Design, Synthesis, and biological evaluation of novel disubstituted dibenzosuberones as highly potent and selective inhibitors of p38 mitogen activated protein kinase

Article abstract of DOI:10.1021/jm300327h

Synthesis, biological testing, structure-activity relationships (SARs), and selectivity of novel disubstituted dibenzosuberone derivatives as p38 MAP kinase inhibitors are described. Hydrophilic moieties were introduced at the 7-, 8-, and 9-position of th

Full text of DOI:10.1021/jm300327h

Article
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of Novel Disubstituted
Dibenzosuberones as Highly Potent and Selective Inhibitors of p38
Mitogen Activated Protein Kinase
Solveigh C. Koeberle,†,∥ Stefan Fischer,†,∥ Dieter Schollmeyer,‡ Verena Schattel,† Christian Grutter,§
̈
Daniel Rauh,§ and Stefan A. Laufer*,†
†Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Eberhard-Karls-University, Auf der Morgenstelle 8,
72076 Tubingen, Germany
̈
‡Department of Organic Chemistry, Johannes Gutenberg-University Mainz, Duesbergweg 10-14, D-55099 Mainz, Germany
§Faculty of ChemistryChemical Biology, Technische Universitat Dortmund, Otto-Hahn-Strasse 6, D-44227 Dortmund, Germany
̈
S
* Supporting Information
ABSTRACT: Synthesis, biological testing, structure−activity
relationships (SARs), and selectivity of novel disubstituted
dibenzosuberone derivatives as p38 MAP kinase inhibitors are
described. Hydrophilic moieties were introduced at the 7-, 8-,
and 9-position of the 2-phenylamino-dibenzosuberones,
improving physicochemical properties as well as potency.
Extremely potent inhibitors were obtained, with half-maximal
inhibitory concentration (IC50) values in the low nM range in
a whole blood assay measuring the inhibition of cytokine
release. The high potency of the target compounds together
with the outstanding selectivity of this novel class of
compounds toward p38 mitogen activated protein (MAP) kinase as compared to other kinases indicate them to be most
applicable as tools in pharmacological research and eventually they may foster a new generation of anti-inflammatory drugs.
INTRODUCTION
■
Therapy for chronic inflammatory diseases like rheumatoid
arthritis (RA) and inflammatory bowel diseases (IBD) has
progressed greatly in recent years. Introduction of biological
agents, particularly monoclonal antibodies which neutralize
Figure 1. VX-745.
TNF-α (e.g., etanercept, infliximab, and adalimumab) or IL-1β
(e.g., anakinra), have afforded many advances in treatment of
these disorders. The main drawbacks of these drugs are their
lack of oral bioavailability and their high cost of production.
Several global players have invested considerable resources to
discover low molecular weight drugs with the same efficacy as
biologic agents in therapy of inflammatory diseases.1−3 One
main target is p38α mitogen activated protein (MAP) kinase, a
key enzyme in the regulation of the pro-inflammatory cytokines
TNF-α and IL-1β.4−6
Until now, no p38α MAP kinase inhibitor has been approved
for therapy of inflammatory diseases.1−3 Since the cloning of
p38α MAP7 was first described and the corresponding
inhibitors were developed, the p38α MAP kinase community
has strived to produce a commercial drug. A serious problem in
the development of p38α MAP kinase inhibitors is the lack of
selectivity. Novel and fast screening methods revealed that even
VX-745 (Figure 1), purported to be a highly selective inhibitor
against p38α MAP kinase, exhibited considerable off-target
activity.8,9
The ATP binding site, the locus where all inhibitors of p38α
MAP kinase at least partially bind, is highly conserved
throughout all kinases. An inhibitor which binds only to this
site will certainly inhibit several other kinases as well.
Therefore, the successful design of p38α MAP kinase inhibitors
must exploit characteristics that are unique to p38α MAP
kinase such as the hydrophobic region I and the glycine
flip.2,10−12 The hydrophobic region I, which is adjacent to the
ATP binding site, is accessible in p38α MAP kinase because the
opening of this area is occupied by the small gatekeeper amino
acid Thr106.10,11 In other types of kinases, the entrance to this
region is blocked by a bulky methionine residue. Inhibitors
occupying hydrophobic region I should therefore have only
weak binding affinities to kinases with large gatekeeper amino
acids. Another p38α MAP kinase structural feature is
Received: March 8, 2012
© XXXX American Chemical Society
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Figure 2. Binding modes of 2-(2-amino-phenylamino)-10,11-dihydro-dibenzo[a,d]cyclohepten-5-one (1) and Skepinone-L (2). Left: Schematic of
the binding mode of 2-(2-amino-phenylamino)-10,11-dihydro-dibenzo[a,d]cyclohepten-5-one (1, PDB ID 3ZYA) in complex with p38α MAP
kinase; Right: Schematic of the binding mode of 2-(2,4-difluoro-phenylamino)-7-(2R,3-dihydroxy-propoxy)-10,11-dihydro-dibenzo[a,d]cyclohepten-
5-one (2, Skepinone-L, PDB ID 3QUE) with p38α MAP kinase.13
exemplified by Gly110 in the hinge region. This small amino
acid at this position enables the enzyme to adjust its
conformation (i.e., to undergo the glycine flip)12 in order to
form a second hydrogen bond with the inhibitor. A carbonyl
oxygen in the inhibitor is most commonly used to induce the
glycine flip. The peptide bond between Met109 and Gly110
rotates and places the NH of glycine at a distance favorable to
the inhibitor (Figure 2). Kinases which feature amino acids with
longer side chains at this position are not able to perform the
glycine flip.
the 2-phenylamino substituent was assembled by Buchwald−
Hartwig reaction,16 a chloro group was introduced in the 2-
position. Oxygen-linked hydrophilic moieties in the 7/8/9-
positions can be accessed via the corresponding phenols. Thus,
2-chloro-7/8/9-hydroxydibenzosuberones were synthesized.
