Biphenyl amide p38 kinase inhibitors 4: DFG-in and DFG-out binding modes

Article abstract of DOI:10.1016/j.bmcl.2008.06.028

The biphenyl amides (BPAs) are a series of p38α MAP kinase inhibitors. Compounds are able to bind to the kinase in either the DFG-in or DFG-out conformation, depending on substituents. X-ray, binding, kinetic and cellular data are shown, providing the most detailed comparison to date between potent compounds from the same chemical series that bind to different p38α conformations. DFG-out-binding compounds could be made more potent than DFG-in-binding compounds by increasing their size. Unexpectedly, compounds that bound to the DGF-out conformation showed diminished selectivity. The kinetics of binding to the isolated enzyme and the effects of compounds on cells were largely unaffected by the kinase conformation bound.

Full text of DOI:10.1016/j.bmcl.2008.06.028

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4433–4437
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Biphenyl amide p38 kinase inhibitors 4: DFG-in and DFG-out binding modes
*
Richard M. Angell , Tony D. Angell, Paul Bamborough , Mark J. Bamford, Chun-wa Chung,
Stuart G. Cockerill , Stephen S. Flack , Katherine L. Jones, Dramane I. Laine, Timothy Longstaff,
Steve Ludbrook, Rosannah Pearson, Kathryn J. Smith, Penny A. Smee, Don O. Somers, Ann L. Walker
GlaxoSmithKline R&D, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The biphenyl amides (BPAs) are a series of p38a MAP kinase inhibitors. Compounds are able to bind to
the kinase in either the DFG-in or DFG-out conformation, depending on substituents. X-ray, binding,
kinetic and cellular data are shown, providing the most detailed comparison to date between potent com-
pounds from the same chemical series that bind to different p38a conformations. DFG-out-binding com-
pounds could be made more potent than DFG-in-binding compounds by increasing their size.
Unexpectedly, compounds that bound to the DGF-out conformation showed diminished selectivity.
The kinetics of binding to the isolated enzyme and the effects of compounds on cells were largely unaf-
fected by the kinase conformation bound.
Received 9 May 2008
Revised 6 June 2008
Accepted 6 June 2008
Available online 12 June 2008
Keywords:
p38 Kinase inhibitors
MAP kinase
Biphenyl amide
Protein kinase X-ray structure
Binding mode
DFG-out
Ó 2008 Elsevier Ltd. All rights reserved.
Selectivity
Kinetics
The biphenyl amides (BPAs) are a novel series of p38a MAP ki-
nase inhibitor.1 Replacing the oxadiazole moiety found in the ear-
liest examples (as in 1) with amides led to compounds with
improved enzyme, cellular and in vivo activity, for example, 2.2,3
Me
Me
a
O
O
5'
NH2
N
H
N
H
An X-ray structure of p38
a complexed with 2 showed binding to
NH
A
the apo-like (or ‘DFG-in’) conformation.3
O
b
Me
O
N
Me
Me
Cl
O
O
5'
NH
N
H
N
N
H
H
O
H
O
1
N
2
NC
O
N
Me
N
N
R1R2N
Prior to the publication of the X-ray structure of p38
BIRB-796, and of Abl with Gleevec, compounds had been prepared
a with
Scheme 1. Intermediate A was prepared as previously described.3 Reagents: (a) 2-
chloropyridine-4-carbonyl chloride, NEt3, DCM, 33%. (b) Amine, heat.
within GSK which revealed the possibility of binding to a rear-
ranged (‘DFG-out’) form of p38a 4–11
Large substituents were
.
attached to the BPA template with the aim of exploiting the
DFG-out pocket. Initial work focused on anilides (4–15), prepared
from the 50-aniline position (Scheme 1).
The phenyl-substituted anilide 3, with p38
a Ki of 12 nM, pro-
vides a benchmark for the activity of 4–15 (Table 1).3 4-Amido
pyridines with small 2-substituents such as 4 were weakly active.
Activity was increased by the introduction of moderate bulk at the
2-position (5). Many compounds with increased bulk at this posi-
tion, particularly aliphatic rings, gave greater activity (6–14), espe-
cially the cyclobutyl amide 13. Cationic charge in this region was
not tolerated (compare 15 to 9).
* Corresponding author. Tel.: +44 1438 763246; fax: +44 1438 763352.
E-mail address: paul.a.bamborough@gsk.com (P. Bamborough).
