Dibenzosuberones as p38 mitogen-activated protein kinase inhibitors with low ATP competitiveness and outstanding whole blood activity

Article abstract of DOI:10.1021/jm301539x

p38α mitogen-activated protein (MAP) kinase is a main target in drug research concerning inflammatory diseases. Nevertheless, no inhibitor of p38α MAP kinase has been introduced to the market. This might be attributed to the fact that there is no inhibitor which combines outstanding activity in biological systems and selectivity. Herein an approach to the development of such inhibitors on the basis of the highly selective molecular probe Skepinone-L is described. Introduction of a "deep pocket" moiety addressing the DFG motif led to an increased activity of the compounds. Hydrophilic moieties, addressing the solvent-exposed area adjacent to hydrophilic region II, conserved a high activity of the compounds in a whole blood assay. Combined with their outstanding selectivity and low ATP competitiveness, these inhibitors are very interesting candidates for use in biological systems and in therapy.

Full text of DOI:10.1021/jm301539x

Article
pubs.acs.org/jmc
Dibenzosuberones as p38 Mitogen-Activated Protein Kinase
Inhibitors with Low ATP Competitiveness and Outstanding Whole
Blood Activity
Stefan Fischer,† Heike K. Wentsch,† Svenja C. Mayer-Wrangowski,‡ Markus Zimmermann,†
Silke M. Bauer,† Kirsten Storch,† Raimund Niess,† Solveigh C. Koeberle,† Christian Grutter,‡
̈
Frank M. Boeckler,† Daniel Rauh,‡ and Stefan A. Laufer*,†
†Institute of Pharmacy, University of Tubingen, Auf der Morgenstelle 8, D-72076 Tubingen, Germany
̈
̈
‡Chemische Biologie, Fakultat Chemie, Technische Universitat Dortmund, Otto-Hahn-Strasse 6, D-44227 Dortmund, Germany
̈
̈
S
* Supporting Information
ABSTRACT: p38α mitogen-activated protein (MAP) kinase is a main target in drug
research concerning inflammatory diseases. Nevertheless, no inhibitor of p38α MAP
kinase has been introduced to the market. This might be attributed to the fact that there
is no inhibitor which combines outstanding activity in biological systems and selectivity.
Herein an approach to the development of such inhibitors on the basis of the highly
selective molecular probe Skepinone-L is described. Introduction of a “deep pocket”
moiety addressing the DFG motif led to an increased activity of the compounds.
Hydrophilic moieties, addressing the solvent-exposed area adjacent to hydrophilic
region II, conserved a high activity of the compounds in a whole blood assay. Combined
with their outstanding selectivity and low ATP competitiveness, these inhibitors are very
interesting candidates for use in biological systems and in therapy.
of the so-called “deep pocket”. As a result of this conformational
change, the entrance to the ATP binding site is blocked by the
activation loop of the enzyme (Figure 1).
INTRODUCTION
■
Kinase inhibitors have proved to be drugs of major therapeutic
and economic interest. As p38α MAP kinase is a key player in the
regulation of inflammatory processes, it has been the target of
many drug discovery and research efforts.1
Other unique features of this DFG-out binding mode are slow
association and dissociation constants due to the necessary
change of conformation. This structural transition increases the
drug−target residence time and therefore improves efficacy of
the inhibitors at low concentrations.13,14
The binding mode does not necessarily dictate selectivity of
the compounds. Rather it has to be established by exploiting
structural features of the kinase.15
There are two traditional approaches to the design of selective
inhibitors of p38α MAP kinase. One of them is the glycine flip,16
and the other one is the occupation of hydrophobic region I.17
Several inhibitors are able to form a second hydrogen bond to
the hinge region of p38α MAP kinase after a conformational
change of the enzyme (Figure 1A). This includes the rotation or
flip of Gly110 and a conformational change of the adjacent
Met109. Only 46 kinases show a glycine at this position. Amino
acids with longer side chains are most probably not able to
perform this flip. Affinity for the respective kinases should
therefore be reduced.
Traditionally, the variety of considered applications covers a
vast number of autoimmune diseases such as rheumatoid arthritis
and inflammatory bowel disease.2,3 Probably due to intolerable
adverse effects of chronic application of many p38α MAP kinase
inhibitors, attention has shifted to more acute inflammatory
disorders, e.g., pulmonary diseases.1 So far, no inhibitor of p38α
MAP kinase has advanced to a final clinical stage.4 Especially
when dosed at high concentrations, many compounds showed
toxic properties, partially due to off-target activity.5−7 Con-
sequently, there is a need for potent and selective p38α MAP
kinase inhibitors with high efficacy, even when administered at a
low dose. However, even compounds which proved to be potent
in a biochemical kinase activity assay often showed poor activity,
e.g., in whole blood assays,8 which are most important to evaluate
the potency of inhibitors in biological systems. The reason for
this could be based on high intracellular concentrations of ATP
competing with the inhibitor.
Occupation of hydrophobic region I improves selectivity for
p38α MAP kinase, as access to this region is only possible
Therefore, research has focused on inhibitors which are less
ATP competitive. These inhibitors change the conformation of
the enzyme into an enzymatically inactive state and are therefore
less easily displaced by ATP from the ATP binding site.9,10
During binding of these inhibitors, the DFG motif is moved out
Received: October 20, 2012
Published: December 27, 2012
© 2012 American Chemical Society
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Figure 1. Crystal structures of p38α MAP kinase. (A) Crystal structure of p38α in complex with [(2-aminophenyl)amino]dibenzosuberone showing the
DFG-in conformation. Phe169 of the DFG motif is located in the deep pocket (PDB code 3ZYA).11 Two hydrogen bonds are formed to the hinge
region by a flip of Gly110. (B) Crystal structure of p38α in complex with N-{4′-[(cyclopropylmethyl)carbamoyl]-6-methylbiphenyl- 3-yl}-2-morpholin-
4-ylpyridine-4-carboxamide stabilizing the DFG-out conformation. Phe169 is located outside of the deep pocket (PDB code 3D83).12.
Figure 2. (R)-2-[(2,4-Difluorophenyl)amino]-7-(2,3-dihydroxypropoxy)-10,11-dihydrodibenzo[a,d]cyclohepten-5-one (1) in complex with p38α
MAP kinase. (A) Crystal structure (PDB code 3QUE).11 (B) Schematic binding mode.
because of the small gatekeeper residue Thr106 (Figure 1).
