong Hu,† Lina Yin,†,‡ Carsten Jagusch,†,§ Ulrike E. Hille,† and Rolf W. Hartmann*,†
†Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C23, and Helmholtz Institute for Pharmaceutical Research Saarland
(HIPS), D-66123 Saarbru€cken, Germany, and ‡ElexoPharm GmbH, Campus A1, D-66123 Saarbru€cken, Germany. §Present address: Dottikon
Exclusive Synthesis AG, 5605 Dottikon, Switzerland.
Received March 30, 2010
CYP17 inhibition is a promising therapy for prostate cancer (PC) because proliferation of 80% of PC
depends on androgen stimulation. Introduction of isopropylidene substituents onto the linker of
biphenylmethylene 4-pyridines resulted in several strong CYP17 inhibitors, which were more potent and
selective, regarding CYP 11B1, 11B2, 19 and 3A4, than the drug candidate abiraterone.
Introduction
To avoid the stimulation, the inhibition of 17R-hydroxylase-
17,20-lyase (CYP17) was proposed as a superior alternative
to CAB. CYP17 catalyzes not only the testicular but also
the adrenal conversion of pregnenolone and progesterone to
the weak androgens DHEA and androstenedione, respec-
tively. Moreover, recent observations suggest that there is
also CYP17 activity in prostate cancer cells.6 Testosterone
subsequently formed from these two weak androgens is in the
prostate converted to DHT, which is the most potent andro-
gen. This final step of androgen activation can be inhibited by
5R-reductase (5RR) inhibitors.8 However, CYP17 inhibition
should be a better strategy than 5RR inhibition, as it totally
blocks not only androgen biosynthesis in testes and adrenals
but also intracellular androgen formation in the cancer cell.
The benefit of PC treatment via CYP17 inhibition was
shown by the off-label administration of the antimycotic
ketoconazole.9 However, ketoconazole had to be withdrawn
because of severe hepatic toxicity resulting from its nonselec-
tive inhibition of other CYP enzymes. Nevertheless, as it has
been demonstrated by the success of aromatase (CYP19)10
and more recently aldosterone synthase (CYP11B2) inhibi-
tors,11 high selectivity can be achieved. As for CYP17, abi-
raterone12 is an outstanding example of a potent and selec-
tive inhibitor among the steroidal compounds synthesized.13
This compound exhibited significant antitumor effects in pa-
tients diagnosed as “castration resistant” in phases II and III
clinical trials.12b However, the potential affinity of steroidal
scaffolds for steroid receptors, which often results in side effects
whether acting as agonists or antagonists, prompted the devel-
opment of nonsteroidal CYP17 inhibitors.14
Prostate cancer (PCa) is the carcinoma with the highest
incidence in male, and it accounts for a quarter of cancer
related deaths each year. Few patients in early stages can be
cured by local therapy, like prostatectomy or radiotherapy.
Most, especially the ones with metastases, are treated with
hormone therapy. Because of severe side effects, chemother-
apy is usually reserved as the last choice. Hormone therapy is
based on the finding that up to 80% of PCs depend on
androgen stimulation for proliferation. Early attempts to
suppress androgen production by estrogen application were
soon replaced by orchidectomy and later by administration of
gonadotropin-releasing hormone (GnRH) analogues.1 GnRH
analogues unfold their activity via the hypothalamic, pituitary,
and gonadal axis, resulting in an annihilation of testicular
androgen production. However, adrenal androgen formation
is not affected. Although ∼90% of androgens are no longer
produced and the plasma testosterone concentration is re-
duced below 50 ng/dL, androgen levels (testosterone and
subsequently dihydrotestosterone, DHT) in the prostate are
higher and maintain cancer cell growth.2,3 This accumulation
is due to the presence of androgen receptor (AR) and steroido-
genic enzymes that convert adrenal steroids into testosterone
and DHT.3,4 Hence, AR antagonists (anti-androgens) are
employed in combination with GnRH analogues to prevent
adrenal androgens from unfolding activity.5 This is the current
standard therapy, the so-called “combined androgen block-
ade” (CAB). However, long-term application of antagonists
induces mutations of AR that render the receptor to be acti-
vated by the anti-androgenic drug6 or by endogenous gluco-
corticoids,7 resulting in resistance to CAB.