Schemes 1−3 outline a synthetic strategy for the assembly of
the target compounds.
Starting from 3-methoxy benzoic acid, 6-methoxyphthalide 3
was obtained by treatment with formaldehyde/acetic acid.17
Despite taking into account the occupation of the hydro-
phobic region I and the induction of the glycine flip in the
design of VX-745, there was still off-target activity. Therefore,
other selectivity features have been sought in order to achieve
selective inhibitors of p38α MAP kinase. The flexible structures
of kinases permit them to undergo conformational changes
during activation. In conjunction with flexible inhibitors, these
conformational changes might provoke undesirable induced fits
that may cause nonspecific effects. Therefore, it was our aim to
design very rigid inhibitors. We started out from monosub-
stituted dibenzosuberones structures, compounds which should
possess the required rigidity. They proved to be quite potent in
the isolated p38α MAP kinase enzyme assay (compound 1,
IC50 = 104 nM) and were demonstrated to occupy hydro-
phobic region I and to induce the glycine flip (Figure 2).13,14
Unfortunately, they were highly lipophilic, which probably
accounted for their low activity in whole blood. In an effort to
improve hydrophilicity of the dibenzosuberone scaffold, we
synthesized aza-analogue structures, but these compounds lost
potency in the p38α MAP kinase enzyme assay.15
Scheme 1. Synthesis of 2-Chloro-7-hydroxydibenzosuberone
and Corresponding Derivatives
a
Docking studies suggested that hydrophilic moieties at
positions 7, 8, and 9 should be well tolerated. This approach
eventually led to Skepinone-L (2, Figure 2, PDB ID 3QUE),
which is highly potent in vivo and, to the best of our
knowledge, is the most selective p38α MAP kinase inhibitor
described in literature.13 Herein, we will discuss optimization
and structure−activity relationships (SARs) of scaffolds
occupying hydrophobic regions I and II, which were
synthesized during the design of Skepinone-L (2).
a
Reagents and conditions: (a) formaldehyde (37%), acetic acid, 90 °C,
64%; (b) (1) NBS, AIBN, chlorobenzene, 90 °C, (2) NaOH, 87%; (c)
(1) PPh3, MeOH, reflux, (2) NaOMe (30%), 0 °C, 40%; (d) H2/Pd/
C, ethyl acetate/acetonitrile, room temperature, 95%; (e) (1) SOCl2,
DCM, (2) AlCl3, 58−93%; (f) HBr (48%), acetic acid, reflux, 65−
95%; (g) K2CO3, acetonitrile, reflux or K2CO3, DMF, reflux, 78−91%.
(h) Compounds 11, 13−16: Pd(OAc)2, X-Phos, NaO-tert-Bu, toluol,
tert-BuOH, 90 °C, 32−87%. Compounds 17: Pd(OAc)2, di-tert-butyl-
X-Phos, K3PO4, toluol, 90 °C, 27−48%.
CHEMISTRY
■
Synthesis of the dibenzosuberone derivatives in this study
required dibenzosuberone scaffolds with two functionalities. As
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Oxidative ring-opening with N-bromo-succinimide/azoisobu-
tyronitrile (NBS/AIBN) yielded the desired aldehyde 4. The
central step of the synthesis is the Wittig reaction, yielding the
respective stilbeno-2-carboxylic acid 5. Reduction of the
etheno-linker using H2 and Pd/carbon provided the corre-
sponding saturated acid (6), which was cyclized by an
intramolecular Friedel−Crafts reaction with AlCl3. Friedel−
Crafts acylations are possible in both the ortho- and para-
positions. We supposed that the acylation in the para-position
would be preferred because the large volume of the chlorine
may hinder the acylation in ortho-position. We detected two
peaks (one very large peak and a very small one) in GC-MS
analysis. The fraction from the large peak was collected during
flash chromatography purification, and crystal structure
confirmed that the isolated product was the desired 2-chloro-
methoxydibenzosuberone (Supporting Information Figure S1).
Treatment of the respective 2-chloro-methoxydibenzosuber-
ones (7, 8) with HBr/acetic acid18 proceeded in high yields to
give the corresponding 2-chloro-7-hydroxy-dibenzosuberone
(9).
Hydrophilic moieties were introduced either by Williamson’s
ether synthesis with alkyl halogenides or with activated alcohols
(R- or S-O-isopropylidene glycerol tosylate). Buchwald−
Hartwig reaction was performed with different anilino-
derivatives, providing the 2-substituted dibenzosuberone
derivatives (11, 13−16) in very good yields. We employed
di-tert-butyl-X-Phos for coupling phenoxy residues (17). Chiral
dihydroxypropoxy derivatives were accomplished by depro-
tection of the acetal with p-toluenesulfonic acid.19 Analogue 12
was prepared by Suzuki coupling20 of 7 with boronic acid as
described in Scheme 2.