Other kinases show a bulky methionine at this position
displacing inhibitors with moieties directed into this region.
Recently, we reported another successful approach to
selectivity: the design of very rigid inhibitors.18 As protein
kinases are quite flexible structures and undergo conformational
changes during activation, there might be room for induced fits,
together with flexible inhibitors. We were able to show that, by
rigidization of kinase inhibitors, induced fits can be reduced and
the selectivity of these inhibitors is therefore improved.18
Combination of these three selectivity features led to
Skepinone-L (1),11 which showed outstanding potency and
selectivity (Figure 2). It was successfully used as a molecular
probe in vitro and in vivo. In the present paper we discuss efforts
to address the deep pocket by inhibitors which were derived from
1. Introduction of novel hydrophilic moieties which are directed
into the solvent-exposed area adjacent to hydrophilic region II
conserved the whole blood activity of the modified compounds,
most probably due to an improved solubility of the compounds.
These modifications might be another step toward a drug in
therapy of inflammatory diseases.
CHEMICAL RESULTS
■
Bristol-Meyers Squibb19 and GlaxoSmithKline12 addressed the
deep pocket by adding amide moieties to the phenyl ring which
occupies hydrophobic region I (Figure 3A). This approach was
transferred to inhibitors developed by our group, showing a
binding mode similar to that of compound 1 (Figure 3B).20 As
there were promising results, this procedure was applied to
compound 1 as well. Furthermore, a novel dibenzosuberone
scaffold was designed to easily introduce novel hydrophilic
moieties, which should improve whole blood activity of the
compounds.
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to the corresponding amides and esters. This way we were able to
introduce functionalities which were unstable or incompatible
with the conditions of the Buchwald−Hartwig reaction.
BIOLOGICAL RESULTS
■
Enzyme Assay and Inhibition of TNF-α Release in
Whole Blood. All compounds were screened in two different
enzyme-linked immunosorbent assays (ELISAs). The first one
was a p38α MAP kinase activity assay, which used the isolated
enzyme.22 In the second one, the inhibition of lipopolysaccharide
(LPS)-stimulated TNF-α (tumor necrosis factor-α) release in
whole blood was analyzed.23 As p38α MAP kinase is a key
enzyme in the regulation of the proinflammatory enzyme TNF-
α, its release can be used as an indicator of inhibitor potency.
At first a small series of compounds, quite similar to compound
1, were prepared. By addition of benzoic acid moieties (16a−
16e), which were suggested to presumably address the deep
pocket by docking them into a related crystal structure, affinity
for the enzyme was considerably improved (Table 1). This series
suggested flexible hydrophilic moieties at position 7 (R4) which
are directed into the solvent-exposed area adjacent to hydrophilic
region II to be favorable for activity in the isolated p38α MAP
kinase enzyme assay.
Nevertheless, inhibition of LPS-stimulated TNF-α release in
whole blood was decreased (1 vs 16e), maybe due to high
molecular mass and lipophilicity. Therefore, it was our primary
aim to improve these parameters.
Exchange of the benzoic acid by tetrahydrofurancarboxylic
acid was detrimental for affinity to the enzyme (16b vs 16f).
Altering the position of the fluoro atom reduced potency in
whole blood even more (16b vs 16g).
By exchanging the benzoic acid for heteroaromatic carboxylic
acids (Table 2), affinity for the enzyme was improved. We
obtained inhibitors with IC50 values below 1 nM by this approach
(16i, 16j, 16l). The 3-thiophenecarboxylic acid derivative (16i)
proved to be most potent compared to its 2-thiophene (16j) and
3-furan (16h) counterparts. Exchange of the ethylmorpholine
(16j) moiety for a diol (16l) moiety did not alter the inhibitory
potential. More importantly, the improvement in whole blood
assays was even higher than that observed for the isolated p38α
MAP kinase enzyme assay (16d vs 16i). This can be ascribed to
higher hydrophilicity of the heterocycles compared to phenyl.
For the first time an improved whole blood activity compared to
that of compound 1 could be detected (1 vs 16i).
Figure 3. (A) Inhibitors developed by GlaxoSmithKline (2a) were able
to address the deep pocket after addition of amide moieties directed into
the deep pocket (2b). (B) A similar approach by our group (2c to 2d).
Synthesis of the novel dibenzosuberone scaffold is described in
Scheme 1. Compound 5 was synthesized according to a modified
synthesis of Anzalone et al.21 and coupled with 3-chlorobenzyl
iodide (6). After hydrolysis of diester 7, ring closure was
performed by intramolecular Friedel−Crafts acylation. The
scaffold 9 was subsequently converted to the respective alkyl
amides 10a and 10b, Scheme 2).
Deep pocket moieties were synthesized according to Scheme 3
and coupled with 10a, 10b, and 15a−e (synthesis is described by
Koeberle et al.18) via Buchwald−Hartwig reaction (Scheme 4).
Small anilines without any additional aryl carboxylic acid
moieties were introduced the same way. Cleavage of the amide
16s under acidic conditions yielded compound 17 (Scheme 5),
which could now again be derivatized with amines and alcohols
a
Scheme 1. Synthesis of 8-Chloro-5-oxo-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-3-carboxylic Acid (9)
a
Reagents and conditions: (a) CuCN, N-methylpyrrolidone, 200 °C, 7 d; (b) NaOH, diethylene glycol, 200 °C, 5 d; (c) H2SO4, MeOH, reflux, 29%
for steps 1−3; (d) LDA, THF, −78 °C, 70%; (e) NaOH, MeOH, reflux, >95%; (f) (1) SOCl2, DCM, reflux; (2) AlCl3, rt, 98%.
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a
Scheme 2. Synthesis of 10a and 10b
a
Reagents: (a) (1) SOCl2, DCM; (2) amino compound, Et3N, DCM, >95%.
a
enzyme, but potency in whole blood. Again, the 3-tetrahydrofur-
ancarboxylic adic derivative 16n showed a quite low inhibition
(16m vs 16n). Derivatives with an additional fluoro atom were
more potent in whole blood than their methyl counterparts (16n
vs 16o). Again the derivative with the 3-thiophenecarboxylic acid
(16r) was most potent.