Our group has designed and synthesized several series of
biphenylmethylene heterocycles15 as CYP17 inhibitors, in
which some imidazoles were found to be very potent.15a-f
During further optimization, it was revealed that the replace-
ment of imidazolyl by 4-pyridyl significantly improved po-
tency and selectivity, whereas 3-pyridyl analogues exhibited
similar activity as imidazoles.15g For the imidazoles, we ob-
served that small alkyl groups, especially ethyl, significantly
increase inhibitory potency, while bulkier substituents reduce
activity.15c This observation inspired us to perform a thorough
*To whom correspondence should be addressed. 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.
a Abbreviations: CYP, cytochrome P450; PC, prostate cancer; CAB,
combined androgen blockade; GnRH, gonadotropin-releasing hor-
mone; AR, androgen receptor; CYP17, 17R-hydroxylase-17,20-lyase;
5RR, 5R-reductase; CPY19, aromatase, estrogen synthase; CYP11B2,
aldosterone synthase; DHEA, dehydroepiandrosterone; DHT, dihydro-
testosterone.
r
2010 American Chemical Society
Published on Web 06/15/2010
pubs.acs.org/jmc

5050 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Chart 1. Design of Synthesized Compounds 1-23
Hu et al.
Scheme 1a
study on the influence of the linker between the biphenyl and
pyridyl moieties, which is described in this article. Besides
freely rotable alkyl groups, ethylidene and isopropylidene,
which rigidify the conformation of the whole molecule, were
also inserted onto the methylene bridge (10-22, Chart 1).
Because different interaction angles between sp2 hybrid N and
heme Fe might account for the disparity in potency between
3- and 4-pyridines, further modifications, such as prolonging
the bridge and inserting various substitutions onto the bridge,
were performed on the 3-pyridyl scaffold (1-9, Chart 1).
Furthermore, optimizations of the A-ring by exchanging
phenyl to naphthyl or thiophenyl were also done, resulting in,
for example, compound 23. In the following, we also describe
the selectivity of selected compounds against CYP11B1,
CYP11B2, CYP19, and hepatic CYP enzyme 3A4 to evaluate
safety.
Results and Discussion
Chemistry. The synthetic routes for the preparation of
1-23 are shown in Scheme 1-4. For the syntheses of the
compounds with the two-membered linker between the
biphenyl and pyridine moieties, the corresponding 4-bromo-
phenylpyridin-3-yl ethanones 2a and 5a were prepared as
building blocks. These bromo compounds were coupled with
the corresponding boronic acids via Suzuki coupling to yield
the ketones 2, 5, and 7, which were then converted to the
alcohols 3, 4, 6, 8, and 9 via Grignard reaction or reduced by
Wolff-Kishner reduction to give 1 (Schemes 3 and 4, see
Supporting Information).
Similarly, the syntheses of the biphenylmethylene pyri-
dines started from (4-bromophenyl)pyridyl methanones,
which were subjected to Suzuki coupling and Grignard
reaction to form the corresponding alcohol intermediates.
After elimination of a H2O molecule under acidic conditions,
the alcohols were converted to the enylpyridines 10, 13, 15,
and 16. The hydrogenation of the double bond led to
saturated analogues 11, 12, and 14 (Scheme 1). Subsequent-
ly, a more efficient strategy was employed by synthesizing
4-(1-(4-bromophenyl)-2-methylprop-1-enyl)pyridine 17a as
a common building block, followed by introduction of the
aryl ring via Suzuki reaction. Via this route, the final com-
pounds 17-23 were conveniently obtained (Scheme 2).