Present address: Arrow Pharmaceuticals, Britannia House,
London SE1 1DB, UK.
7 Trinity Street,
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.028

4434
R. M. Angell et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4433–4437
Table 1
Table 3
Ki (nM), MW and BEI of pyridinyl anilides12
Ki (nM), MW and BEI of aryl benzamides12
Me
Me
Me
Me
NC
O
O
O
4'
O
5'
N
N
H
N
H
3
N
H
H
H
N
H
NH
NH
N
O
O
O
O
3
4-15
20-21
22-24
2
R
R
R
N
Compound
R
Ki
MW
BEI
Compound
R
Ki
MW
BEI
20
21
22
23
24
H
73
1.9
11
79
730
384
441
509
489
462
19
20
16
15
13
12
23
33
4
5
6
7
8
9
10
11
12
13
14
15
n/a
n/a
n/a
Cl
240
12
12
150
14
1.9
2.5
4.4
15
5.2
6.3
4.6
0.6
3.0
300
394
348
384
420
429
455
469
471
483
441
455
457
455
483
484
17
23
21
16
18
19
18
18
16
19
18
18
20
18
13
3-tBu
3-(2-Pyridyl)
3-(NHCOMe)
4-OMe
NMe2
1-Pyrrolidine
1-Piperidine
1-Morpholine
(4-Methyl)-1-piperidine
NH-cyclopropyl
NH-methylcyclopropyl
NH-isobutyl
NH-cyclobutyl
NH-cyclohexyl
(4N-methyl)-1-piperazine
The crystal structure of p38a complexed with 8 was solved and
found to adopt a rearranged DFG-out conformation.14 The back-
bone around the DFG motif reorganises in conjunction with the
activation loop. The Phe169 sidechain moves some 12 Å relative
to the complex with 2, to a new location around the ATP-site sugar
pocket. Figure 1 shows the binding site in the region of the DFG-
out pocket. The morpholine ring of 8 fills the lipophilic space that
is occupied by Phe169 in the complex with compound 2 (the DFG-
pocket). It makes direct contact with the sidechains of residues
including Leu74, Met78, Val83, Ile141 and Ile166. The motion of
Phe169 out of this pocket is accompanied by a small inward move-
ment of Met78 towards the inhibitor morpholine. Figure 1 also
shows the details of the hydrogen-bonding interactions between
the protein and 50-amide of 8. Two hydrogen-bonds are conserved
in 2 and 8, between the backbone NH of Asp168 and the amide car-
bonyl, and between the Glu71 sidechain and the amide NH.
The interactions of the hinge-binding parts of 8 and 2 with the
protein are similar, but the biphenyl amides of 8 and 2 are shifted
relative to one another (Fig. 2). The backbone around Met109 and
Gly110 in the complex with 8 has some features of the apo-like
conformation seen with 2 and some of the flipped conformation
seen with 1.1,3 In complex with 8, the carbonyl of Met109 points
towards the ATP-site as in the apo structure. However, the overall
shape of the hinge is more like that seen with 1 than that with 2.
The movement of the hinge-binding amide of 8 relative to 2 prob-
Substituted phenyls showed similar trends to the pyridines (Ta-
ble 2).13 Compared to the unsubstituted phenyl 3, and the furan 16,
even small 4-substituents led to reduced activity (17). Acids cho-
sen to introduce bulky groups meta to the amide (e.g., 18, 19) in-
creased the activity.
The same trends were seen in the benzamide series (Table 3).13
Although the phenyl 20 was less potent than the corresponding
anilide 3, introduction of bulky meta substituents (21) gave the
same increase in activity.
The 40-amide group could be varied using previously described
chemistry.13 The cyanophenyl was known to give good potency in
DFG-in binding BPAs.3 With the same 40-group, 22 showed good
activity (Table 3). The relatively poor inhibition of the 3-acetamide
analogue (23) illustrates again the importance of a meta group with
sufficient size and lipophilicity. As in the anilide series, para substi-
tution (24) reduced the potency.
Table 2
Ki (nM), MW and BEI of aryl anilides12
Me
O
N
H
NH
O
R
Compound
R
Ki
MW
BEI
33
Phenyl
3-Furan
4-Methyl phenyl
12
15
100
384
374
399
21
21
18
163
17
18
19
N
2.1
1.6
478
451
18
20
*
Figure 1. Overlaid p38a X-ray structures of 8 (green) and 2 (orange), focusing on
O
the part of the inhibitors around the DFG-pocket. H-bonds are shown as dotted
lines, in cyan from 8 and magenta from 2. The arrow shows the movement of
Phe169.