may have enhanced selectivity compared to their more flexible
counterparts as well as increased potency. This hypothesis is
based on the fact that the kinase may also induce conforma-
tional changes at a flexible inhibitor which is not possible with a
rigid compound.22,23 Unlike what was suggested by Ottosen et
al.21 (Figure 3, left), our docking studies with both compounds
suggested that the amino-linked phenyl residue occupies the
hydrophobic pocket one (Figure 3, middle). We crystallized the
2-amino-phenyl-amino-benzophenone in complex with p38α
MAP kinase (PDB ID 3QUD, Figure 3) as well as the 2-
aminophenylaminodibenzosuberon to gain insights into its
binding mode. In contrast to the binding mode suggested by
Ottosen et al.,21 the aminophenylamino residue of 2-amino-
phenyl-amino-benzophenone occupies the hydrophic region I
(Figure 3, right). The rigidized compound 1 confirmed a
binding mode isostructural to that of the benzophenone and
has been previously published (Figure 2, PDB ID 3ZYA).13,14
A first optimization step was performed with the phenyl-
amino moieties which occupy the hydrophobic region I (see
Figures 2 and 3), and all compounds were primarily screened in
a p38α MAP kinase activity assay.24
Because of the small dimensions of this pocket, small
substituents like NH2, F, or CH3 at the phenyl-amino moiety
are better tolerated than larger residues like CF3. Among the
monosubstituted derivatives, the 2-amino moiety (11a) was
quite efficient, with an IC50 value of 40 nM in the kinase assay
(Table 1). The small 2-fluoro moiety (11g) was moderately
active, with an IC50 value of 70 nM in the kinase assay. Larger
substituents like methoxy- (11d), chloro- (11e), or trifluor-
omethyl (11f) led to less active compounds. The unsubstituted
analogue 11h was moderately active, resulting in a 2-fold lower
potency compared to compound 11a. 2,4-Difluorophenylamino
derivative 11b was slightly superior to all target compounds,
and target compounds with 2-chloro-4-fluoro (11i) and 2-
methyl-4-fluoro-(11j)-substituents were good inhibitors of
p38α MAP kinase. Larger substituents in the para-position
were not tolerated very well, thus resulting in less active
compounds (for example, 2-amino-4-trifluoromethyl, 11k).
Furthermore, 3-methyl-4-fluoro- (11l) and 2-,3-,5-trifluoro-
substituents (11m) could be introduced without loss of activity,
whereas the 2-,3-,4-trifluoro- substitution (11n) led to a 50-fold
loss of activity as compared to 11m. Because the gatekeeper
Thr106 refuses access to the direct-linked phenyl-substituent to
the hydrophobic region I, replacement of the 2-phenylamino-
substituent with a phenyl-substituent (compound 12) resulted
in an almost complete loss of activity (32% inhibition at 10
μM). The target compound with an etheno-linker (13) also
resulted in a complete loss of activity.
a
Scheme 2. Suzuki Coupling
a
Reagents and conditions: Pd(OAc)2, K2CO3, PEG-400, 90 °C, 32%.
Starting from the commercially available methoxy-methyl-
benzoic acids 18a and 18b, the bromomethyl derivatives 19a
and 19b can be readily formed by treatment with N-
bromosuccinimide (NBS)/azo-isobutyronitrile (AIBN). Wittig
reaction provided the analogues 20a and 20b, and subsequent
hydrogenation of the unsaturated etheno-linker, using H2 and
either catalytic palladium on carbon (20a) or palladium on
BaSO4 (20b), afforded derivatives 21a and 21b. Ring closure
was performed using SOCl2/AlCl3, resulting in 8-methoxy-
dibenzosuberone 22a or 9-methoxy-dibenzosuberone 22b.
Treatment of the respective 2-chloro-methoxydibenzosuber-
ones (22a, 22b) with HBr/acetic acid18 proceeded in high
yields to give the corresponding 2-chloro-hydroxy-dibenzosu-
berones (23, 24).
Introduction of hydrophilic moieties (25, 26) and
Buchwald−Hartwig (27−30) reaction were performed as
described in Scheme 3.
Lead Compound Optimization and Biological Testing.
We started from substituted benzophenones as initial lead
structures which had been reported as inhibitors of p38α MAP
kinase in 2003 (Figure 3).21 We supposed that rigid inhibitors
To test the compounds under physiologically more relevant
conditions in the presence of serum albumin and other
proteins, inhibition of cytokine release (TNF-α) from immune
cells was observed using diluted fresh human whole blood.25
Compounds 11a, 11b, 11c, and 11l exhibited the best
potency in blocking the release of TNF-α. Most of the other
compounds (11d, 11e, 11h, 11i, 11j, 11m) were less potent in
this assay, with IC50 values of 3.2−7.6 μM. The lipophilic
compounds 11f, 11k, and 11n, which showed low activity in
the p38 MAP kinase activity assay, exhibited no activity in the
whole blood assay, with IC50 values in the double-digit μM
range. Cleavage of the methoxy-group resulted in more
hydrophilic 7-hydroxydibenzosuberones 14 (Table 1). The
IC50 values in the p38 MAP kinase activity assay were
comparable to the methoxy compounds. In the whole blood
C
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a
Scheme 3. Synthesis of 2-Chloro-8/9-hydroxydibenzosuberones
a
Reagents and conditions: (a) NBS, AIBN, chlorobenzene, 90−120 °C, 25−55%; (b) (1) P(Ph)3, acetone, reflux, (2) NaOMe (30%), MeOH, 3-
chlorobenzaldehyde, reflux, 28−48%; (c) H2/Pd/C, ethyl acetate, acetonitrile, MeOH room temperature, 83% (21a) or H2/Pd, acetonitril/ethyl
acetate/BaSO4, MeOH, HCl, room temperature, 87% (21b); (d) (1) SOCl2, DCM, (2) AlCl3, DCM, 47−90%; (e) HBr (48%), acetic acid, 91−
95%; (f) K2CO3, DMF, reflux, 94−98%; (g) PPh3, azodiisopropylcarboxylate, DCM, 0 °C, 45%; (h) K2CO3, DMF, 90 °C, 53−97%; (i) Pd(OAc)2,
X-Phos, NaO-tert-Bu, toluol, tert-BuOH, 90 °C, 20−62%.
assay, however, potency was improved by ∼1.7-fold over that
with the corresponding 7-methoxy variants, probably due to
lower lipophilicity. Because the higher hydrophilicity of the 2-
aminophenylamino-derivative (11a) resulted in activity in the
whole blood assay comparable to that of compound 11b, both
were selected for further optimization of the hydrophilic
moieties at the 7-position.