CYP17 Inhibitory Activity. Inhibition of CYP17 by the
synthesized compounds was determined using the 50 000
sediment after homogenation of E.coli coexpressing human
a Reagents and conditions: Compounds were synthesized from the
corresponding a, b, c, or d intermediate unless annotated otherwise. (i)
Method A: Pd(OAc)2, corresponding boronic acid, TBAB, EtOH,
Na2CO3 aq, toluene, 110 °C, 4 h. (ii) Method B: i-PrMgCl or EtMgCl,
THF, room temp, 8 h. (iii) Method D: HCOOH, Pd(OAc)2, reflux, 16 h.
(iv) Method E: H2, Pd/C, THF, rt, 3 h. (v) Method F: BBr3, DCM. (vi)
Method G: HBr, reflux, 4 h.
CYP17 and cytochrome P450 reductase.16 The assay was run
with progesterone (25 μM) as substrate and NADPH as
cofactor.15a Separation of substrate and product was accom-
plished by HPLC using UV detection. IC50 values are pre-
sented in comparison to ketoconazole, abiraterone, and
reference compounds 24-2815g in Table 1. All 3-pyridyl
compounds (detailed structures can be found in Schemes 3
and 4, see Supporting Information) were only weakly active
(IC50 > 5000 nM), whether furnished with a two-membered
linker (1-9) or ethylidene substituted methylene (10).
In contrast, the 4-pyridyl compounds were potent CYP17
inhibitors and the substituents on the methylene bridge
showed profound influence on the inhibitory potency. It is
apparent that for the A-ring m-OH analogues, the introduc-
tion of an ethyl (11) and a propyl (12) group decreased
activities to 189 and 783 nM, respectively, compared to
the corresponding nonsubstituted reference compound 24
(IC50 = 97 nM). However, the isopropylidene substitution
increased activity to 56 nM. A similar observation can be made

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 5051
Scheme 2a
Table 1. Inhibition of CYP17 by 11-23 and Reference Compounds
24-28
compd
X
R
IC50 (nM)b
24c
11
m-OH
m-OH
m-OH
m-OH
H
Et
97
189
783
56
12
i-Pr
13
isopropylidene
H
Et
25c
14
m-F, p-OH
m-F, p-OH
m-F, p-OH
m, p-di-OH
m, p-di-OH
m-BocNH
p-BocNH
186
343
75
15
isopropylidene
H
26c
16
52
37
isopropylidene
isopropylidene
isopropylidene
isopropylidene
isopropylidene
H
17
18
1458
493
852
>10000
226
38
a Reagents and conditions. (i) Method B: i-PrMgCl, THF, room
temp, 8 h. (ii) Method G: HBr, reflux, 4 h. (iii) Method A: Pd(OAc)2,
corresponding boronic acid, TBAB, EtOH, Na2CO3 aq, toluene, 110 °C,
4 h. (iv) Method F: BBr3, DCM.
19
m-F, p-BocNH
m, p-di-BocNH
m-NH2
20
27c
21
m-NH2
m, p-di-NH2
m, p-di-NH2
isopropylidene
H
isopropylidene
28c
22
337
75
for the A-ring m-F, p-OH series: the ethyl compound 14 is less
potent than the nonsubstituted reference compound 25 (IC50
of 343 nM vs 186 nM), whereas the isopropylidene 15 once
again was the most active one exhibiting an IC50 of 75 nM.
More examples for the enhancement of inhibitory potency
rendered by isopropylidene were observed with the remaining
couples: the di-OH analogues (reference compounds 26 and
16, IC50 of 52 nM vs 37 nM), the amines (reference compounds
27 and 21, IC50 of 226 nM vs 38 nM), and the diamino
compounds (reference compounds 28 and 22, IC50 of 337
nM vs 75 nM). The change in geometry from sp3 to sp2 leading
to a planar compound with less conformational flexibility is a
plausible explanation for the observed increase in activity.
