N-(Pyridin-3-yl)benzamides as selective inhibitors of human aldosterone synthase (CYP11B2)

Article abstract of DOI:10.1016/j.bmcl.2010.11.040

A series of 23 N-(Pyridin-3-yl)benzamides was synthesized and evaluated for their potential to inhibit human steroid-11β-hydroxylase (CYP11B1) and human aldosterone synthase (CYP11B2). The most potent and selective CYP11B2 inhibitors (IC₅₀ valu

Full text of DOI:10.1016/j.bmcl.2010.11.040

Bioorganic & Medicinal Chemistry Letters 21 (2011) 186–190
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-(Pyridin-3-yl)benzamides as selective inhibitors of human aldosterone
synthase (CYP11B2)
⇑
Christina Zimmer, Marieke Hafner, Michael Zender, Dominic Ammann, Rolf W. Hartmann ,
Carsten A. Vock
⇑
Pharmaceutical and Medicinal Chemistry, Saarland University & Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), PO Box 15 11 50, D-66041 Saarbrücken, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 23 N-(Pyridin-3-yl)benzamides was synthesized and evaluated for their potential to inhibit
human steroid-11b-hydroxylase (CYP11B1) and human aldosterone synthase (CYP11B2). The most
potent and selective CYP11B2 inhibitors (IC50 values 53–166 nM) were further evaluated for their
potential to inhibit human CYP17 and CYP19, and no inhibition was observed. Clear evidence was shown
for N-(Pyridin-3-yl)benzamides to be a highly selective class of CYP11B2 inhibitors in vitro.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 3 November 2010
Accepted 4 November 2010
Available online 12 November 2010
Keywords:
CYP11B2
Aldosterone synthase
Selective inhibition
Metyrapone
Aldosterone
Congestive heart failure
Myocardial fibrosis
Aldosterone synthase (CYP11B2) is the key enzyme in the human
biosynthesis of mineralocorticoids, catalysing the three-step inter-
conversion of 11-deoxycorticosterone to aldosterone via corticoste-
rone and18-hydroxycorticosterone.1 Due toitspivotal role, CYP11B2
is claimed as an useful target for the treatment of hyperaldosteron-
ism, myocardial fibrosis and congestive heart failure.2 After having
successfully developed selective inhibitors of crucial steroidogenic
enzymes involved in the endocrine (CYP193–5 and CYP176–8) as well
In an effort to develop and evaluate new classes of compounds
as CYP inhibitors, we based our investigations on the structure of
the well-known CYP inhibitor Metyrapone 2, which has been used
for the treatment of hypercortisolism and Cushing’s syndrome for
several decades.23–27 We decided to substitute the bridging 2,2-
dimethylethanone moiety in 2 by a much easier accessible amide
linker to generate N-(Pyridin-3-yl)benzamides 3. In addition—and
with respect to activity studies carried out with the bovine CYP11B
enzyme by Hays et al.28 resp. Tobes et al.29—we also investigated
the exchange of the keto-substituted pyridine system of Metyra-
pone 2 against substituted phenyl moieties in order to improve
the potency of the compounds. We were very pleased by the activ-
ity and astonishingly high CYP11B2 selectivity of these small and
structurally quite simple compounds in vitro, and we would like
to communicate our detailed observations in this Letter.
as intracrine (5a
-reductase9–11 and 17b-HSD112–14) stimulation
of hormone dependent diseases, we more recently have been
concerned with the development of selective CYP11B2
inhibitors.2,15–21 Thus, wehave discoveredcompound1 tobea highly
potent and selective inhibitor of CYP11B2.15 We were able to verify
the lowering of plasma aldosterone levels by 1 in rats, which can
be considered as validation of the target and proof-of-principle.22
Compounds of type 3 were prepared by a routine amide
coupling procedure using commercially available acid chlorides 4
and 3-aminopyridine 5 in pyridine at room temperature or with
application of gentle heating (Scheme 1). The products were
obtained as solids in moderate to good yields.
Routinely, all synthesized compounds 3 were evaluated for their
potential to inhibit human CYP11B2 and CYP11B1 at an inhibitor
concentration of c = 500 nM. Tests were carried out using our estab-
lished comparative in-house test system (V79 Chinese hamster cells
stably transfected with either human CYP11B2 or CYP11B1; sub-
strate: 11-deoxycorticosterone for both enzymes; substrate concen-
tration: c = 100 nM).30 The results are shown in Table 1. As can be
seen from data, none of compounds 3 showed significant inhibition
N
N
N
R1
O
O
R2
R3
N
H
X
Y
R4
O
N
H
N
1
2
3
Metyrapone
⇑
Corresponding authors. Tel.: +49 681 302 70300; fax: +49 681 302 70308
(R.W.H.).
E-mail address: rwh@mx.uni-saarland.de (R.W. Hartmann).
URL: http://www.pharmmedchem.de.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.040