*

R. M. Angell et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4433–4437
4435
It is difficult to obtain accurate Ki values for compounds with
very slow kinetics in the catalytic activity assay. Comparative di-
rect binding data for 2, 8 and BIRB-796 to immobilised unphos-
phorylated p38
a
were obtained using surface plasmon
resonance.16 Both on- and off-rates of 8 are slightly slower than
those of 2, but both are much faster than BIRB-796 (Fig. 4). The rate
constants (Table 4) are comparable to published values for the lit-
erature compounds.7,8,17
It has been suggested that the slow association is rate-limited
by structural reorganisation of p38a from DFG-in to DFG-out,
influenced by features of the inhibitor that hinder access to the
bioactive conformation.6,7 However, despite the similarity of the
protein structures in complex with 8 and BIRB-796, 8 shows a
much more rapid on-rate. This suggests that there may be a barrier
due to conformational changes in the protein, but this is relatively
minor compared to the effects of the characteristics of the inhibi-
tor. One possible explanation is that the ATP-site part of 8 is less
flexible, and so may spend more time in its bioactive conformation.
Whilst a slow on-rate is unlikely to be beneficial, a slow off-rate
may be. BIRB-796 has a very slow koff (Fig. 4). The off-rate of BIRB-
796 depends largely on the interactions between protein and
ligand in the bound state.7 For example, replacing the tolyl ring
of BIRB-796 with methyl led to a 400-fold increase in koff. This is
consistent with our results. 8 binds in the DFG-out mode but does
not fill the tolyl space (Fig. 3) and has a faster koff. More extensive
interactions with the site might decrease the dissociation rate.
Figure 2. Overlaid X-ray structures of 8 (green) and 2 (orange), focusing on the part
of the inhibitors around the hinge region. H-bonds to the hinge are shown as dotted
lines.
ably results from the DFG-out position of Phe169, which would
clash with 2 in the overlaid structures.
The binding modes of BIRB-796 and 8 are shown inFigure 3. The
binding interactions of 8 are comparable to this and other pub-
lished DFG-out crystal structures.4 The amide of 8 makes the same
interactions to Asp168 and Glu71 as the urea of BIRB-796, and the
morpholine occupies the same pocket as the tert-butyl group of
BIRB-796. The SAR in Tables 1–3 can be explained as follows.
Groups with small amide substituents (like 2, 3 and 16) are highly
Given the lipophilic nature of the p38a active site, this is likely
to require compounds with increased molecular weight.
In consequence, DFG-out BPA compounds can reach higher po-
tency than DFG-in compounds, but they are larger molecules, with
MW ꢀ500, regarded as disadvantageous for oral drugs.18 Molecular
weight and BEI, a measure of binding efficiency, are shown in
Tables1–3 and 5.12 DFG-out compounds do not gain enough activ-
ity from their extra mass to give them the efficiency of potent
DFG-in compounds (compare compound 2 to 13).
potent and bind to p38a in the DFG-in conformation. Compounds
with large meta-substituted aryl amides (e.g. 8) would clash with
Phe169 in this conformation. Instead, they take advantage of the
alternative DFG-out conformation. Compounds such as 4 are of
intermediate size and are weaker inhibitors than smaller or larger
ones. In the DFG-in conformation, they are large enough to clash
with Phe169, but not large enough to fill the pocket vacated by
Phe169 in the DFG-out conformation. One interpretation is that
adopting the DFG-out conformation incurs an energetic penalty
which can only be overcome by making significant lipophilic inter-
actions with the Phe-out pocket.
1
0.8
0.6
0.4
0.2
0
As well as being competitive with the fluorescent ATP-site li-
gand used to generate the SAR reported here, compounds 2 and
8 were both competitive with ATP in an assay measuring the cata-
lytic activity of activated p38a, with Ki of 9 and 6 nM, respec-
tively.15 In the same assay, the Ki of BIRB-796 decreased 2.5-fold
from 46 to 19 nM after 90 min of compound preincubation, indi-
cating a slow on-rate, consistent with the literature.4,8 Compound
8 showed no time-dependence under the same conditions.