To optimize the physiological properties of the compounds,
docking studies were performed using the crystal structure of
the 2-aminophenyl-aminodibenzosuberone (PBD ID 3ZYA)13
as a template (Figure 4). The results from our docking studies
indicated that introduction of substituents in 7-, 8-, and 9-
position should be tolerated by the protein. In addition, these
moieties can be used to improve potency by providing
additional interaction possibilities to the protein backbone
proximal to the hydrophobic region II or to its surrounding
solvent-exposed area. Introduction of polar substituents should
improve water solubility as well as potency by providing
additional interaction possibilities. Orientation of the OH-
groups toward the surrounding solvent-exposed area may favor
the formation of additional hydrogen bonds and result in more
potent compounds. The hydrophobic region II is mostly
defined by the amino acids Val30, Gly110, Ala111, Arg49, and
Leu108. Except for Arg49, all side chains are hydrophobic (see
PBD 3ZYA). Therefore, compounds which are able to generate
hydrophobic interactions in this region were also synthesized to
evaluate whether this approach is valid to increase potency.
As small alkyl-moieties can be well incorporated into the
hydrophobic region II and form additional interactions, flexible,
linear hydroxyl-alkoxy moieties were first introduced. The 2,3-
dihydroxypropoxy moiety increased potency 2-fold in the
enzyme assay compared to the methoxy residue (11a). Potency
was increased ∼20-fold (15b vs 11a) in the whole blood assay,
indicating that these compounds are highly potent under
D
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Figure 3. Binding mode of 2-amino-phenyl-amino-benzophenone. Left: Schematic binding mode according to Ottosen et al.21 Middle: Schematic
binding mode suggested by our group. Right: Crystal structure of 2-amino-phenyl-amino-benzophenone in complex with p38α MAP kinase (PDB
ID 3QUD), confirming the binding mode suggested by our group.
physiological conditions. Acetal analogues with increased spatial
dimension were synthesized in order to obtain additional
hydrophobic interactions in the hydrophobic region II.
Furthermore, the acetal analogues (15a, 15c) provided only
hydrogen bond acceptors, whereas the dihydroxypropoxy
compounds could act as hydrogen bond donors as well as
acceptors. Although both compounds with acetal residues (15a,
15c) were comparable to the methoxy derivative (11a) in
respect to potency in the enzyme assay, they showed ∼8-fold
higher potency in whole blood. Morpholino-ethoxy- (15e)
provided a hydrogen bond donor (protonated nitrogen) and a
hydrogen bond acceptor (oxygen). The saturated ring offered
the possibility of additional hydrophobic interactions similar to
the acetals. The morpholino derivative was very potent both in
enzyme and whole blood assays with IC50 values in the low nM
range, exhibiting similar inhibitory efficiency as the 2,3-
dihydroxypropoxy target compounds.
Taken together, particularly the very hydrophilic derivatives
(dihydroxypropoxy and (15b, 15d)), were very potent, with
IC50 values in the double-digit nM range, suggesting them to be
promising drug candidates. Interestingly, the more lipophilic
acetals (15a, 15c) did not show improved potency in the
enzyme assay but displayed superior activity in whole blood
when compared to the methoxy derivatives.
Encouraged by these findings, we conducted a similar
optimization strategy for the 2,4-difluorophenylamino deriva-
tive 11b. We tested an even larger panel of candidate
compounds. To optimize the spacer length for the hydroxyl-
group, hydroxyethoxy- (16a) and hydroxypropoxy- (16b)
derivatives were synthesized. The potencies of both these
derivatives increased by 2−2.5-fold compared to the methoxy
derivative 11b. The spacer was made rigid by introducing a
tetrahydropyran-4-yl-moiety (16c); however, this change
abolished the increase in potency. This result indicated the
need for either a flexible, adaptable substituent or a hydrogen
bond donor functionality (which was lost in the tetrahydropyr-
an-4-yl ring). Both the 3-hydroxypropoxy and 2-hydroxyethoxy
moieties were beneficial for potency; therefore, we introduced a
2,3-dihydroxypropoxy moiety that combined both structural
features. 2,3-Dihydroxypropoxy-derivatives (2 = Skepinone-L,
16f) showed potencies similar to the monohydroxyalkoxy
compounds. The two configurations (2 vs 16f) were not
substantially different in potency. The crystal structure of
Skepinone-L (2) in complex with p38α MAP kinase revealed
that an additional hydrogen bond was formed between the
dihydroxypropoxy moiety and the backbone carbonyl of
Gly110.13 This additional interaction could explain the superior
binding affinity of the dihydroxyalkoxy-substituted dibenzosu-
berones (Skepinone-L, 2) compared to the methoxy derivative
(11b). The dihydroxybutanoxy derivative 16h was also
extremely potent, with an IC50 of 10 nM in the in vitro
enzyme assay. In contrast to 16c, all compounds with acetal
residues (16d, 16e, 16g) had potencies that were comparable
to that of 11b. The potencies increased according to the
sequence: 16d, 16e < 16g, which corresponded to increasing
compound flexibility. As observed in the diols (Skepinone-L,
2), the configuration did not affected potency (16d vs 16e).
