Novel nanomolar imidazo[4,5-b]pyridines as selective nitric oxide synthase (iNOS) inhibitors: SAR and structural insights

Article abstract of DOI:10.1016/j.bmcl.2011.05.073

Inducible arginine oxidation and subsequent NO production by correspondent synthase (iNOS) are important cellular answers to proinflammatory signals. Prolonged NO production has been proved in higher organisms to cause stroke or septic shock. Several classes of potent NOS inhibitors have been reported, most of them targeting the arginine binding site of the oxygenase domain. Here we disclose the SAR and the rational design of potent and selective iNOS inhibitors which may be useful as anti-inflammatory drugs.

Full text of DOI:10.1016/j.bmcl.2011.05.073

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4228–4232
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel nanomolar imidazo[4,5-b]pyridines as selective nitric oxide synthase
(iNOS) inhibitors: SAR and structural insights
Ulrich Grädler a, , Thomas Fuchß a, Wolf-Rüdiger Ulrich a, Rainer Boer a, Andreas Strub a,
⇑
Christian Hesslinger a, Céline Anézo a, Kay Diederichs b, Andrea Zaliani a
a Nycomed GmbH, Byk-Gulden-Str. 2, D-78467 Konstanz, Germany
b Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Inducible arginine oxidation and subsequent NO production by correspondent synthase (iNOS) are
important cellular answers to proinflammatory signals. Prolonged NO production has been proved in
higher organisms to cause stroke or septic shock. Several classes of potent NOS inhibitors have been
reported, most of them targeting the arginine binding site of the oxygenase domain. Here we disclose
the SAR and the rational design of potent and selective iNOS inhibitors which may be useful as anti-
inflammatory drugs.
Received 1 April 2011
Revised 18 May 2011
Accepted 20 May 2011
Available online 30 May 2011
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Nitric oxide synthase
Inflammation
Crystal structure
Structure-based design
Selectivity
Inhibitor
Nitric oxide synthase (NOS; EC 1.14.13.39) generates the impor-
tant neurotransmitter and cytotoxic agent NO via oxidation of
such as stroke and septic shock. Therefore, selective inhibition of
the inducible isoform can serve as a suitable strategy to target both
acute and chronic inflammation.6
L
-arginine.1 Three major isoforms have been identified in mam-
mals: neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial
NOS (eNOS).2,3 They share 50–60% sequence identity and have
identical overall architecture. The known NOS enzymes are active
as a homodimer, and each monomer is comprised of a catalytic
N-terminal oxygenase domain, a C-terminal reductase domain
Several classes of potent NOS inhibitors have been reported,
most of them targeting the arginine binding site of the oxygenase
domain (Table 1).7 One major class of inhibitors are
L-arginine ana-
logues bearing a guanidine, amidino or isothiourea group that mi-
mic the recognition of the guanidinium group of the substrate as
confirmed by X-ray crystallography.8
and a calmodulin binding linker. The oxygenase domain (NOSOX
)
binds the substrates -arginine and O2 and contains the two cofac-
L
Among these, the weakly selective iNOS inhibitor N-iminoethyl-
tors heme and 5,6,7,8-(6R)-tetrahydrobiopterine (BH4) and a struc-
tural zinc atom. Electrons are supplied to the O2 bound heme from
the NADPH-binding reductase domain involving the cofactors FAD
and FMN.4
The constitutively expressed isoforms nNOS and eNOS are reg-
ulated by Ca2+/calmodulin and produce low levels of NO
(100–500 nmol/mg min) predominantly for both nerve function
and blood pressure regulation, respectively. In contrast, the induc-
ible isoform is controlled at the transcription level and expressed
in response to proinflammatory signals.5 Because iNOS is often ex-
pressed at high levels over longer time periods, the overproduction
of NO (1500 nmol/mg min) can lead to undiserable pathologies
L-lysine (1a, L-NIL) as its tetrazole-amide acid prodrug (L-NIL-TA,
SC-51) (1b, Fig. 1) has been demonstrated to reduce exhaled breath
nitric oxide in patients with mild asthma.9,10 The cyclic amidine
derivative ONO-1714 2 belongs to the most potent iNOS-inhibitors
(Ki = 2 nM).9
Table 1
Published NOS-affinity data (given as pIC50
)
Entry
iNOS (human)
eNOS (human)
nNOS (human)
ref
1a
1b
2
3
4
5.33
<4
pKi = 8.73
5.85
7.43
<4
<4
4.21
<4
ND
3.84
6.13
ND
9
9
pKi = 7.73
3.33
<4
7.35
<4
11
15
16
17
18
⇑
Corresponding author. Tel.: +49 (0)178 4575455.