Scheme 3. Synthesis of Deep Pocket Moieties
As heterocycles in general and especially 3-thiophene might
lead to toxic metabolites,24 we had to provide backup structures.
In the past, we had been successful in improving whole blood
activity of dibenzosuberones by addition of a hydrophilic moiety
at position 7 of the dibenzosuberone scaffold.11 Therefore, we
figured it possible to improve whole blood activity of the novel
compounds by altering these hydrophilic moieties, yielding
structures potent in whole blood using no heterocycles.
A dibenzosuberone scaffold was synthesized (Scheme 1)
which showed a carboxylic acid at position 7. Using this
functional group, new hydrophilic moieties could easily be
introduced. We synthesized amides (Tables 4 and 5) and several
esters (Table 6) to investigate interactions in and adjacent to
hydrophobic region II with the aim of an improved whole blood
performance. No deep pocket moieties were used for this
optimization, but small moieties occupying only hydrophobic
region I. This should ensure an easy and fast synthesis of multiple
derivatives. The combination with deep pocket moieties should
be performed afterward.
a
Reagents and conditions: (a) (1) corresponding carboxylic acid,
(COCl)2, DCM or THF; (2) corresponding nitroaniline, Et3N, DCM
or THF, 12−33%; (b) H2, Pd/C, ethyl actetate or MeOH, 32−95%;
(c) SnCl2, EtOH, 93−95%.
a
Scheme 4. Synthesis of 16a−u
The carboxylic acid derivative 17 (Table 4) showed potency in
the biochemical kinase activity assay comparable to that of the
unsubstituted amide 18a, but was inferior in the whole blood
assay. This may be attributed to a reduced cell permeability of
acid 17, which may be deprotonated and therefore charged under
physiological conditions.
a
Reagents: (a) Pd(OAc)2, X-Phos, KO-t-Bu, toluene, t-BuOH,
corresponding aniline (14c,d,i,k,l−q), 3−95%.
A methyl-substituted amide (18b) already showed affinity for
the enzyme comparable to that of compound 1. No further
increase in potency could be achieved by longer moieties
featuring H-bond donor functions (18d−18h). Sterically
hindered derivatives even led to less active compounds (16s,
18c).
Scheme 5. Synthesis of Amides and Esters, Unstable under
Buchwald−Hartwig Conditions
a
In the next series of compounds, derivatives with H-bond
donor functions were synthesized (Table 5) including hydroxyl
groups (18i−18k) and basic nitrogens (16t, 18l−18n). While
this strategy did not improve potency of the compounds in the
isolated enzyme assay, activity in whole blood was increased,
probably due to the higher hydrophilicity of the inhibitors.
Furthermore, we synthesized a couple of compounds with
derivatized amino acids. Again, the impact on enzyme activity
was minor. Lipophilic derivative 18r showed low activity in
whole blood. Nevertheless, the quite lipohilic compound 18p
was extraordinarily potent, maybe due to a specific amino acid
transporter, which recognized the inhibitor as a substrate.
Finally, a series of esters was synthesized (Table 6). The
present derivatives were predominantly made to investigate
structure−activity relationships in the solvent-exposed area
adjacent to hydrophilic region II. Nevertheless, the use of esters,
a
Reagents and conditions: (a) HCl(concd), reflux, >95%; (b) various
methods; see the Supporting Information.
Preliminary results18 suggested an additional substitution of
the phenyl which occupies hydrophobic region I to be beneficial.
Therefore, the corresponding compounds were synthesized
(Table 3). The addition of a methyl group (16m vs 16h) and of a
fluoro atom (16p vs 16d) did not improve affinity for the
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Table 1. Biological Activity of Derivatives Featuring Deep Pocket Moieties and Different Hydrophilic Moieties at Position 7
a
b
c
d
n = 3. n = 2. n = 1. n = 6.
especially as prodrugs, might be considered. In general, the ester
derivatives showed a slightly increased affinity for the enzyme
(19a vs 18b, 19e vs 18i, 19f vs 16t) when compared to the
corresponding amides. Consequently, the amides most likely do
not act as H-bond donors. Otherwise, they would show superior
potency. The inverted ester 19d showed a decrease of potency,
emphasizing the importance of the position and probably the
electron density of the carboxyl group. Whole blood activity
decreased for all ester compounds accordingly to their higher
lipophilicity.
We combined both optimization strategies: novel hydrophilic
moieties, improving activity in whole blood, and deep pocket
moieties, which improved affinity for the enzyme. This approach
led to 16u (Table 7), which showed an IC50 value of 0.2 nM and
was highly active in the whole blood assay, while the compound
was rid of any heterocyclic structures which could be subject to
metabolic pathways and lead to toxic intermediates.
Binding Mode of the Novel Compounds. Optimizing
whole blood activity of the novel compounds was only one part
of our work. Furthermore, we had to confirm our initial thesis
that the deep pocket is accessible by introduction of novel
moieties.
Recently, Simard et al.10 developed FLiK (fluorescent labels in
kinases), a fluorescence-based direct binding assay which is able
to detect ligand-induced conformation changes in protein
kinases,25 including p38α MAP kinase. For this purpose, the
enzyme is genetically altered and labeled with the environ-
mentally sensitive fluorophore acrylodan. Binding of DFG-out
ligands to the kinase domain of p38α induces conformational
changes in the activation loop, moves the attached acrylodan to a
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Table 2. Biological Activity of Derivatives Featuring Deep Pocket Moieties with Heterocyclic Structures
a
b
n = 3. n = 2.
more polar environment, and increases fluorescence emission at
514 nm, while emission at 468 nm is decreased. This way it is
possible to determine both Kd values and Kon rates. Compound
16e was subjected to this assay (Table 8).
For comparison, Kd and Kon of the known DFG-out binder 1-
(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-
ylethoxy)naphthalen-1-yl]urea (BIRB796) is shown in Table 8
too.10 It was possible to determine a Kd value for 16e, therefore
suggesting that it induces a conformational change of the
enzyme. Nevertheless, Kon of 16e was 1000 times faster than that
of BIRB796. We concluded that 16e is able to stabilize the DFG-
out conformation to some extent.