Morpholino-ethoxy- (16i) and morpholino-propoxy- (16j)
derivatives were very potent, with IC50 values in the low nM
range; the inhibitory efficiency was similar to that observed for
the diol target compounds (Skepinone-L, 16f, 16h). Because
the hydrogen bond donor function in 16i and 16j was located
at the same distance as the OH functions of the alcohol
moieties, the arrangements of these residues were expected to
be broadly similar to the arrangements observed in the alcohols
and to form a hydrogen bond with Gly110, like the interaction
revealed in the X-ray structure of Skepinone-L (2, Figure 1).13
In addition to the gain in potency offered by these additional
interactions, we reasoned that disubstituted compounds might
also benefit from higher hydrophilicity. Indeed, in the whole
blood assay, the potency of the disubstituted compounds
(Skepinone-L, 16f, 16i) showed an overall improvement of
∼15−40-fold compared to compound 11b. The potencies of
these compounds decreased in the order from monohydrox-
yalkoxy compounds 16a and 16b to acetals and tetrahydropyr-
anyl species (16c, 16d, 16e) according to their increases in
lipophilicity.
Both series of compounds (2-aminophenylamino-derivatives,
Table 2 and 2,4-difluorophenylamino-derivatives, Table 3)
showed comparable results. Small flexible substituents with
polar groups were more advantageous than lipophilic moieties,
especially if they were conformational hindered. Generally,
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corresponding phenylamino derivatives (17a vs 11b, 17c vs
16f, 17d vs 16i) . We ascribe this decrease in activity to the loss
of a hydrogen bond. The crystal structure of Skepinone-L (2)13
suggests that the phenylamino nitrogen might form a (water-
mediated) hydrogen bond to Asp169, which is located in an
allosteric pocket of the enzyme. This interaction could be lost
due to the exchange of the nitrogen atom for oxygen. Therefore
compounds 17a, 17c, and 17d support this mode of binding
(Table 4). As expected, potency in whole blood likewise
decreased. We attributed this loss of potency in the ex vivo
assay to the decreased hydrophilicity.
Table 1. Biological Activity of Different Phenylamino-
Substituted 7-Methoxy- and 7-Hydroxy-dibenzosuberones
Dibenzosuberones with moieties in the 8- and 9- positions
(Table 5) were slightly less active in the isolated p38α MAP
kinase assay than the corresponding derivatives with residues at
position 7. The SARs of the different residues were similar to
the ones of the 7-position. In contrast to this, potency in the
whole blood assay dramatically decreased. There are several
possible explanations for this phenomenon. The position of the
residues might alter the binding to plasma proteins or may
prevent the inhibitor from crossing through the cell
membranes. The participation of a specific membrane trans-
porter could also play a role in this process.
Kinase Selectivity Profiling. After we improved potency
in the ex vivo whole blood assay, we had to confirm that
introduction of residues at position 7/8/9 of the dibenzosuber-
one scaffold was not detrimental to the selectivity of the
compounds. Therefore selectivity profiling of 32 compounds
against a panel of 16 different kinases (Table 6) was performed
by an independent laboratory.27 All compounds tested revealed
no significant inhibition of the 16 tested kinases, even at a
concentration as high as 10 μM. As this result suggested high
selectivity of the whole structural class, we tested Skepinone-L
(2) against a panel of 402 kinases.13 Only p38α and p38β MAP
kinase were inhibited in a significant manner.
CONCLUSION
■
In summary, we have synthesized a large number of
disubstituted phenylamino-dibenzosuberones. The recently
published cocrystal structure of 2-(2-aminophenylamino)-
dibenzosuberone with p38 MAP kinase13 guided our efforts
to improve potency as well as to select the physicochemical
properties of our initial lead compound. Specifically, hydro-
philic moieties at the 7-position resulted in highly potent
compounds both in the p38 MAP kinase enzyme assay and in
the whole blood assay with IC50 values in the single-digit
(enzyme assay) and double-digit (whole blood assay) nM
range. The use of the dihydroxypropoxy and the morpholin-4-
yl-ethoxy moiety resulted in molecules with excellent drug-like
properties while increasing potency toward p38α MAP kinase.
Kinase selectivity profiling of 32 compounds against a panel of
16 kinases suggested the whole structural class to be highly
selective. Our efforts finally yielded Skepinone-L (2), which is
highly potent and shows an outstanding selectivity, both in
vitro and in vivo.13 Therefore it is an excellent tool which
should lead to a better understanding of p38α MAP kinase and
may foster the next generation of anti-inflammatory drugs.
compounds of the 2,4-difluorophenylamino series are more
potent than the corresponding inhibitors of the 2-amino-
phenylamino series (15e vs 16i) in the enzyme activity assay
but are less active in whole blood, probably due to the higher
hydrophilicity of the amino residue. These differences are only
minor as the activities of both compounds are very high.
In addition to the series shown in Table 1 and Table 2,
several phenoxy-substituted dibenzosuberones (Table 3) were
also synthesized. All of them were less active than their
EXPERIMENTAL SECTION
■
General. 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). Melting points were determined with
a Buchi Melting Point B-545 melting point apparatus, and they were
̈
F
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Figure 4. Suggested binding mode of selected dibenzosuberones. The molecules have been docked in the p38 MAP kinase active center by using the
Induced Fit docking tool.26 As a protein model, the X-ray structure of the 2-aminophenylaminodibenzosuberone (1) has been used. (A) Binding
mode of compound 1. (B) Binding mode of compound 2. (C) Binding mode of compound 27g. (D) Binding mode of compound 29f.