E-mail addresses: ugraedler@hotmail.com, ulrich.graedler@merck.de (U.
5
6
6.96
7.15
5.18
Grädler).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.073

U. Grädler et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4228–4232
4229
Table 2
NH
NH2
O
H
R 1
N
NH
Inhibitory activities of compounds from the first screening campaign against human
NOS
N
Cl
H
1a: R1=O
Entry
Structure
pIC50
iNOS
H
H
eNOS
4.27
nNOS
5.47
2
N
N
1b: R1=
N
O
N
O
N
N
F
H
N
NH2
N
H
N
S
S
S
OH
7
6.94
N
N
N
N
CN
N
N
H
NH
O
F
NH2
N
3
4
O
N
O
N
O
8
9
4.31
4.97
<4
<4
N
N
NH2
N
H
N
H
N
CN
N
5
6
O
Figure 1. Published iNOS inhibitors.
4.58
4.57
S
S
N
O
N
H
However, ONO-1714 shows only 10-fold selectivity over eNOS
and no selectivity over nNOS.11 Since the arginine binding-site is
highly conserved among the NOS isoenzymes, structure-based de-
sign of selective inhibitors remains a challenging problem.12–14
However, several substrate mimetics, like GW-274150 3, achieve
isoform selectivity through additional interactions outside of the
10
11
12
6.81
<4
5.97
<4
6.35
<4
N
O
O
catalytic site.15,16 Both
L-arginine analogues L-NIL-TA 1b and
S
N
GW-274150 3, respectively, have been tested in clinical phase II
for the oral treatment of migraine, rheumatoid arthritis (RA),
osteoarthritis (OA), chronic obstructive pulmonary disease (COPD),
asthma and allergic rhinitis. However, no further development has
been reported for both compounds. A series of spirocyclic amidines
such as 4 (AR-C102222) were reported as highly potent and selec-
tive iNOS-inhibitors with good oral activity.17 Several groups have
identified 2-aminopyridines such as 5 as potent NOS inhibitors
revealing their potential to mimic the basic amidine or guanidine
O
O
<4
<4
<4
S
N
In the first optimization loop, a methylene group replaced the
sulfur atom of 7, because of its susceptibility for oxidation in phase
I metabolism. Synthesis of 2-[2-(4-Methoxy-pyridin-2-yl)-ethyl]-
3H-imidazo[4,5-b]pyridine (17, BYK191023) and its selected deriv-
atives 17–26 are shown in Scheme 1. Malonester synthesis using 4-
methoxy-pyridine-2-carbaldehyde 13, which was synthesized in a
three-step procedure starting from commercially available 2-
methoxy-4-nitro-pyridine 1-oxide, yields exclusively (E)-3-(4-
Methoxy-pyridin-2-yl)-acrylic acid methyl ester 14.24,25 Subse-
quent standard hydrogenation on palladium/charcoal and hydroly-
sis of the methyl ester furnished the key intermediate 3-(4-
methoxy-pyridin-2-yl)-propionic acid 15. iNOS inhibitors 17–26
are readily obtained by the final condensation of the corresponding
2,3-diaminopyridines and 14 in fresh polyphoshoric acid at ele-
vated temperatures to afford the common 3H-imidazo[4,5-b]pyri-
dine moiety in a straightforward manner.
The resulting analogue BYK191023 17 (Table 3) showed both an
improved iNOS-affinity (pIC50 = 7.09) and selectivity profile (nNOS:
pIC50 = 4.86; eNOS: pIC50 = 3.95), respectively.24 A slightly reduced
potency was observed for the 6-bromine- (18; iNOS: pIC50 = 6.73)
and the 7-methyl-derivative (20; iNOS: pIC50 = 6.84) of 17. The
drop in binding affinity was even more pronounced by the intro-
duction of a 5-methoxy group (19; iNOS: pIC50 = 6.42) and by
5,7-dimethyl substitution (21; iNOS: pIC50 = 5.64). Taken together,
these results indicate limited space in the 5-position for further
introduction of substituents. Therefore, we focused our efforts to
improve potency on the 6-and 7-position of 17.