To confirm our observations, a cocrystallized structure of 16e
with the enzyme was examined by X-ray crystallography (Figure
4). The glycine flip is present and hydrophobic region I is
occupied by the fluorophenyl moiety. The interaction geometry
found in the crystal structure confirmed the ligand pose
suggested initially by docking 16e into crystal structure
3UVP.20 The RMSD of the ligand’s coordinates after structural
alignment of both protein−ligand complexes was 0.91 Å (see the
Supporting Information, SI-Figure 1). The crystal structure
shows an active conformation of p38α, but the electron density
indicates flexibility of the activation loop, and the inhibitor is
therefore most probably able to stabilize the DFG-out
conformation. The carbonyl oxygen of the amide is able to
form a direct hydrogen bond to Asp168, which might be
responsible for the stabilization of the DFG-out conformation
observed in the fluorescence assay. Therefore, the results from
both observations are coherent.
These results raised the question of whether the novel
compound 16e is less easily displaced by ATP than a traditional
DFG-in binder. Although the inhibitor does stabilize the DFG-
out conformation to some extent, the deep pocket is almost not
addressed by the inhibitor.
Therefore, we modified our enzyme assay and applied
increased concentrations of ATP. A DFG-out binder should
show a reduced loss of potency when compared to classical DFG-
in binders, if the amount of ATP is increased.
Even at high concentrations of ATP (300 μM, Table 9), 16e is
extremely potent. In contrast, the traditional DFG-in binder 4-
{4-(4-fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-1H-imidazol-
5-yl}pyridine (SB203580) shows only a moderate inhibition at
300 μM ATP.
This experimental setup confirmed a low ATP competitive-
ness of compound 16e. Competition with ATP might be reduced
further by introduction of residues even longer than described
herein, but whole blood activity would certainly suffer under
these modifications.
Selectivity Screen. To confirm that our modifications of
compound 1 were not detrimental with respect to selectivity,
compounds 16u and 18j were screened against 451 kinases (see
the Supporting Information, SI-Table 2).26 As observed for
compound 1 only p38α and p38β were inhibited in a relevant
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Table 3. Biological Activity of Derivatives Featuring Deep Pocket Moieties with Additional Substitutions
a
b
c
n = 3. n = 2. n = 4.
manner by 18j. Compound 16u additionally inhibited CSNK1E,
but can still be considered as extremely selective.
concentrations of compound 16e might mark the transition to
a therapeutically relevant drug.
EXPERIMENTAL SECTION
CONCLUSION
■
■
General Information. All commercially available reagents and
solvents were used without further purification. Flash chromatography
was performed with a LaFlash system (VWR) and Merck silica gel
(PharmPrep 60 CC 25−40 μm). Analytical HPLC analyses were run
(method 1) on a Merck HPLC instrument (autosampler AS-2000,
interface module D-6000, pump L-6200, detector L-4250) equipped
with a LiChrospher RP18 column (5 μm) (Merck) or (method 2) on a
LaChrome Ultra HPLC instrument (autosampler, interface module,
oven L2300, pump L-216OU, detector diode array C-2455U) equipped
with a Symmetry column (150 mm × 4.6 mm; 5 μm) (Waters). Melting
In conclusion, we were able to design novel inhibitors of p38α
MAP kinase based on 1 displaying subnanomolar IC50 values
with respect to p38α MAP kinase and low nanomolar IC50 values
with respect to TNF-α release in whole blood. The moiety
occupying hydrophobic region I was optimized. Additional
moieties which were intended to address the deep pocket were
introduced. Crystallization experiments revealed that these
moieties do not occupy the deep pocket, but form a hydrogen
bond to the DFG motif. A fluorescence-based direct binding
assay showed that the compounds are able to stabilize the DFG-
out conformation to some extent. For further stabilization effects,
longer moieties most probably have to be chosen. Novel
hydrophilic moieties, which are directed toward the solvent-
exposed area adjacent to hydrophilic region II, improved
performance in the physiologically relevant human whole
blood assay (16u, 18j). Selectivity screens confirmed that none
of these modifications were detrimental for selectivity (16u, 18j).
The outstanding activity and low dependency on ATP
points were determined with a Buchi B-545 melting point apparatus, and
̈
they were thermodynamically corrected. IR data were recorded with a
Nicolet 380 FT-IR (ATR) spectrometer. Mass spectra were acquired on
a Hewlett-Packard HP 6890 Series GC system equipped with an HP-
5MS capillary column (0.25 μm film thickness, 30 m × 0.25 mm i.d.)
(Hewlett-Packard) and an HP 5973 mass-selective detector (70 eV)
(Hewlett-Packard). High-resolution measurements (Fourier transform
ion cyclotron resonance) were obtained on a Bruker APEX II with
electron spray ionization. 1H NMR and 13C NMR spectroscopies were
performed on a Bruker Advance 200 MHz or a Bruker Ultra Shield 400
MHz spectrometer. The chemical shifts are reported in parts per million.
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Table 4. Biological Activity of Amide Derivatives
a
b
c
d
e
n = 3. n = 2. n = 4. Residual activity (μM) at a concentration of 10 μM. n = 1.
The purity of tested compounds (>95%) was confirmed by HPLC
analysis (see above, methods 1 and 2, and Supporting Information).
Three compounds showed less than 95% purity (16h, 86%; 18b, 92%;
19e, 93%). The numbering of atoms for analytical purposes (e.g., NMR
data) is defined in the Supporting Information for each compound as the
direction of counting is different for some compounds compared to that
proposed by IUPAC to maintain consistency.
General Procedure D. Chlorsuberone, aniline, X-Phos, KO-t-Bu, t-
BuOH, and toluene are mixed in a three-necked flask under an argon
atmosphere, followed by addition of Pd(OAc)2. The mixture is heated to
90−100 °C for 15 min to 6 h. Complete conversion is determined by
TLC. The reaction mixture is poured into water and extracted with ethyl
acetate or diethyl ether. Solvents are removed in a rotary evaporator.
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Table 5. Biological Activity of Amide Derivatives with Additional Hydrophilic Moieties
a
b
c
d
n = 3. n = 2. n = 4. No inhibition at a concentration of 1 μM.
The crude product is purified by automated column chromatography
(SiO2). The product can be further purified by recrystallization.
4-Methyl-isophthalonitrile (3). A 200 g (2.23 mol) sample of CuCN
in 100 g (0.62 mol) of 2,4-dichlorotoluene and 500 mL of N-
methylpyrrolidone are mixed. The suspension is stirred for 7 days at 180
°C, cooled to 110 °C, and poured carefully into 1500 mL of NH3 (25%).