C. Zimmer et al. / Bioorg. Med. Chem. Lett. 21 (2011) 186–190
187
N
R1
O
R1
O
R2
R3
Δ
Pyridine, RTor
N
R2
R3
Cl
N
H
+
X
X
H2N
Y
R4
Y
R4
4
5
3
Scheme 1. Synthesis of N-(Pyridin-3-yl)benzamides 3.
Table 1
Inhibition of human CYP11B2 and CYP11B1 enzymes by compounds 3
No.a
R1
R2
R3
R4
X
Y
CYP11B2 inhibition @ 500 nMb (%)
CYP11B1 inhibition @ 500 nMb (%)
log P (calcd)c
1
2
—
—
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
—
—
H
H
Br
H
H
H
H
H
H
H
H
H
H
H
F
—
—
H
—
H
H
F
Cl
Br
OMe
OCF3
CF3
Me
Ph
CN
NO2
H
—
—
—
H
—
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
—
—
C
N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
—
—
N
C
N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
94
79
8
2
4
9
7
3
2
3
4
8
3
4
1
4
2
5
3
91
94
3
4
1
2
5
3.03
1.78
0.81
0.81
1.70
1.98
2.19
2.76
2.91
2.10
3.21
2.99
1.80
3.87
1.58
1.88
2.19
2.76
1.78
2.29
1.75
2.55
2.03
3a
3b
3c
3d
3e
3f
3g
3h
3i
39
29
30
86
88
64
19
3
1
25
6
81
64
47 11
26
34
83
2
51
70
4
0.4 0.5
2
7
5
0
0
3
1
0
0
9
0
1
0
1
6
2
5
7
3
1
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
4
2
Cl
H
F
OMe
Cl
H
H
H
F
OMe
H
F
7
6
2
1
3
2
2
6
H
H
Cl
F
0.1 0.1
4
2
5
2
3u
a
The purity of all tested compounds was P95%.
Test system: V79 Chinese hamster cells stably transfected with either human CYP11B2 or CYP11B1 enzyme; substrate: 11-deoxycorticosterone for both enzymes;
b
substrate concentration: c = 100 nM.
c
log P values were calculated using the add-on ChemProp implemented in ChemDraw Ultra, Version 10.0, CambridgeSoft.
of human CYP11B1 at c = 500 nM, while a number of derivatives
strongly inhibit human CYP11B2. Concerning the two direct ana-
logues 3a and 3b of Metyrapone 2, isonicotinamide 3b shows weak
inhibition of CYP11B2, whereas nicotinamide 3a is not active. Addi-
tion of a bromo substituent in meta-position of the nicotinamide
moiety leads to the weakly active CYP11B2 inhibitor 3c, but no
improvement is achieved in comparison with 3b. The unsubstituted
benzamide3d is similarly active as 3b. Introductionof halogenosub-
stituents in para-position within the benzamide moiety (3e, 3f, 3g)
leads to stronginhibitors ofCYP11B2. Interestingly, similar activities
could be observed with strongly electron-withdrawing substituents
(-M-effect) like cyano derivative 3m and nitro compound 3n,
whereas trifluoromethoxy-substituted 3i and trifluoromethyl-
substituted 3j (-I-effect) showed no activity at all. Introduction of
halogeno substituents in meta- (3o, 3p) and ortho-position (3q) of
the benzamide leads to selective CYP11B2 inhibitors with reduced
activity in comparison with the para-substituted derivatives. The
difluoro derivative 3r shows similar activity to the monohalogenat-
ed compounds 3e, 3f and 3g, whereas a slight drop of activity is
observedfor the trifluoroderivative 3u. Compound 3t bearing chloro
substituents in ortho- and meta-position of the benzamide moiety is
less active compared with 3u.
For the seven most active inhibitors of CYP11B2 (3e, 3f, 3g, 3m,
3n, 3r, 3u) IC50 values were determined in order to quantify the
Table 2
Inhibition of human CYP11B2 and CYP11B1 enzymes (IC50 values) for selected compounds of type 3
No.a
Structure
CYP11B2 IC50 (nM)
CYP11B1 IC50
Selectivity factor IC50 (CYP11B1)/IC50 (CYP11B2)
b
b
N
1
0.2015
33 nM15
18715
O
N
H
N
O
2
72 17
14.6 2.1 nM
0.23
N
(continued on next page)