group in L
-arginine analogues.18 In another study, N-substituted
4-methoxy-2-aminopyridines such as 6 (AR-C133057XX) demon-
strated both high potency and selectivity as iNOS inhibitors.19 La-
tely, several papers showed renewed interest on selective iNOS
inhibitors.20–22
In a screening campaign conducted on our corporate compound
library, 2-(4-methoxypyridin-2-ylmethylsulfanyl)-3H-imidazo[4,
5 -b] pyridine (7) has been identified as potent iNOS inhibitor
(pIC50 = 6.94, Table 2). NOS activity was measured by quantification
of [3H] -arginine conversion to [3H] -citrulline.23 The selectivity
L L
profile of 7 (nNOS: pIC50 = 5.47; eNOS: pIC50 = 4.27) provided an
ideal starting point for further hit-to-lead optimization. Radioligand
binding experiments demonstrated, that 13, a carbo analogue of 7
(Table 3), acts as a competitive inhibitor with respect to
This observation suggests that 7, and the entire SAR family, interact
L
-arginine.23
with theL-arginine-binding site within iNOSOX as exemplifiedby dif-
ferent classes of NOS inhibitors.12–16
Binding affinity for iNOS was also observed for the simple 7
derivative
4-methoxy-2-methylsulfanylmethyl-pyridine
(10;
pIC50 = 6.81). As shown in Table 2, the introduction of a second
methoxy group in the 3- or 5-position of the pyridine ring resulted
in a complete loss of iNOS-affinity (11, 12; iNOS: pIC50 <4). Inter-
estingly, modifications of the imidazo[4,5-b]pyridine ring system
also significantly affect the iNOS affinity as demonstrated by the
imidazo[4,5-c]pyridine derivative 8 (iNOS: pIC50 = 4.31) and the
imidazobenzene analogue 9 (iNOS: pIC50 = 4.97). This initial struc-
ture–activity relationship (SAR) prompted us to use 4-methoxy-
pyridine in combination with the imidazo[4,5-b]pyridine as
templates for further optimization.
We next determined the binding position of 17 within the
L-arginine binding site by X-ray crystallography using murine
8,26
iNOSOX
The 4-methoxypyridine ring is inserted into the guanidinium
binding site bearing -stacking interactions to the heme (Fig. 2).
.
p

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U. Grädler et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4228–4232
Table 3
Inhibitory activities of selected compounds against human NOS
Entry
Structure
pIC50
iNOS
eNOS
3.95
nNOS
4.86
O
N
17
7.09
6.73
N
N
N
N
H
N
N
N
O
N
18
19
20
21
4.06
4.00
4.34
< 4
5.08
4.58
4.73
4.9
N
H
Br
Figure 2. Crystal structure of murine iNOS in complex with 17 (PDB Code 3NW2).
O
N
[4,5-b]pyridine ring of 17. This water molecule is part of a four-
membered water-cluster (wat1–4) forming a polar contact area
at the bottom side of the imidazo[4,5-b]pyridine ring. In agreement
with the SAR, the 4-methoxy substituent of 17 is accomodated in a
small hydrophobic pocket formed by Val346 (murine iNOS num-
bering) and Phe363. The different active site volumes of the three
isoforms (eNOS > iNOS > nNOS) in this region have been discussed
as an important factor for inhibitor selectivity.27,28 In the observed
binding position of 17, a second methoxy group in either the 3- or
5-position of the pyridine ring would result in steric clashes with
the backbone of Trp372 and the side chain of Val352 respectively.
This might explain the decrease in iNOS-affinity of the dimethoxy
substituted pyridine derivatives 11 and 12 (pIC50 <4) compared to
10 (pIC50 = 6.81) with only one methoxy group in the 4-position.
The distance between the nitrogen atom of the 4-methoxypyr-
idine and both carboxylic O-atoms of Glu371 (OE1-atom: 2.7 Å and
OE2-atom: 3.3 Å) suggests a hydrogen bond and therefore a pro-
tonation of this pyridine N-atom. Potentiometric titration experi-
6.42
6.84
5.64
N
H
O
O
N
N
N
H
N
O
N
N
N
H
N
ments performed with
7 (pKa2 = 5.31) and 17 (pKa2 = 6.25)
O
suggested, that the inhibitors are likely to be neutral in solution
at the applied assay conditions. However, pKa values of ligands
can be shifted in protein binding sites due to the electrostatic influ-
ence of the protein’s titratable groups in the direct environment.29
For the complex of methotrexate (MTX) with dihydrofolate reduc-
tase (DHFR), a pKa-shift from 5.7 (apo) to 7.9 (complexed) was pre-
dicted in agreement with previous experimental results.30 In a
study from Astra-Zeneca researchers, in fact, the X-ray structures
of iNOS in complex with 4-methyl-2-aminopyridine and the 2-
amino-4-methoxypyridine-derivative 6 (AR-C133057XX) revealed
a bidendate charge-interaction of the (protonated) 2-aminopyri-
dine-group to Glu371.19 Furthermore, the presence of a basic gua-
nidine, amidino or isothiourea group has been proposed as a key
recognition element in NOS inhibitors targeting the guanidine
binding region.31 We therefore predict, that the pKa assigned to
the 4-methoxypyridine N-atom of 17 is shifted from 6.25 in solu-
tion towards higher values in the binding pocket of iNOS stabiliz-
ing appropriate protonation for salt-bridge interaction to Glu371.