Moreover, the conjugation of the biphenyl moiety and the
pyridyl ring facilitated by isopropylidene increases the electron
density in the heteroaromat, which might additionally con-
tribute to the enhanced affinity to the enzyme. In our previous
studies on biphenylmethylene imidazoles, introduction of an
ethyl group into the methylene bridge was found to signifi-
cantly increase activity.15c The opposite impact of ethyl ex-
hibited with the pyridines presented in this paper compared to
those imidazoles is probably due to the longer distance be-
tween the sp2 hybrid N and the methylene group in the
pyridines, which might prevent the ethyl group from binding
into the hydrophobic pocket.15c Furthermore, Boc-amido was
again15g proven to be not tolerated: the corresponding ana-
logues 17-20 showed modest to no inhibition of CYP17 (IC50
ranging from 493 nM to more than 10 000 nM). Besides the
introduction of isopropylidene, the exchange of the A-ring
from phenyl to 6-OH naphthyl also led to a highly potent
CYP17 inhibitor, 23 (IC50 = 62 nM).
23
62
KTZa
ABTa
2780
72
a KTZ: ketoconazole. ABT: abiraterone. b Concentration 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 Reference compounds 24-28 were taken from ref 15g.
Table 2. Selectivity Profiles of Selected Compounds toward CYP11B1,
CYP11B2, CYP19, and CYP3A4
IC50 (nM)b
compd
CYP11B1c
CYP11B2c
CYP19d
CYP3A4e
24 f
13
342
3100
351
261
3450
110
663
12000
1670
538
730
25 f
15
896
2000
1400
37470
287
1870
948
5750
2440
17650
7580
464
26 f
16
974
921
9500
2830
27 f
21
1520
1330
ndg
7300
502
3830
368
5060
28 f
22
24560
49500
48700
>50000
>50000
7180
5060
127
1130
49500
67
1770
ndg
23
KTZa
ABTa
57
2700
1610
1750
a KTZ: ketoconazole. ABT: abiraterone. b Standard deviations were
within (5%. All the data are mean values of at least three tests. c Hamster
fibroblasts expressing human CYP11B1 or CYP11B2 are used with deox-
ycorticosterone as the substrate at 100 nM. d Human placental CYP19 is
used with androstenedione as the substrate at 500 nM. e Microsomal
fraction of recombinantly expressed enzyme from baculovirus-infected
insect is used with 7-benzoyloxytrifluoromethyl coumarin as the substrate
at 50 μM. f Reference compounds 24-28 were taken from reference 15g. g n.
d.: not determined.
Selectivity. As a criterion to evaluate safety, the inhibition
values of the most potent CYP17 inhibitors (all of them
isopropylidene compounds) toward CYP11B1, CYP11B2,
and CYP19 were determined (Table 2). CYP11B1 and
CYP11B2 catalyze the crucial final steps in cortisol and
aldosterone biosynthesis, respectively. Inhibition of these
enzymes could lead to hyponatremia, hyperkalemia, adrenal
hyperplasia, and hypovolemic shock.17a CYP19 catalyzes
the formation of estrogens, which have been proven to
be capable of reducing the incidence of heart disease.17b

5052 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Hu et al.
Furthermore, estrogen deficiency resulting from CYP19
inhibition causes osteoporosis, increased fracture risk, and
memory loss.17c It can be seen that all the isopropylidene
analogues tested are much more selective compared to the
corresponding nonsubstituted compounds. For CYP11B1,
only weak inhibition (IC50 of around 2000-3000 nM for 13
and 15) or no inhibition (IC50 of more than 5000 nM for 16,
21-23) was observed, which makes these compounds supe-
rior to abiraterone (IC50 = 1610 nM). A similar selectivity
pattern was achieved for CYP11B2: most of the tested
compounds did not interfere with this enzyme with IC50 of
more than 3000 nM (13, 21, and 23). The only exception was
15 exhibiting weak inhibition with an IC50 of 1870 nM, which
is comparable to that of abiraterone (IC50 = 1750 nM).