0
100
Time in seconds
200
300
Figure 4. Normalised sensorgrams of compounds 2 (blue), 8 (red) and BIRB-796
(black) at 3.7
M.16 Compounds were injected at 0 s. The traces from 0 to 90 s show
l
the association phase. Compound 2 shows the most rapid on-rate, closely followed
by 8, whilst BIRB-796 barely reached equilibrium during the 90 s. The traces from
90 to 300 s show the dissociation phase of these compounds and a similar hierarchy
in off-rates, 2 > 8 ꢁ BIRB-796.
Table 4
On- and off-rates from SPR measurements16
Compound
DFG
kon (Mꢂ1 sꢂ1
)
koff (sꢂ1
)
koff/kon (nM)
SB-203580
2
8
In
In
Out
Out
1.1 ꢃ 106
5.8 ꢃ 106
1.8 ꢃ 106
3.5 ꢃ 104
0.016
0.023
0.0084
15
4
5
BIRB-796
<0.00001
<0.5
Figure 3. p38a X-ray structures of 8 (green) and BIRB-796 (orange).

4436
R. M. Angell et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4433–4437
All reported crystal structures of p38
a
have used inactive pro-
LOK or RAF1. BIRB-796 reportedly inhibited STK10, the JNKs,
p38
, SLK, TEK and TNIK with sub-micromolar Kd.26 Whilst signif-
icant activity against Lck and JNK was not seen with 7, some other
tein, unphosphorylated on T180 and Y182. The DFG-out conforma-
tion appears to be incompatible with phosphate transfer, and so
has been described as an ‘inactive conformation’.19 Nevertheless,
c
DFG-out BPAs did show these activities. This complex profile con-
BIRB-796 binds to phosphorylated and unphosphorylated p38
a
,
trasts with that of the selective DFG-in p38a/b inhibitor 2, which
suggesting that both states of the kinase sample the DFG-out con-
has shown no significant inhibition of any other kinase.3
formation.8 The ability of compounds to bind to p38
a
and block its
It is sometimes stated that binding to a DFG-out kinase confor-
mation can be used to gain improved selectivity. This assertion was
supported by screening data from relatively small numbers of
kinases, and relied on comparisons between different chemical
series. This made it difficult to distinguish between differences
caused by the binding mode and those due to the compound clas-
ses. Incorporating a DFG-out binding group into a DFG-in template
changes its selectivity, as was found by others studying Abl inhib-
itors with DFG-in and DFG-out binding modes.19 However, these
changes are not always for the better. DFG-out BPAs were less
selective than DFG-in compounds when tested against an exten-
sive panel of kinases (Table 6).
activation has been reported in vitro and in cells.8,20–23 BIRB-796
has shown this activity in all studies. One interpretation is that
DFG-out compounds can lock p38a into a state that cannot be acti-
vated, giving them a potential advantage. However, results for
DFG-in compounds in the same studies have been inconsistent.
To investigate this, two DFG-in and two DFG-out BPAs were
tested in IL-1-stimulated HLF cells for their ability to inhibit phos-
phorylation of a downstream marker of the p38 pathway, Hsp27,
and of p38
a
itself.24 All compounds showed potent inhibition of
both readouts, as did two standards, BIRB-796 and SB-242235 (Ta-
ble 5).25 The ratio of the two readouts should normalise for com-
pounds with different cell permeability, and is comparable for all
compounds irrespective of their binding conformation. If anything,
In conclusion, potent biphenyl amides have been prepared.
Crystallography has been used to determine their binding mode
and to rationalise the activity in terms of protein conformational
flexibility. Minor changes in substitution determine whether
for reasons that are unclear, the BPAs inhibit p38
a phosphorylation
to a greater extent than Hsp27 phosphorylation. The effect on p38
a
phosphorylation differs from some published data, perhaps be-
cause of different cell types, stimuli or incubation times, but others
p38
a
is bound in the DFG-in or DFG-out conformation.