The mixture is extracted 10 times with ethyl acetate. The organic layers
are separated by decantation. Ethyl acetate is removed in a rotary
evaporator. The crude product is normally used in the next step without
removal of the remaining N-methylpyrrolidone. The crude product can
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Table 6. Biological Activity of Ester Derivatives
a
b
n = 3. n = 2.
Table 7. Combination of Both Strategies: Novel Hydrophilic Moieties and Deep Pocket Moieties
be purified further by recrystallization (ethyl acetate/hexane). The yield
is determined after step 3 (compound 5). H NMR (DMSO-d6) δ
(−CH3), 110.2 (C1), 113.6 (C3), 116.6 (CN), 117.6 (CN), 131.9 (C5),
136.7 (C2), 136.7 (C6), 147.6 (C4).
1
(ppm) (f = 200 MHz): 2.55 (s, 3 H, −CH3), 7,68 (dd, 1H, J1 = 0.6 Hz, J2
= 8.1 Hz, C5H), 8.06 (dd, 1 H, J1 = 1.8 Hz, J2 = 8.1 Hz, C6H), 8.39 (d, 1
H, J = 1.8 Hz, C2H). 13C NMR (DMSO-d6) δ (ppm) (f = 50 MHz): 20.7
4-Methylisophthalic Acid (4). A 90 g (633 mmol) sample of 3, 120 g
of NaOH (powder), 500 mL of diethylene glycol, and 30 mL of H2O are
mixed and refluxed for 5 d. Evaporated H2O is replaced. The mixture is
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min. A 5 g (24.0 mmol) sample of 5, dissolved in 40 mL of THF, is
slowly added at −78 °C. Stirring is continued for 1 h. Subsequently, 3-
chlorobenzyl iodide (6) is added (during 30 min). The reaction mixture
is allowed to warm to 0 °C during the night. After addition of NH4Cl
solution (satd), the product can be extracted using ethyl acetate.
Solvents are removed in a rotary evaporator. The crude product is
purified by automated column chromatography (SiO2, petroleum ether
(bp 60−90 °C)/ethyl acetate (95:5 to 90:10)) in 1 h. Yield: 70−75%. 1H
NMR (DMSO-d6) δ (ppm) (f = 400 MHz): 2.85 (t, 2 H, J = 7.8 Hz,
−CH2CH2−), 3.24 (t, 2 H, J = 7.8 Hz, −CH2CH2−), 3.87 (s, 6 H,
−CH3), 7,16 (d, 1 H, J = 7.3 Hz, C6′H), 7.22−7.35 (m, 3 H, C2′/C4′/
C5′), 7.53 (d, 1 H, J = 8.1 Hz, C3H), 8.04 (d, 1 H, J = 8.1 Hz, C4H), 8.37
(s, 1 H, C6H). 13C NMR (DMSO-d6) δ (ppm) (f = 100 MHz): 35.4
(−CH2CH2−), 36.4 (−CH2CH2−), 52.3 (−CH3), 52.3 (−CH3), 126.0
(C6′), 127.1 (C4′), 127.8 (C5), 128.2 (C3), 129.6 (C1), 130.1 (C2′),
130.9 (C6), 131.8 (C5′), 132.3 (C4), 132.9 (C3′), 143.7 (C1′), 147.9
(C2), 165.3 (−COO−), 166.3 (−COO−).
Table 8. Kinetic Constants of 16e in Comparison to BIRB796
compd
16e
BIRB796
Kd (nM)
Kon (M−1 s−1
)
1.1 × 106
4.3 × 103
17
12
4-[2-(3-Chlorophenyl)ethyl]isophthalic Acid (8). A 6 g (18.0 mmol)
sample of 7 is dissolved in 100 mL of methanol and 5 mL of H2O. A 10 g
sample of KOH is added and the resulting solution refluxed for 6 h
(monitored by TLC). The reaction mixture is poured into water and
extracted at pH 1 (HCl(concd)) using ethyl acetate. Solvents are
removed in a rotary evaporator. Yield: >95%. 1H NMR (DMSO-d6) δ
(ppm) (f = 400 MHz): 2.86 (t, 2 H, J = 7.9 Hz, −CH2CH2−), 3.26 (t, 2
H, J = 8.0 Hz, −CH2CH2−), 7.15−7.35 (m, 4 H, C6′/2′/4′/5′), 7,46 (d, 1
H, J = 8.1 Hz, C3H), 7.99 (d, 1 H, J = 7.8 Hz, C4), 8.40 (s, 1 H, C6H),
13.14 (s, 2 H, −COOH). 13C NMR (DMSO-d6) δ (ppm) (f = 100
MHz): 35.6 (−CH2CH2−), 36,5 (−CH2CH2−), 125.9 (C6′), 127.0
(C4′), 128.2 (C3), 128.8 (C5), 130.1 (C2′), 130.5 (C1), 131.4 (C6), 131.5
(C5′), 132,1 (C4), 132,9 (C3′), 144.0 (C1′), 147.4 (C2), 166.5
(−COO−), 167.9 (−COO−).
8-Chloro-5-oxo-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-3-
carboxylic Acid (9). A 6.5 g (21.3 mmol) sample of 8 is suspended in
150 mL of DCM and the suspension heated to reflux temperature. 8.5
mL (117.2 mmol) SOCl2 are added dropwise. The reaction mixture is
refluxed until a clear solution is visible. It might be necessary to add
catalytic amounts of DMF. A 15.53 g (116.5 mmol) sample of AlCl3 is
added at 0 °C. After 20 min the mixture is poured onto ice, stirred for 30
min, and extracted with DCM. Solvents are removed in a rotary
evaporator. Yield: 98%. 1H NMR (DMSO-d6) δ (ppm) (f = 400 MHz):
3.08−3.31 (m, 4 H, −CH2CH2−), 7.35−7.54 (m, 3 H, C1/3/9), 7.90 (d, 1
H, J = 8.5 Hz, C4H), 8.03 (dd, 1 H, J1 = 1.9 Hz, J2 = 7.8 Hz, C8), 8.44 (d, 1
H, J = 1.8 Hz, C6H), 13.0 (s, 1 H, −COOH). 13C NMR (DMSO-d6) δ
(ppm) (f = 100 MHz): 33.7 (C11), 34.2 (C10), 127.1 (C3), 129.6 (C1),
129.6 (C9), 130.7 (C6), 131.7 (C7), 132.7 (C4), 133.3 (C8), 136.6 (C4a),
137.8 (C5a), 137.9 (C2), 144.7 (C11a), 147,2 (C9a), 166.8 (−COO−),
192.8 (C5).