The bottom part of the heterocyclic binding pocket is completed
by the side chain of Arg382, which is hydrogen bonded to wat4
through its NH1-atom. Interestingly, the C5-atom of the imi-
dazo[4,5-b]pyridine is in close contact (3.3 Å) to the NH2-atom of
Arg382 and readily explains the decrease in binding affinity of
the 5-methoxy- (19; iNOS: pIC50 = 6.42) and of the 5,7-dimethyl-
derivative (21; iNOS: pIC50 = 5.64) of 17. The upper part of the
imidazo[4,5-b]pyridine binding pocket is less well defined, since
the side chains of the proximal residues Ser256 and Gln257 are dis-
ordered in the electron density map. Therefore, the exact role of
the Ser256 and Gln257 side chains in the recognition of the imi-
dazo[4,5-b]pyridine ring remains unclear.
O
N
a
13
O
N
O
N
b
O
O
R2
O
O
14
15: R2 = CH3
16: R2 = H
c
O
N
d
N
R3
R4
N
H
N
17 - 26
R3, R4 = H, Br, CH3, OCH3, O(CH2)2OCH3, O(CH2)2Ph, phenyl
Scheme 1. Reagents and conditions: (a) monoethyl malonate potassium salt
(1.8 equiv), pyridine hydrochloride (2.0 equiv), piperidine (0.12 equiv), pyridine,
120 °C, 2 h, 68%; (b) H2, Pd/C (10%), MeOH, 96%; (c) NaOH (1.0 M, 1.2 equiv), THF,
r.t., 85%; (d) 2,3-diaminopyridine derivative, polyphosphoric acid (PPA), 110–
160 °C, 1–24 h, 650%.
An additional hydrogen bond to Glu371 is formed between the
OE1-atom and an adjacent water molecule (wat1), which is also in-
volved in an H-bond (3.1 Å) to the N3-atom of the imidazo

U. Grädler et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4228–4232
4231
Table 4
Inhibitory activities of further selected compounds against human NOS
Entry
Structure
pIC50
iNOS
eNOS
3.69
nNOS
O
N
O
O
O
22
6.11
6.29
6.14
7.26
7.41
4.23
3.91
4.86
5.72
5.92
N
N
N
N
N
N
H
N
N
N
N
N
O
N
O
23
24
25
26
3.69
4.18
4.06
4.74
N
H
Figure 3. Superimposition of crystal structures of murine iNOS (magenta), human
iNOS (grey, PDB-Code: 1NSI and green, PDB-Code: 3EZG), human eNOS (cyan, PDB-
Code: 3NOS) and rat nNOS (orange, PDB-Code: 1ZVL) demonstrate conformational
differences of the amino acids in the vicinity of 17. The imidazo[4,5-b]pyridine ring
of 17 points towards the Gln specificity pocket in human iNOS involving an induced
fit of Gln263 from ‘closed’ towards an ‘open’ position.
O
N
N
H
O
N
Both murine and human iNOS oxygenase domains show a high
level of sequence identity (89%) and the amino acids involved in
substrate and 17 binding are strictly conserved. In agreement with
these structural similarities, the experimental Ki of 17 towards the
murine (pKi = 6.78) and human iNOS (pKi = 7.07) appears to be in
the same range.23 When comparing the ligand binding sites of
N
H
O
N
murine iNOSOXꢀ17 and human iNOSOX
ꢀ
L-arginine, significant con-
formational differences of Arg260 (hiNOS: Arg266) and Gln257 (hi-
NOS: Gln263) adjacent to the imidazo[4,5-b]pyridine ring can be
observed (Fig. 3).