and Gleevec
The DFG-out binding modes of BIRB-796 to p38
a
have also found that DFG-in compounds can block p38
phosphorylation.21,23
a
to Abl provoked much interest in this conformation. Compari-
sons between these molecules and DFG-in inhibitors from differ-
ent series have revealed differences in selectivity, kinetic and
cellular properties. Whilst these may arise from their binding
modes, they could simply be properties of the different com-
pound classes. A cleaner comparison can be made between
DFG-in and DFG-out compounds from within one series, such
as the BPAs presented here. Both DFG-in and DFG-out biphenyl
Ki values for binding to the p38
a enzyme are compared to IC50
for inhibition of TNF-
a
production in PBMC cells in Table 5.12 The
ratio between enzyme and PBMC activities is comparable for all
four BPAs. Binding to different enzyme conformations does not af-
fect TNF-a production in LPS-stimulated PBMC cells over the time-
scales used in the assay (18–20 h). It is unclear why lower ratios
are seen for BIRB-796 and SB-242235. These clinical candidates
may simply have better cell permeability, but other causes may
be slow kinetics in the case of BIRB-796 or inhibition of off-target
kinases.
amides are potent ATP-competitive inhibitors of active p38
a that
bind with similar affinity to inactive p38 . DFG-out binding-
a
mode BPAs achieve greater potency at the expense of higher
molecular weight, lower binding efficiency and lower selectivity.
The kinetic behaviour of compounds is more complex than ‘DFG-
in quick, DFG-out slow’, depending greatly on the compounds
The selectivity profiles of exemplar DFG-in and DFG-out BPAs
are summarised in Table 6. Out of 35 protein kinases tested, 7
inhibited cRaf (80 nM), Lyn (4
lM) and murine Lck (5
lM). It had
themselves. BPA compounds inhibit the cellular p38
a pathway,
IC50 > 10 M against the others, including JNKs 1 and 3. In addition,
l
activation of p38 itself, and cytokine release to a similar extent
a
compounds were screened in the KinomeScan binding assay panel
of 203 kinases.26 7 displaced ATP-site ligands from several kinases
(Table 6). Some of these overlap with published activities of BIRB-
796 against a smaller panel of 116 kinases, which did not include
regardless of their binding conformation. This suggests that some
of the perceived advantages of targeting the DFG-out binding
mode based on measurements with isolated p38a are not rele-
vant in cells.
Table 5
DFG-in and DFG-out compounds behaviour in cells
Compound DFG mode p38a a a
bindinga MW Binding BEIb p38 activityc HLF Hsp27d HLF p38ae HLF Hsp27/p38af PBMC TNF-ag PBMC TNF- /enzymeh
SB-242235 In
36
12
15
2.5
4.4
6.4
353 21
348 23
374 21
469 18
471 18
528 16
N.T.
9
6
9
6
160
53
88
6
27
58
130
18
16
2
6
47
1.2
2.9
5.5
3.0
4.5
1.2
170
250
650
90
270
30
5
21
43
36
61
>5
2
In
16
In
7
8
Out
Out
Out
BIRB-796
<46
(a) Ki (nM) for binding to isolated enzyme.12 (b) BEI (from Ki, not including preincubation, which may be significant for BIRB-796).12 (c) Ki (nM) for inhibition of enzyme
activity.15 IC50 (nM) for inhibition in IL-1-stimulated HLF cells of phosphorylation of (d) Hsp27 and (e) p38 24 (f) The ratio between the two HLF readouts. (g) IC50 (nM) for
TNF- IC50 in LPS-stimulated PBMC cells and enzyme binding Ki.
inhibition in PBMC cells.12 (h) The ratio between TNF-
a
.
a
a
Table 6
Kinases inhibited by DFG-in and DFG-out compounds, and the literature results for BIRB-7964,26
Compound
DFG
<1
l
M
P1
l
M
KinomeScan26
2
In
p38b
—
Only p38b
7
Out
Out
cRAF, p38b
mLCK, LYN
KIT, LCK, LOK, p38b/c, PDGFRa/b, RAF1, STK10
BIRB-796
JNK24
cRAF, FYN, LCK4
JNKs, p38b/c
, SLK, STK10, TEK, TNIK26

R. M. Angell et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4433–4437
4437
an ADSC Q4R CCD detector. The data were processed with the HKL package
(Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276:Macromol. Crystallogr.
A, 307) and CCP4 program suite (Bailey, S. Acta Crystallogr., 1994, D50, 760). The
structure was solved using the native p38 coordinates (PDB entry 1WFC) as the
initial model in refinement by REFMAC (Murshudov, G.; Vagin, A.; Dodson, E.
Acta Crystallogr. 1997, D53, 240). The final R-factor achieved for the complex
was 17.5%. Coordinates have been deposited in the PDB as entry 3D83. Figures
were produced using Pymol (DeLano, W. L. DeLano Scientific, Palo Alto, CA,
USA. http://www.pymol.org).