2-Chloro-7-(morpholin-4-ylcarbonyl)-10,11-dihydrodibenzo[a,d]-
cyclohepten-5-one (10a). A 2.5 g (8.7 mmol) sample of 9 is dissolved in
10 mL of DCM, 3 mL (41.4 mmol) of SOCl2 is added dropwise, and the
solution is refluxed for 1 h. The reaction mixture is poured into a
solution of 21.2 mL (243.3 mmol) of morpholine in 10 mL of DCM at 0
°C. Stirring is continued overnight at room temperature. Afterward the
mixture is poured into water and extracted with ethyl acetate. Solvents
are removed in a rotary evaporator. The yellow oil is washed with water
and dried in vacuo. Yield: 3.0 g (97%). 1H NMR (DMSO-d6) δ (ppm) (f
= 400 MHz): 3.09−3.22 (m, 4 H, −CH2CH2−), 3.51−3.83 (m, 8 H,
morpholine), 7.15−7.30 (m, 3 H, C1/3/9H), 7.46 (d, 1 H, J = 7.6 Hz,
C4H), 7.92 (d, 1 H, J = 8.6 Hz, C8H), 7.97 (s, 1 H, C6H). 13C NMR
(DMSO-d6) δ (ppm) (f = 100 MHz): 34.6 (C10), 34.7 (C10), 66.9 (4 C,
morpholine), 127.1 (C3), 129.3 (C1), 129.6 (C9), 129.9 (C6), 131.4
(C8), 132.7 (C4), 134.0 (C4a), 136.2 (C5a), 138.2 (C7), 138.8 (C2), 143.5
(C9a), 143.7 (C11a), 169.4 (−COO−), 192.9 (C5).
8-[[3-(Benzoylamino)-4-fluorophenyl]amino]-5-oxo-10,11-dihy-
dro-5H-dibenzo[a,d]cycloheptene-3-carboxylic Acid (2-Morpholin-
4-ylethyl)amide (16u). The compound is synthesized according to
general procedure D. A 0.37 g (0.93 mmol) sample of 8-chloro-5-oxo-
10,11-dihydro-5H-dibenzo[a,d]cycloheptene-3-carboxylic acid (2-mor-
pholin-4-ylethyl)amide (10b), 0.20 g (0.87 mmol) of N-(5-amino-2-
fluorophenyl)benzamide (14c), 0.05 g (0.22 mmol) of Pd(OAc)2, 0.14
Figure 4. Crystal structure of 16e in complex with p38α MAP kinase
(PDB code 3UVQ). The DFG-in conformation is shown, but the
electron density indicates flexibility of the activation loop.
Table 9. Connection of Inhibitory Activity and Concentration
of ATP
a
IC50(p38α) SEM (μM)
compd
16e
SB203580
100 μM ATP
300 μM ATP
0.001 0.000
0.048 0.004
0.003 0.000
0.273 0.001
a
n = 3.
poured into 1000 mL of H2O, the product is precipitated using
HCl(concd), and filtered. Remaining diethylene glycol is removed by
washing the crude product with water at pH 1. The yellow powder is
dried in vacuo. The yield is determined after step 3 (compound 5). 1H
NMR (DMSO-d6) δ (ppm) (f = 400 MHz): 2.58 (s, 3 H, −CH3), 7.43
(d, 1 H, J = 7.8 Hz, C5H), 7.97 (d, 1 H, J = 7.8 Hz, C6H), 8.38 (s, 1 H,
C2H), 12.98 (s, 2 H, −COOH). 13C NMR (DMSO-d6) δ (ppm) (f =
100 MHz): 21.3 (−CH3), 128.5 (C1), 130.6 (C3), 131.1 (C5), 132.1
(C2), 132.1 (C6), 144.2 (C4), 166.5 (−COO−), 167.8 (−COO−).
4-Methylisophthalic Acid Dimethyl Ester (5). A 55 g (305 mmol)
sample of 4 is dissolved in 400 mL of methanol. A 10 mL volume of
H2SO4 is added, and the mixture is refluxed for 6 h (monitored by TLC).
Afterward the mixture is poured into water and extracted with ethyl
acetate. The crude product is purified by automated column
chromatography (SiO2, petroleum ether (bp 60−90 °C)/ethyl acetate
1
(95:5 to 90:10)) in 1 h. Yield: 29% (calcd for steps 1−3). H NMR
(DMSO-d6) δ (ppm) (f = 400 MHz): 2.57 (s, 3 H, −CH3), 3.71−3.99
(m, 6 H, −COOCH3), 7.46 (d, 1 H, J = 8.1 Hz, C5H), 7.99 (d, 1 H, J =
7.8 Hz, C6H), 8.34 (s, 1 H, C2H). 13C NMR (DMSO-d6) δ (ppm) (f =
100 MHz): 21.3 (−CH3), 52.1 (−COOCH3), 52.2 (−COOCH3), 127.4
(C1), 129.5 (C3), 130.7 (C5), 132.2 (C2), 132.2 (C6), 144.8 (C4), 165.3
(−COO−), 166.3 (−COO−).
4-[2-(3-Chlorophenyl)ethyl]isophthalic Acid Dimethyl Ester (7). An
18 mL (45.0 mmol) sample of n-BuLi (2.5 M in hexane) is added
dropwise during 15 min to a mixture of 6.35 mL (45.2 mmol) of
diisopropylamine and 80 mL of THF at 0 °C. Stirring is continued for 15
251
dx.doi.org/10.1021/jm301539x | J. Med. Chem. 2013, 56, 241−253

Journal of Medicinal Chemistry
Article
g (0.29 mmol) of 2-(dicyclohexylphosphino)-2′,4′,6′-triisopropylbi-
phenyl, 0.40 g (3.6 mmol) of KO-t-Bu, 15 mL of toluene, and 3 mL of t-
BuOH are reacted at 100 °C for 30 min. The reaction mixture is
extracted with ethyl acetate. The crude product is purified by automated
column chromatography (SiO2, DCM/ethanol (95:5)). The product is
further purified by recrystallization (ethyl acetate/hexane). Yield: 0.04 g
(7%). HRMS (m/z): [M + H]+ 593.255492 (calcd 593.255860). IR
A.; Swinamer, A.; Torcellini, C.; Tsang, M.; Moss, N. Structure−activity
relationships of the p38α MAP kinase inhibitor 1-(5-tert-butyl-2-p-tolyl-
2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-ylethoxy)naphthalen-1-yl]urea
(BIRB 796). J. Med. Chem. 2003, 46, 4676−4686.