N
H
This region has been described as ‘Gln specificity pocket’ pro-
moting isoform selectivity by sequence specific plasticity of the
binding site.32 In this study, bulky derivatives of 6 induced confor-
mational changes in human iNOS involving the rotation of Gln263
interactions displayed by these 7-substituents in the correspond-
ing part of the binding site as seen in our docking studies.
In contrast, the exemplified 6-derivatives of 17 having a n-butyl
(25; iNOS: pIC50 = 7.26) and phenyl substituent (26; iNOS:
pIC50 = 7.41) both demonstrate a gain in iNOS affinity. The murine
iNOSOXꢀ17 structure indicates a hydrophobic contact area formed
from a ‘Gln-closed’ position (L-arginine bound) towards a ‘Gln-
open’ position (inhibitor bound). Although the corresponding
Gln257 is not defined in the murine iNOSOXꢀ17 structure, the side
chain conformation of Arg260 corresponds to Arg266 in the ‘Gln-
open’ structure of human iNOS (Fig. 3). The induced fit of Arg266
is supplemented by the rotation of Asn283 (nNOS: Asn498, eNOS:
Asn267), which is supported by less bulkier 3rd shell amino acids
in human iNOS (Val/Phe) compared to eNOS (Leu/Ile) and nNOS
(Leu/Phe). In the murine iNOSOXꢀ17 structure, the rotation of
Arg260 is tolerated by the presence of Thr277 and Ser256 in the
vicinity resembling a ‘Gln-open’ conformation as in human iNOS.
Taken together, we would expect distinct conformations of the
imidazo[4,5-b]pyridine ring in eNOS and nNOS with disfavoured
interactions, explaining the observed isoform selectivity. The crys-
tal structures of human NOS isoforms in complex with 17 would be
of great value to address this hypothesis. We have recently demon-
strated, that 17 irreversibly inactivates murine iNOS by an NADPH-
and time-dependent mechanism leading to uncoupled electron
transfer and heme loss.33 However, this time-dependent inhibition
was not observed for nNOS. Structural differences in the binding
site of 17 between the NOS isoforms might contribute in this inac-
tivation mechanism.
by Trp84 (human iNOS: Trp90), Met114 (Met120), Ser256
0
(Ala262), Arg260 (Arg266) within 8 ÅA distance to the 6-position
of the imidazo[4,5-b]pyridine ring (Fig. 4). This hydrophobic region
is probably addressed by the n-butyl and phenyl substituents of 25
Selected results of our subsequent SAR evaluation are summa-
rized in Table 4. In the series of 7-substituted 17 derivatives, a sig-
nificant drop in iNOS binding affinity was observed. The
introduction of a 7-methoxy group resulted in a 10-fold reduction
of potency (22; iNOS: pIC50 = 6.11) as well as the 7-ethoxy-meth-
oxy- (23; iNOS: pIC50 = 6.29) and the 7-phenethyloxy-derivative
(24; iNOS: pIC50 = 6.14) of 17. This might be due to less favourable
Figure 4. Connolly surface coloured by lipophilic potential (green: polar, brown:
hydrophobic) calculated for the X-ray structure of murine iNOS (magenta) in
complex with 17 indicates a hydrophobic contact area formed by Trp84, Met114,
Ser256, Arg260. This area (dotted circle) is adressed by, for example, n-butyl and
phenyl substituents in the 6-position of the imidazo[4,5-b]pyridine (marked by an
arrow) leading to improved iNOS affinity.

4232
U. Grädler et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4228–4232
Table 5
Pharmacokinetic data and CYP P450 isoform profile (pIC50
)
Entry
Structure
Pharmacokinetic data (rat)
Cmax [mg/l]
Fabs [%]
CL [l/h kg]
2.21
O
N
17
2.24
48
N
N
H
N
CYP 2C9
5.5
All other CYP isoforms
<4.0
12. Fischmann, T. O.; Hruza, A.; Xiao, D. N.; Fossetta, J. D.; Lunn, C. A.; Dolphin, E.;
Prongay, A. J.; Reichert, P.; Lundell, D. J.; Narula, S. K.; Weber, P. C. Nat. Struct.
Biol. 1999, 6, 233.
and 26 leading to enhanced iNOS affinity and demonstrating a
guideline for further structure-based optimization.
BYK 191023 17 as representative of the discussed imidazo
[4,5-b]pyridines displayed favorable pharmacokinetic parameters
along with a clean CYP P450 isoform inhibition profile (Table 5)
and drug-like physicochemical properties (data not shown). A
comprehensive pharmacological assessment for 17 had been pub-
lished elsewhere.23,34,35
13. Li, H.; Shimizu, H.; Flinspach, M.; Jamal, J.; Yang, W.; Xian, M.; Cai, T.; Wen, E.
Z.; Jia, Q.; Wang, P. G.; Poulos, T. L. Biochemistry 2002, 41, 13868.