Advance 200 MHz instrument using TMS as internal standard. The
chemical shifts were reported in ppm. The purity of the final
compounds was determined by HPLC on a (method 1) Merck HPLC
Table 2. Biological Activity of 2-Aminophenylamino-
Substituted Dibenzosuberones with Hydrophilic Moieties in
the 7-Position
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) employing a gradient of 0.01 M
KH2PO4 (pH 2.3) and methanol as the solvent system with a flow rate
of 1.0 mL/min (method 1) or 1.5 mL/min (method 2). All final
compounds have a purity of >95% (purity 17d = 94.1).
General Procedure E. A mixture of the corresponding
dibenzsouberone, aniline deriviative, Pd(OAc)2, 2-(dicyclohexylphos-
phino)-2′-,4′-,6′-triisopropyl-biphenyl, KOtert-Bu/NaOtert-Bu, tol-
uene, and tert-BuOH was heated at 90 °C under argon. After stirring
at this temperature for 2 h, the reaction mixture was poured into water
and extracted with 3 × 200 mL ethyl acetate. The residue was purified
by flash chromatography.
General Procedure F. Water and p-toluenesulfonic acid
monohydrate were added to a solution of the acetal in methanol.
The solution was warmed to 50 °C under an argon atmosphere. After
6 h, the reaction mixture was cooled to room temperature and
concentrated in vacuo to a yellow oil. This oil was redissolved in ethyl
acetate and 5% sodium bicarbonate solution (50 mL). The organic
layer was separated, dried over magnesium sulfate, and concentrated in
vacuo. Recrystallization from dichloromethane/hexane gave the pure
diol.
General Procedure H. A mixture of the corresponding
dibenzsouberone, aniline deriviative, Pd(OAc)2, 2-di-tert-butylphos-
phino-2′,4′,6′-triisopropylbiphenyl, K3PO4, and toluene was heated at
90 °C under argon. After stirring at this temperature for 30 min, the
reaction mixture was poured into water and extracted with 3 × 200 mL
ethyl acetate. The residue was purified by flash chromatography.
2-(2-Aminophenylamino)-7-methoxy-10,11-dihydro-
dibenzo[a,d]-cyclohepten-5-one (11a). 11a was synthesized
according to general procedure E using 0.52 g (1.9 mmol) of 7,
1.00 g (9.2 mmol) of 1,2-phenylenediamine, 0.05 g of Pd(OAc)2, 0.15
g of 2-(dicyclohexylphosphino)-2′-,4′-,6′-triisopropyl-biphenyl, and
1.60 g (17.0 mmol) of NaOtert-Bu in 10 mL of toluene and 2 mL
of tert-BuOH. The crude product was purified by flash chromatog-
raphy (SiO2; hexane/ethyl acetate 8 + 2). Yield 78%; purity ≥95%; mp
106,5 °C. IR (cm−1) 3418−3343 (C−H)arom, 3320 (N−H), 2976−
2939 (C−H), 1546, 1516, 1494, 1323, 1284, 1147, 1026, 828, 751,
thermodynamically corrected. IR data were recorded with a Perkin-
Elmer Spectrum One (ATR technique) spectrometer. Mass spectra
were acquired on a Hewlett-Packard HP 6890 Series GC system
equipped with a HP-5MS capillary column (0.25 μm film thickness, 30
m × 0.25 mm i.d.) (Hewlett-Packard) and a HP 5973 mass selective
detector (70 eV) (Hewlett-Packard). High-resolution measurements
(Fourier transform ion cyclotron resonance) were obtained on a
1
Bruker APEX II with electron spray ionization. H NMR (200 MHz)
and 13C NMR (50 MHz) spectroscopy was performed on a Bruker
740. H NMR (DMSO-d6) δ in ppm: 2.96 (s, 4 H, −CH2−CH2−),
1
G
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Table 3. Biological Activity of 2,4-Difluorphenylamino-
Substituted Dibenzosuberones with Hydrophilic Moieties in
the 7-Position
Table 4. Biological Activity of Phenoxy-Substituted
Dibenzosuberones with Hydrophilic Moieties in the 7-
Position
16d was synthesized according to general procedure E using 0.67 g
(1.8 mmol) of 10d, 0.25 g (1.9 mmol) of 2,4-difluoroaniline, 0.05 g
(0.22 mmol) of Pd(OAc)2, 0.10 g (0.21 mmol) of 2-(dicyclohex-
ylphosphino)-2′-,4′-,6′-triisopropyl-biphenyl, and 0.60 g (6.2 mmol) of
NaOtert-Bu in 10 mL of toluene and 2 mL of tert-BuOH. The crude
product is purified by flash chromatography (SiO2; hexane/ethyl
acetate 7 + 3). Yield 0.57 g (68%); purity ≥95%; mp 114.6 °C. IR
(ATR) (cm−1): 3352 (N−H), 2997−2944 (C−H), 1560, 1509, 1289,
1262, 1140, 843, 814, 802. 1H NMR (DMSO-d6) δ in ppm: 1.29 (s, 3
H, −CH3), 1.34 (s, 3 H, −CH3), 3.31 (s, 4 H, −CH2−CH2−), 3.72−
4.45 (m, 5 H, C1/3glyceryl−H, C2isopropyliden−H), 6.60 (s, 1 H, C1−H),
6.73 (d, 1 H, J = 9.3 Hz, C3−H), 7.00−7.47 (m, 6 H, C6−H, C8−H,
C9−H, C3′−H, C5′−H, C6′−H), 7.94 (d, 1 H, J = 8.6 Hz, C4−H), 8.54
(s, 1 H, −NH−). 13C NMR (DMSO-d6) δ in ppm: 25.8 (−CH3), 26.9
(−CH3), 33.5 (C11), 36.2 (C10), 66.0 (C3glyceryl), 69.2 (C1glyceryl), 74.1
(C2glyceryl), 105.3 (dd, 1 C, J1 = 24.2 Hz, J2 = 26.6 Hz, C3′), 109.2
(C2isopropyliden), 112.2 (dd, 1 C, J1 = 3.9 Hz, J2 = 22.0 Hz, C5′), 112.6
(C6), 113.9 (C3), 115.4 (C1), 119.1 (C8), 125.2 (dd, 1 C, J1 = 3.5 Hz,
J2 = 12.1 Hz, C1′), 126.5 (dd, 1 C, J1 = 3.5 Hz, J2 = 9.8 Hz, C6′), 127.7
C4a), 130.6 (C9), 133.8 (C5a), 134.9 (C4) 140.1 (C9a), 145.7 (C11a),
149.5 (C2), 156.0 (dd, J1 = 12.6 Hz, J2 = 247.9 Hz, C4′), 157.2 (C7),
158.8 (dd, J1 = 11.5 Hz, J2 = 243.0 Hz, C2′), 190.7 (C5).