(3) Zhang, H.; Nei, H.; Dougherty, P. M. A p38 mitogen-activated
protein kinase-dependent mechanism of disinhibition in spinal synaptic
transmission induced by tumor necrosis factor-α. J. Neurosci. 2010, 30,
12844−12855.
(4) www.clinicaltrials.gov (accessed March 1, 2012), 2012.
(5) Cohen, S. B.; Cheng, T. T.; Chindalore, V.; Damjanov, N.; Burgos-
Vargas, R.; Delora, P.; Zimany, K.; Travers, H.; Caulfield, J. P. Evaluation
of the efficacy and safety of pamapimod, a p38 MAP kinase inhibitor, in a
double-blind, methotrexate-controlled study of patients with active
rheumatoid arthritis. Arthritis Rheum. 2009, 60, 335−344.
(6) Dominguez, C.; Powers, D. A.; Tamayo, N. p38 MAP kinase
inhibitors: many are made, but few are chosen. Curr. Opin. Drug
Discovery Dev. 2005, 8, 421−430.
(7) Hill, R. J.; Dabbagh, K.; Phippard, D.; Li, C.; Suttmann, R. T.;
Welch, M.; Papp, E.; Song, K. W.; Chang, K. C.; Leaffer, D.; Kim, Y. N.;
Roberts, R. T.; Zabka, T. S.; Aud, D.; Dal, P. J.; Manning, A. M.; Peng, S.
L.; Goldstein, D. M.; Wong, B. R. Pamapimod, a novel p38 mitogen-
activated protein kinase inhibitor: preclinical analysis of efficacy and
selectivity. J. Pharmacol. Exp. Ther. 2008, 327, 610−619.
(8) Goldstein, D. M.; Gabriel, T. Pathway to the clinic: inhibition of
P38 MAP kinase. A review of ten chemotypes selected for development.
Curr. Top. Med. Chem. 2005, 5, 1017−1029.
1
(ATR): 329, 1576, 1525, 1261, 1114, 861, 786, 706 cm−1. H NMR
(DMSO-d6) δ (ppm) (f = 200 MHz): 2.27−2.62 (m, 6 H + DMSO,
C2/6morpholinylH/NCH2CH2NOC), 2.99−3.21 (m, 4 H, −CH2CH2−),
3.24−3.48 (m, 2 H + H2O, NCH2CH2NOC), 3.49−3.62 (m, 4 H,
C3/5morpholinylH), 6.86 (s, 1H, C1H), 6.93−7.44 (m, 4 H, C3/6 /5 /2
′ ′ ′H),
7.47−7.62 (m, 4 H, C4/3″/5″/4″H), 7.86−8.06 (m, 4 H, C9/2″/6″/8H),
8.32 (d, 1 H, J = 1.1 Hz, C6H), 8.45−8.61 (m, 1 H, −CONHCH2−),
8.89 (s, 1 H, −CONH−), 10.1 (s, 1H, −NH−). 13C NMR (DMSO-d6)
δ (ppm) ( f = 100 MHz): 33.8 (C10), 35.5 (C11), 36.4
(−NCH2 CH2 NH−), 53.2 (2 C, C2 / 6 m o r p h o l i n y l ), 57.2
(−NCH2CH2NH−), 66.1 (2 C, C3/5morpholinyl), 112.6 (C3), 113.9
(C1), 116.2 (d, J = 20.4 Hz, C5′), 118.1 (d, J = 6.5 Hz, C6′), 118.2 (s,
C1′), 126.2 (d, 1 H, J = 13.8 Hz, C3′), 127.2 (C4a), 127.8 (2 C, C2″/6″),
128.4 (2 C, C3″/5″), 128.9 (C9), 129.1 (C6), 130.4 (C4) 131.8 (C8),
133.5 (C4″), 134.0 (C7), 137.0 (C5a), 137.1 (C1″), 138.9 (C2), 144.5
(C11a), 148.7 (C9a), 150.9 (d, J = 241.4 Hz, C4′), 165.5 (2 C,
−CONH−), 190.3 (C5).
ASSOCIATED CONTENT
* Supporting Information
■
S
(9) Rabiller, M.; Getlik, M.; Kluter, S.; Richters, A.; Tuckmantel, S.;
Simard, J. R.; Rauh, D. Proteus in the world of proteins: conformational
changes in protein kinases. Arch. Pharm. (Weinheim, Ger.) 2010, 343,
193−206.
(10) Simard, J. R.; Getlik, M.; Grutter, C.; Pawar, V.; Wulfert, S.;
Rabiller, M.; Rauh, D. Development of a fluorescent-tagged kinase assay
system for the detection and characterization of allosteric kinase
inhibitors. J. Am. Chem. Soc. 2009, 131, 13286−13296.
Kinetics of 16e, crystallization and structure determination of
p38α in complex with 16e, analytical equipment and methods,
synthetic procedures, 1H NMR, 13C NMR, HPLC, HRMS, and
IR data, melting points, selectivity data of 16u and 18j, and
molecular modeling. This material is available free of charge via
the Internet at http://pubs.acs.org.
(11) Koeberle, S. C.; Romir, J.; Fischer, S.; Koeberle, A.; Schattel, V.;
Albrecht, W.; Grutter, C.; Werz, O.; Rauh, D.; Stehle, T.; Laufer, S. A.
Skepinone-L is a selective p38 mitogen-activated protein kinase
inhibitor. Nat. Chem. Biol. 2012, 8, 141−143.