Acknowledgements
The authors thank Nicola Aston, John Christopher, Duncan
Holmes, Rachel Hosking, Gareth Wayne and all members of the
p38 team for their many contributions.
References and notes
15. Inhibition of phosphorylation of ATF-2 by activated p38
described in Ref. 3.
a was measured as
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16. Experiments were performed at 25 °C on a BIAcore S51 instrument. Human
unphosphorylated p38 (as used in the crystallography) was immobilised
a
using random amine coupling onto a CM5 chip in the presence of SB-203580 at
pH 5.3 in 50 mM NaAc at normally 3-4kRU. Compounds were titrated over
p38
a
in 50 mM Hepes pH 7.4, 150 mM NaCl, 1% DMSO, typically in doubling or
M. BIAcore software and Grafit were used to
tripling dilutions from 3.7
l
extract on- and off-rates to ensure consistency and reproducibility.
17. Kroe, R.; Regan, J.; Proto, A.; Peet, G.; Roy, T.; Landro, L.; Fuschetto, N.; Pargellis,
C.; Ingraham, R. J. Med. Chem. 2003, 46, 4669.
18. Lipinski, C.; Lombardo, F.; Dominy, B.; Feeney, P. Adv. Drug Deliv. Rev. 1997, 23,
3.
19. Okram, B.; Nagle, A.; Adrian, F.; Lee, C.; Ren, P.; Wang, X.; Sim, T.; Xie, Y.; Wang,
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24. Human lung fibroblast (HLF) cells (Lonza, CC-2512) were plated into 96-well
tissue culture plates (15,000 cells/well). After overnight incubation, media
were replaced with serum-free media. After overnight serum starvation,
11. Schindler, T.; Bornmann, W.; Pellicena, P.; Miller, W.; Clarkson, B.; Kuriyan, J.
Science 2000, 289, 1938.
12. Enzyme Ki determinations, using displacement of
compounds were added and incubated for 1 h, followed by addition of IL-1
(R&D Systems, 200-LA-010) to a final concentration of 0.09 ng/ml for 40 min.
Liquid was removed from the wells, replaced with 40 l/well Meso Scale
Discovery complete lysis buffer, and 35 l transferred to immunoassay plates
for the detection of phospho-p38 (pT180/pY182) or Hsp27 (pS15). Detection
a
a
fluorescent ATP-
l
competitive inhibitor, and IC50s for inhibition of TNF
a release from LPS-
l
stimulated peripheral blood mononuclear cells were carried out as described in
Ref. 2. BEI (Binding Efficiency Index) = (pKi/MW)*1000, as defined by Abad-
Zapatero, C.; Metz, J. Drug Discovery Today 2005, 10, 464.
a
and quantification of the level of phosphorylated analyte was performed
according to the supplier protocol (MSD), with raw data analysed and IC50
values determined using ActivityBase software (IDBS). In addition to BIRB-796,
SB-242235 was used as a standard. SB-203580 gave variable results in the HLF
assays, perhaps due to its physicochemical properties. For example, from 40
13. Compounds given in Table 2 are representative of an array prepared from
substituted benzoic acids as shown in Scheme 2 of Ref. 3, using several amide
coupling conditions. Compounds given in Table 3 were prepared as shown in
Scheme 1 of Ref. 3, also using a number of different amide coupling conditions.
For 22–24, cyclopropylmethyl-amine in step
aminobenzonitrile.
14. An apo crystal of unphosphorylated human p38
tests in the phospho-p38 assay, it gave IC50 > 10
times.
lM 7 times and <250 nM 9
a
was replaced with 3-
25. Adams, J.; Boehm, J.; Gallagher, T.; Kassis, S.; Webb, E.; Hall, R.; Sorenson, M.;
Garigipati, R.; Griswold, D.; Lee, J. Bioorg. Med. Chem. Lett. 2001, 11, 2867.
26. KinomeScan assays were carried out at Ambit Biosciences and interpreted as
described in Ref. 3. Data for BIRB-796 are taken from Fabian, M. et al Nat.
Biotechnol. 2005, 23, 329.
a
(expressed, purified and
crystallised as previously described) was soaked with 2 mM 8 for 5 days and
cryoprotected.1,3 X-ray diffraction data were collected from the crystal at 100 K
(using an Oxford Cryostream) at Daresbury Laboratory SRS (station 9.6) using