2-(2,4-Difluorophenylamino)-7-[2R-,3-dihydroxypropoxy]-
10,11-dihydro-dibenzo[a,d]-cyclohepten-5-one (2). 2 was syn-
thesized according to general procedure F using 1.0 g (2.1 mmol) of
16d, 0.25 g (1.3 mmol) of p-toluensulfonic acid monohydrate, 40 mL
of methanol, and 10 mL of H2O. Yield 0.85 g (95%); purity ≥95%; mp
130.3 °C. IR (ATR) (cm−1): 3305 (N−H), 2934 (C−H), 1629, 1607,
1569, 1510, 1327, 1278, 1218, 1097, 847, 781. 1H NMR (DMSO-d6) δ
in ppm: 3.32 (s, 4 H, −CH2−CH2−), 3.40−3.46 (m, 2 H,
−C1/3propoxy−H), 3.77−4.05 (m, 3 H, C1//2/3propoxy−H), 4.66 (t, 1 H,
J = 5.7 Hz, −OH), 4.94 (d, 1 H, J = 4.9 Hz, −OH), 6.60 (s, 1 H, C1−
H), 6.73 (d, 1 H, J = 8.9 Hz, C3−H), 7.01−7.10 (m, 2 H, C3′−H, C6′−
H), 7.22 (d, 1 H, J = 8.3 Hz, C4′−H), 7.30−7.47 (m, 3 H, C6−H, C8−
H, C9−H), 7.94 (d, 1 H, J = 8.8 Hz, C4−H), 8.54 (s, 1 H, −NH−).
13C NMR (DMSO-d6) δ in ppm: 33.6 (C11), 36.3 (C10), 63.1
3.76 (s, 3 H, −OCH3), 4.84 (s, 2 H, −NH2), 6.46 (s, 1 H, C1−H),
6.54−6.64 (m, 2 H, C4′−H, C5′−H), 4.78 (d, 1 H, J = 7.8 Hz, C3−H),
6.91−7.04 (m, 3 H, C8−H, C3′−H, C6′−H), 7.20 (d, 1 H, J = 8.3 Hz,
C9−H), 7.39 (s, 1 H, C6−H), 7.92−7.97 (m, 2 H, C4−H, −NH−).
13C NMR (DMSO-d6) δ in ppm: 33.6 (C10), 36.6 (C11), 55.5
(C3propoxy), 70.2 (C1propoxy), 70.3 (C2propoxy), 105.3 (dd, 1 C, J1 = 24.1
Hz, J2 = 27.0 Hz, C3′), 112.2 (dd, 1 C, J1 = 3.8 Hz, J2 = 22.5 Hz, C5′),
112.6 (C6), 113.8 (C3), 115.4 (C1), 119.1 (C8), 125.3 (dd, 1 C, J1 =
3.4 Hz, J2 = 11.9 Hz, C1′), 126.4 (dd, 1 C, J1 = 3.1 Hz, J2 = 9.8 Hz,
C6′), 127.7 (C4a), 130.6 (C9), 133.8 (C5a), 134.6 (C4), 139.9 (C9a),
145.7 (C11a), 149.4 (C2), 156.0 (dd, 1 C, J1 = 12.6 Hz, J2 = 248.5 Hz,
C4′), 157.6 (C7), 158.8 (dd, 1 C, J1 = 12.3 Hz, J2 = 243.8 Hz, C2′),
190.8 (C5).
́
(−OCH3), 112.1 (C6), 113.1 (C3), 114.8 (C1), 115.8 (C3), 116.8
(C4′), 118.3 (C8), 125.2 (C5′), 126.2 (C6′), 126.3 (C1′), 126.4 (C4a),
130.4 (C9), 133.9 (C5a), 134.5 (C4), 140.3 (C9a), 144.0 (C2′), 145.7
(C11a), 151.1 (C2). 158.0 (C7), 190.3 (C5).
2-(2,4-Difluorophenylamino)-7-(S-1,2-isopropylidenglycer-
3-yl)-10,11-dihydro-dibenzo-[a,d]-cyclohepten-5-one (16d).