(12) Angell, R. M.; Angell, T. D.; Bamborough, P.; Bamford, M. J.;
Chung, C. W.; Cockerill, S. G.; Flack, S. S.; Jones, K. L.; Laine, D. I.;
Longstaff, T.; Ludbrook, S.; Pearson, R.; Smith, K. J.; Smee, P. A.;
Somers, D. O.; Walker, A. L. Biphenyl amide p38 kinase inhibitors 4:
DFG-in and DFG-out binding modes. Bioorg. Med. Chem. Lett. 2008, 18,
4433−4437.
(13) Copeland, R. A.; Pompliano, D. L.; Meek, T. D. Drug-target
residence time and its implications for lead optimization. Nat. Rev. Drug
Discovery 2006, 5, 730−739.
AUTHOR INFORMATION
Corresponding Author
*E-mail:stefan.laufer@uni-tuebingen.de. Phone: +49-7071-
2972459. Fax: +49-7071-295037.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank M. Goettert and K. Bauer for biological testing. D.R. is
grateful for funds by the German federal state North Rhine
Westphalia (NRW) and the European Union (European
Regional Development Fund: Investing In Your Future) and
the German Federal Ministry for Education and Research
(NGFNPlus) (Grant No. BMBF 01GS08104). S.F. is grateful for
advice and help of Annette Kuhn concerning the synthesis of the
compounds.
(14) Simard, J. R.; Grutter, C.; Pawar, V.; Aust, B.; Wolf, A.; Rabiller,
M.; Wulfert, S.; Robubi, A.; Kluter, S.; Ottmann, C.; Rauh, D. High-
throughput screening to identify inhibitors which stabilize inactive
kinase conformations in p38α. J. Am. Chem. Soc. 2009, 131, 18478−
18488.
(15) Davis, M. I.; Hunt, J. P.; Herrgard, S.; Ciceri, P.; Wodicka, L. M.;
Pallares, G.; Hocker, M.; Treiber, D. K.; Zarrinkar, P. P. Comprehensive
analysis of kinase inhibitor selectivity. Nat. Biotechnol. 2011, 29, 1046−
1051.
(16) Fitzgerald, C. E.; Patel, S. B.; Becker, J. W.; Cameron, P. M.;
Zaller, D.; Pikounis, V. B.; O’Keefe, S. J.; Scapin, G. Structural basis for
p38α MAP kinase quinazolinone and pyridol-pyrimidine inhibitor
specificity. Nat. Struct. Biol. 2003, 10, 764−769.
(17) Lee, M. R.; Dominguez, C. MAP kinase p38 inhibitors: clinical
results and an intimate look at their interactions with p38α protein. Curr.
Med. Chem. 2005, 12, 2979−2994.
(18) Koeberle, S. C.; Fischer, S.; Schollmeyer, D.; Schattel, V.; Grutter,
C.; Rauh, D.; Laufer, S. A. Design, synthesis, and biological evaluation of
novel disubstituted dibenzosuberones as highly potent and selective
inhibitors of p38 mitogen activated protein kinase. J. Med. Chem. 2012,
55, 5868−5877.
ABBREVIATIONS USED
■
ATP, adenosine triphosphate; BuOH, butanol; CSNK1E, casein
kinase 1 isoform ε; DCM, dichloromethane; ELISA, enzyme-
linked immunosorbent assay; EtOH, ethanol; LDA, lithium
diisopropylamide; LPS, lipopolysaccharide; MAP, mitogen-
activated protein; MeOH, methanol; THF, tetrahydrofuran;
TNF, tumor necrosis factor
REFERENCES
■
(1) Fischer, S.; Koeberle, S. C.; Laufer, S. A. p38α mitogen-activated
protein kinase inhibitors, a patent review (2005−2011). Expert Opin.
Ther. Pat. 2011, 21, 1843−1866.
(2) Regan, J.; Capolino, A.; Cirillo, P. F.; Gilmore, T.; Graham, A. G.;
Hickey, E.; Kroe, R. R.; Madwed, J.; Moriak, M.; Nelson, R.; Pargellis, C.
252
dx.doi.org/10.1021/jm301539x | J. Med. Chem. 2013, 56, 241−253

Journal of Medicinal Chemistry
Article
(19) Sack, J. S.; Kish, K. F.; Pokross, M.; Xie, D.; Duke, G. J.; Tredup, J.
A.; Kiefer, S. E.; Newitt, J. A. Structural basis for the high-affinity binding
of pyrrolotriazine inhibitors of p38 MAP kinase. Acta Crystallogr., D:
Biol. Crystallogr. 2008, D64, 705−710.
(20) Martz, K. E.; Dorn, A.; Baur, B.; Schattel, V.; Goettert, M. I.;
Mayer-Wrangowski, S. C.; Rauh, D.; Laufer, S. A. Targeting the hinge
glycine flip and the activation loop: novel approach to potent p38α
inhibitors. J. Med. Chem. 2012, 55, 7862−7874.
(21) Anzalone, L.; Hirsch, J. A. Substitient effects on hydrogenation of
aromatic rings: hydrogenation vs. hydrogenolysis in cyclic analogues of
benzyl ethers. J. Org. Chem. 1985, 50, 2128−2133.
(22) Laufer, S.; Thuma, S.; Peifer, C.; Greim, C.; Herweh, Y.; Albrecht,
A.; Dehner, F. An immunosorbent, nonradioactive p38 MAP kinase
assay comparable to standard radioactive liquid-phase assays. Anal.
Biochem. 2005, 344, 135−137.
(23) Linsenmaier, S. Entwicklung und optimierung von in vitro
testverfahren zur evaluierung von hemmstoffen der p38α MAP kinase
und JNK3. Ph.D. Thesis, Universitat Tubingen, 2006.
̈
̈
(24) Walsh, J. S.; Miwa, G. T. Bioactivation of drugs: risk and drug
design. Annu. Rev. Pharmacol. Toxicol. 2011, 51, 145−167.
(25) Simard, J. R.; Kluter, S.; Grutter, C.; Getlik, M.; Rabiller, M.;
Rode, H. B.; Rauh, D. A new screening assay for allosteric inhibitors of
cSrc. Nat. Chem. Biol. 2009, 5, 394−396.
(26) Ambit Biosciences Home Page. www.ambitbio.com (accessed
March 1, 2012), 2012.
253
dx.doi.org/10.1021/jm301539x | J. Med. Chem. 2013, 56, 241−253