Compounds 16 and 22 were less selective with IC50 of ∼1000
nM. Regarding CYP19, all of the tested compounds 13, 15,
16, and 21-23 showed no inhibition with IC50 of more than
5000 nM. Furthermore, the inhibition of the hepatic CYP
enzyme 3A4 was also determined because it accounts for
most of the drug metabolism and is therefore to a large
extent involved in drug-drug interactions. Although 13 and
16 were less selective, showing IC50 of around 500-
700 nM, 21 and 22 exhibited only weak inhibition with
IC50 of ∼1500 nM. The most selective compound 15 exhib-
ited almost no inhibition of CYP3A4 with an IC50 of more
than 17 600 nM, which is much better than the drug candi-
date abiraterone (IC50 = 2700 nM).
TMS was used as an internal standard. 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. Purities were greater than 95%.
Column chromatography was performed using silica gel 60 (50-
200 μm), and reaction progress was determined by TLC analysis
on Alugram SIL G/UV254 (Macherey-Nagel).
Method A: Suzuki Coupling. See Supporting Information for
details.
40-[2-Methyl-1-(pyridin-4-yl)prop-1-enyl]biphenyl-3-amine (21).
21 was synthesized according to method A using 17a (0.50 g, 1.74
mmol) and 3-aminophenylboronicacid(0.36 g, 2.60 mmol). Yield,
0.39 g (75%); white solid; mp 131-132 °C; Rf = 0.21 (DCM/
MeOH, 20:1); δH (CDCl3, 500 MHz) 1.85 (s, 3H, CH3), 1.86 (s,
3H, CH3), 3.73 (s, br, 2H, NH2), 6.66 (dd, J = 2.1, 7.8 Hz, 1H),
6.90 (t, J = 2.0 Hz, 1H), 6.98 (dt, J = 1.4, 7.9 Hz, 1H), 7.09 (dd,
J = 1.6, 6.1 Hz, 2H), 7.14 (d, J = 8.3 Hz, 2H), 7.21 (t, J = 7.8 Hz,
1H), 7.49 (d, J = 8.3 Hz, 2H), 8.51 (dd, J = 1.6, 6.0 Hz, 2H); MS
(ESI), m/z = 301 [Mþ þ H].
Method B: Grignard Reaction. See Supporting Information
for details.
1-(4-Bromophenyl)-2-methyl-1-(pyridin-4-yl)propan-1-ol (17b).
17b was synthesized according to method B using (4-bromo-
phenyl)(pyridin-4-yl)methanone (2.00 g, 7.63 mmol) and 2.0 M
isopropylmagnesium chloride solution in THF (4.20 mL, 8.39
mmol). Yield, 1.35 g (58%); amber oil;δH (CDCl3, 500 MHz) 0.84
(d, J = 6.7 Hz, 3H, CH3), 0.88 (d, J = 6.7 Hz, 3H, CH3), 2.79 (q,
J = 6.7 Hz, 1H, CH), 3.25 (s, br, 1H, OH), 7.36-7.42 (m, 6H),
8.41 (d, J = 6.1 Hz, 2H); MS (ESI), m/z = 307 [Mþ þ H].
Method C: Reduction with NaBH4. See Supporting Informa-
tion for details.
Conclusion
Method D: Dehydroxylation with HCOOH. See Supporting
Information for details.
Method E: Hydrogenation. See Supporting Information for
details.
Method F: Ether cleavage with BBr3. See Supporting Informa-
tion for details.