188
C. Zimmer et al. / Bioorg. Med. Chem. Lett. 21 (2011) 186–190
Table 2 (continued)
b
b
No.a
Structure
CYP11B2 IC50 (nM)
CYP11B1 IC50
Selectivity factor IC50 (CYP11B1)/IC50 (CYP11B2)
N
O
3e
3f
82 11
65 12
104 20
78 20
14.3 3.3
10.2 2.2
16.7 2.8
7.5 1.1
l
l
l
l
M
174
N
H
F
N
O
M
M
M
157
161
96
N
H
Cl
N
O
3g
3m
N
H
Br
N
O
N
H
NC
O2N
N
O
3n
3r
145 24
53.5 2.0
166 23
24.8 3.4
l
M
171
111
97
N
H
N
O
F
F
5.92 0.89
l
M
N
H
N
O
F
F
F
3u
16.1 2.8
lM
N
H
a
The purity of all tested compounds was P 95%.
Test system: V79 Chinese hamster cells stably transfected with either human CYP11B2 or CYP11B1 enzyme; substrate: 11-deoxycorticosterone for both enzymes;
b
substrate concentration: c = 100 nM.
selectivity toward CYP11B1 ( Table 2). As can be seen, all com-
pounds are highly selective CYP11B2 inhibitors, the most selective
one being 3e with a selectivity factor of 174. Concerning this
parameter, the compounds are comparable with reference com-
pound 1 being developed in our group before.15,22 However, with
regard to activity, the N-(Pyridin-3-yl)benzamides 3 turned out
to be far less potent inhibitors of CYP11B2. For the most active
compound 3r, an IC50 value of 53.5 nM has been determined,
which means an approximately 250-fold lower potency in compar-
ison with 1. However, some of the compounds are exhibiting
similar (3e, 3m) or slightly higher (3f, 3r) CYP11B2 inhibitory
activity compared to Metyrapone 2.
For all synthesized compounds 3 and the references 1 and 2,
log P values were calculated (Table 1).31 There is no obvious corre-
lation between the potency as CYP11B2 inhibitors and the lipophil-
icity of the compounds, which might have been expected due to
the location of both CYP11B1 and CYP11B2 enzymes on the inner
side of the mitochondrial membrane of adrenal cortex cells. For
example, compounds 3e and 3m are equally potent inhibitors of
CYP11B2, but differ by approximately 0.5 units in log P. However,
for almost all compounds log P values of 1.5–3.0 have been calcu-
lated, showing them to be equally or more lipophilic than Metyra-
pone 2, but less lipophilic than reference compound 1.
inhibitory effect on these enzymes. Regarding the inhibition of
CYP17 and CYP19 as potential unwanted side effect for the applica-
tion of CYP11B2 inhibitors as drugs, the structural motifs of all seven
compounds 3e, 3f, 3g, 3m, 3n, 3r and 3u can therefore deliver useful
information to overcome residual CYP17 and CYP19 activity, as
observed for the highly potent inhibitor FAD286A.35,36 Interestingly,
the latter compound has shown activity in several animal models
relevant for heart failure and organ damage caused by elevated plas-
ma aldosterone level, thus demonstrating the importance of the
drug target CYP11B2.37–40
In summary, we synthesized a panel of 23 N-(Pyridin-3-
yl)benzamides 3 and evaluated them for their activity as inhibitors
of CYP11B1, CYP11B2, CYP17 and CYP19. All compounds did not
show any significant inhibition of CYP11B1, and several derivatives
(3e, 3f, 3g, 3m, 3n, 3r, 3u) turned out to be potent inhibitors of
CYP11B2. Those compounds exhibited no inhibitory activity on
CYP17 and CYP19. The highest selectivity was observed for 3e con-
taining a fluoro substituent in para-position of the benzamide moi-
ety, whereas the most active compound was the difluoro
compound 3r. Although the CYP11B2 potency of the compounds
is by far lower in comparison to reference 1, compounds 3 offer a
highly interesting selectivity profile. Further investigations con-
cerning metabolic stability and in vivo activity are pending.
In order to obtain a more detailed selectivity profile for com-
pounds 3 toward steroidogenic CYP enzymes, we additionally
determined the ability of the seven most active inhibitors of
CYP11B2 (3e, 3f, 3g, 3m, 3n, 3r, 3u) to inhibit CYP1732,33 and
CYP1934 (Table 3). None of the compounds showed any significant
Acknowledgements
We are indebted to Jeannine Jung, Gertrud Schmitt and Jannine
Ludwig for performing the bioassays. Furthermore, we would like