H
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Journal of Medicinal Chemistry
Article
(m, 4 H, −CH2−CH2−), 3.77 (s, 3 H, −OCH3), 6.84−6.93 (m, 2 H,
C3′/5′−H), 7.08 (dd, 1 H, J1 = 2.9 Hz, J2 = 8.3 Hz, C3−H), 7.12−7.29
(m, 2 H, C1/6′−H), 7.34−7.59 (m, 3 H, C8/6/9−H), 7.95 (d, 1 H, J =
9.5 Hz, C4−H). 13C NMR (DMSO-d6) δ in ppm: 33.4 (C10), 35.1
(C11), 55.6 (−OCH3), 106.3 (dd, J1 = 22.5 Hz, J2 = 27.4 Hz, C3′),
112.8 (dd, J1 = 3,8 Hz, J2 = 22.9 Hz, C5′), 114.2 (C3), 114.3 (C6),
116.4 (C1), 119.4 (C8), 124.8 (dd, J1 = 1.9 Hz, J2 = 9.9 Hz, C6′), 131.2
(C9), 132.8 (C4a), 133.8 (C4), 134.7 (C5a), 138.2 (dd, J1 = 3,6 Hz, J2 =
12.0 Hz, C1′) 139.1 (C9a), 145.8 (C11a), 154.2 (dd, J1 = 13.0 Hz, J2=
248.0 Hz, C2′), 158.1 (C7), 159.3 (dd, J1 = 10.7 Hz, J2 = 242.7 Hz,
C4′), 160.7 (C2), 192.4 (C5).
Table 5. Biological Activity of Different Phenylamino-
Substituted Dibenzosuberones with Hydrophilic Moieties at
the 8-Position and 9-Position
2-(2,4-Difluorophenylamino)-8-methoxy-10,11-dihydro-
dibenzo[a,d]cyclohepten-5-one (27a). 27a is synthesized accord-
ing to general procedure E using 0.52 g (1.9 mmol) of 22a, 0.30 g (2.3
mmol) of 2,4-difluoroaniline, 0.04 g of Pd(OAc)2, 0.16 g of 2-
(dicyclohexylphosphino)-2′-,4′-,6′-triisopropyl-biphenyl, and 0.86 g
(8.9 mmol) of NaOtert-Bu in 10 mL of toluene and 2 mL of tert-
BuOH. The crude product is purified by flash chromatography (SiO2;
1
hexane/ethyl acetate 9 + 1); mp 157 °C. H NMR (DMSO-d6) δ in
ppm: 3.02 (dd, 4H, J1 = 13.73 Hz, J2 = 8.96 Hz, −CH2−CH2−), 3.82
(s, 3H, −OCH3), 6.61 (s, 1H, C1−H), 6.73 (d, 1H, J = 8.64 Hz, C3−
H), 6.84−6.92 (m, 2H, C3′−/C6′−H), 7.04−7.13 (m, 1H, C5′−H),
7.34−7.42 (m, 2H, C7−/C9−H), 7.96 (d, 2H, J = 8.64 Hz, C4−/C6−
H), 8.49 (s, 1H, −NH−). 13C NMR (DMSO-d6) δ in ppm: 35.2
(C11), 35.8 (C10), 55.7 (−OCH3), 105.3 (dd, J1 = 26.5 Hz, J2 = 24.0
Hz, C3′), 112.1 (dd, J1 = 21.5 Hz, J2 = 3.7 Hz, C5′), 112.5 (C7), 112.7
(C9), 113.6 (C3), 113.9 (C1), 125.4 (dd, J1 = 12.1 Hz, J2 = 3.6 Hz, C1′),
126.3 (dd, J1 = 9.1 Hz, J2 = 3.3 Hz, C6′), 128.2 (C4a), 131.3 (C5a),
133.7 (C4), 133.8 (C6), 145.1 (C2), 145.3 (C11a), 149.1 (C9a), 155.9
(dd, J1 = 246.4 Hz, J2 = 12.5 Hz, C4′), 158.7 (dd, J1 = 241.6 Hz, J2 =
11.4 Hz, C2′), 162.4 (C8′), 189.1 (C5).
ASSOCIATED CONTENT
■
S
* Supporting Information
1
Crystal structure of compound 7, synthetic procedures, H
NMR, 13C NMR, HPLC, HRMS, IR, melting points molecular
modeling, eExpression and purification of p38α, crystallization
and structure determination of p38α in complex with 2-amino-
phenylamino-benzophenone. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +49-7071-2972459. Fax: +49-7071-295037. E-mail:
stefan.laufer@uni-tuebingen.de.
2-(2,4-Difluoro-phenoxy)-7-methoxy-10,11-dihydro-
dibenzo[a,d]cyclohepten-5-one (17a). 17a was synthesized
according to general procedure H using 0.5 g (1.8 mmol) of 2-
chloro-7-methoxy-10,11-dihydro-dibenzo[a,d]cyclohepten-5-one (7),
0.28 g (2.2 mmol) of 2,4-difluorophenol, 0.013 g (0.058 mmol) of
Pd(OAc)2, 0,024 g (0.057 mmol) of 2-di-tert-butylphosphino-2′,4′,6′-
triisopropylbiphenyl, 0.78 g (3.7 mmol) of K3PO4, and 10 mL of
toluene. The crude product is purified by flash chromatography (SiO2;
hexane/ethyl acetate 7 + 3). Yield (39%); purity ≥95%; mp 113 °C. IR
(ATR) (cm−1): 3054, 2906, 1501, 1423, 1293, 1248, 1236, 1186, 1136,
Author Contributions
∥These authors contributed equally to the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank S. Luik, M. Goettert, and K. Bauer for biological
testing.
1
1090, 961, 890, 834, 786. H NMR (DMSO-d6) δ in ppm: 2.98−3.13
Table 6. Kinases and Target Compounds Tested for Selectivity by an Independent Laboratory
AKT1
SAK
ARK5
TRK-B
Aurora-B
SRC
B-RAF VE
VEGF-R2
INS-R
IGF1-R
MET
FAK
PLK1
AXL
PRK1
CK2-alpha1
kinase no.
11e
15a
16j
11f
15b
27d
29f
11i
11j
16a
27f
30b
11k
16b
27g
30c
11l
16c
29a
11m
16d
29b
12
13
15e
27e
30a
16f
29c
16g
29d
29e
I
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