14. Babu, B. R.; Griffith, O. W. Curr. Opin. Chem. Biol. 1998, 2, 491.
15. (a) Young, R. J.; Beams, R. M.; Carter, K.; Clark, H. A.; Coe, D. M.; Chambers, C. L.;
Davies, P. I.; Dawson, J.; Drysdale, M. J.; Franzman, K. W. Bioorg. Med. Chem. Lett.
2000, 10, 597; (b) Alderton, W. K.; Angell, A. D.; Craig, C.; Dawson, J.; Garvey, E.;
Moncada, S.; Monkhouse, J.; Rees, D.; Russel, R. J.; Schwartz, S. Br. J. Pharmacol.
2005, 145, 301.
In conclusion, we have demonstrated valuable structural in-
sights for identifying and evaluating structure–activity relation-
ships of potent and isoform selective iNOS inhibitors starting
from initial HTS results. Binding mode of 17 guided us to rational-
ize our experimental SAR and to drive the design of inhibitors.
iNOS inhibitors 17–26 represent selected structures capable of
illustrating rationals and conclusions. Moreover, we described a
straightforward synthetic access, favorable pharmacokinetic and
CYP P450 data of the lead structure 17 as representative of the
introduced iNOS inhibitor class.
16. Fedorow, R.; Hartmann, E.; Ghosh, D. K.; Schlichting, I. J. Biol. Chem. 2003, 278,
45818.
17. Tinker, A. C.; Beaton, H. G.; Boughton-Smith, N.; Cook, T. R.; Cooper, S. L.;
Fraser-Rae, L.; Hallam, K.; Hamley, P.; McInally, T.; Nicholls, D. J.; Pimm, A. D.;
Wallace, A. V. J. Med. Chem. 2003, 46, 913.
18. Hagmann, W. K.; Caldwell, C. G.; Chen, P.; Durette, P. L.; Esser, C. K.; Lanza, T. J.;
Kopka, I. E.; Guthikonda, R.; Shah, S. K.; MacCoss, M.; Chabin, R. M.; Fletcher, D.
Bioorg. Med. Chem. Lett. 2000, 10, 1975.
19. Connolly, S.; Aberg, A.; Arvai, A.; Beaton, H. G.; Cheshire, D. R.; Cook, A. R.;
Cooper, S.; Cox, D.; Hamley, P.; Mallinder, P.; Millichip, I.; Nicholls, D. J.;
Rosenfeld, R. J.; St-Gallay, S. A.; Tainer, J.; Tinker, A. C.; Wallace, A. V. J. Med.
Chem. 2004, 47, 3320.
20. Bonnefous, C.; Payne, J. E.; Roppe, J.; Zhuang, H.; Chen, X.; Symons, K. T.;
Nguyen, P. M.; Sablad, M.; Rozenkrants, N.; Zhang, Y.; Wang, L.; Severance, D.;
Walsh, J. P.; Yazdani, N.; Shiau, A. K.; Noble, S. A.; Rix, P.; Rao, T. S.; Hassig, C. A.;
Smith, N. D. J. Med. Chem. 2009, 52, 3047.
Acknowledgment
21. Zhou, D.; Lee, H.; Rothfuss, J. M.; Chen, D. L.; Ponde, D. E.; Welch, M. J.; Mach, R.
H. J. Med. Chem. 2009, 52, 2443.
22. Martin, N. I.; Beeson, W. T.; Woodward, J. J.; Marletta, M. A. J. Med. Chem. 2008,
51, 924.
We thank B. Bössenecker for determination of the pKa-values.
23. Strub, A.; Ulrich, W.-R.; Hesslinger, C.; Eltze, M.; Fuchß, T.; Strassner, J.; Strand,
S.; Lehner, M. D.; Boer, R. Mol. Pharmacol. 2006, 69, 328.
Supplementary data
24. Boer, R.; Marx, D.; Eltze, M.; Klein, T.; Nave, R.; Graedler, U.; Fuchss, T.; Barsig,
J.; Ulrich, W-R. PCT Int. Appl. 2003, WO 2003080607 A1.
25. Ashimori, A.; Ono, T.; Uchida, T.; Ohtaki, Y.; Fukaya, C.; Watanabe, M.;
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