C. Zimmer et al. / Bioorg. Med. Chem. Lett. 21 (2011) 186–190
189
Table 3
Inhibition of human CYP17 and CYP19 enzymes for selected compounds of type 3
No.
Structure
CYP17 inhibition @ 2000 nMa
CYP19 inhibition @ 500 nMb
N
O
2
0%
0%
N
N
O
3e
3f
0.3 0.2%
1.3 2.3%
4.3 2.7%
0.2 0.3%
1.1 1.3%
N
H
F
N
O
0%
N
H
Cl
Br
N
O
O
3g
3m
0.1 0.1%
0.2 0.3%
N
H
N
N
H
N
C
N
O
3n
1.0 0.9%
0%
N
H
O2N
N
O
F
F
3r
1.0 1.7%
0.6 1.1%
0.5 0.8%
N
H
N
O
F
F
F
3u
0%
N
H
a
CYP17 test system: 50,000 g sediment of a homogenate of E. coli recombinantly expressing human CYP17; substrate: progesterone
(c = 25 M); inhibitor concentration: c = 2.0 M; Ketoconazole, IC50 = 2780 nM.
CYP19 test system: human placental CYP19; substrate: androstenedione (c = 500 nM); inhibitor concentration: c = 500 nM; Fadrozole,
l
l
b
IC50 = 30 nM.
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to thank Martina Jankowski and Dr. Stefan Boettcher for the HPLC-
MS measurements. We would like to thank Professor Dr. J. J. Rob
Hermans, University of Maastricht, The Netherlands, for generous
supply of the V79 CYP11B1 cells and Professor Dr. Rita Bernhardt,
Saarland University, for the V79 CYP11B2 cells.
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Supplementary data
Supplementary data (experimental details as well as spectral
and HPLC-MS data for all compounds 3) associated with this article
can be found, in the online version, at doi:10.1016/j.bmcl.2010.
11.040.
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