CYP17 inhibitionisapromisingtherapy for prostatecancer
because it blocks not only the androgen biosyntheses in testes
and adrenals but also intracellular androgen formation in the
cancer cell.3,4 Biphenylmethylene heterocycles, especially pyr-
idines, have been proven to be potent CYP17 inhibitors.15 In
the present study, modifications of the linker between biphe-
nyl and pyridine moieties were described. Variations on the
3-pyridyl scaffold, such as prolongation of the linker and
introduction of different substituents onto the bridge, resulted
in only weak inhibitors. In contrast, modifications of 4-pyr-
idyl analogues led to potent and selective CYP17 inhibitors.
The differing substituents on the methylene bridge had a
profound influence on the inhibitory potency: flexible alkyl
groups reduced activity, whereas conformation rigidifying
isopropylidene groups significantly improved activity and
selectivity. Among the nine isopropylidene substituted biphe-
nylmethylene 4-pyridines synthesized, six compounds (13, 15,
16, 21, 22, and 23, IC50 between 37 and 75 nM) were more
potent than or comparable to the drug candidate abiraterone
(IC50 = 72 nM). Most of these potent compounds also exhi-
bited better selectivity profiles toward CYP11B1, CYP11B2,
CYP19, and hepatic CYP3A4 than the parent compounds
and abiraterone. Thus, this study presents an example that
a single substituent can be the key for selectivity among seve-
ral CYP enzymes. Several compounds of this investigation
can be considered as promising drug candidates after further
validation in vivo.
6-[4-(2-Methyl-1-pyridin-4-yl-propenyl)phenyl]naphthalen-2-ol
(23). 23 was synthesized according to method F using 23a (0.25 g,
0.68 mmol) and 1 M BBr3 in DCM (2.05 mL, 2.05 mmol). Yield,
0.18 g (73%); yellow oil; Rf = 0.52 (DCM/MeOH, 19:1); δH
(DMSO-d6, 500 MHz) 1.98 (s, 3H, CH3), 2.02 (s, 3H, CH3),
7.19(d, J = 8.2Hz, 2H), 7.31 (d, J = 8.5Hz, 1H), 7.65 (d, J = 6.4
Hz, 2H), 7.72 (d, J = 8.2 Hz, 2H), 7.72 (d, J = 8.5 Hz, 2H),
7.83 (dd, J = 1.8, 8.5 Hz, 1H), 8.00 (d, J = 1.8 Hz, 1H),
8.14 (d, J = 8.5 Hz, 1H), 8.60 (d, J = 6.4 Hz, 2H); MS (ESI),
m/z = 352 [Mþ þ H].
Method G: Dehydroxyltion and Ether Cleavage with HBr. See
Supporting Information for details.
4-[1-(4-Bromophenyl)-2-methylprop-1-enyl]pyridine (17a). 17a
was synthesized according to method G using 17b (1.00 g, 3.27
mmol). Yield, 0.89 g (95%); white solid; Rf = 0.21 (DCM/
MeOH, 10:1); δH (CDCl3, 500 MHz) 1.85 (s, 3H, CH3), 1.86
(s, 3H, CH3), 7.09 (dd, J = 1.6, 6.1 Hz, 2H), 7.14 (d, J = 8.3 Hz,
2H), 7.49 (d, J = 8.3 Hz, 2H), 8.51 (dd, J = 1.6, 6.0 Hz, 2H); MS
(ESI), m/z=289 [Mþ þ H].
Acknowledgment. The authors thank Professor J. Hermans
(Cardiovascular Research Institute, University of Maastricht,
The Netherlands) and Professor R. Bernhardt (Saarland Uni-
versity, Germany) for providing us with V79MZh11B1 and
V79MZh11B2 cells, respectively.
Experimental 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 million (ppm), and
Supporting Information Available: Synthetic procedures,
characterization and HPLC purities of the intermediates and
final compounds, and the biological assays for CYP17,
CYP11B1, CYP11B2, CYP19, and CYP3A4. This material is
available free of charge via the Internet at http://pubs.acs.org.

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 5053
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