The discovery and initial optimisation of pyrrole-2-carboxamides as inhibitors of p38α MAP kinase

Article abstract of DOI:10.1016/j.bmcl.2010.05.011

A novel pyrrole-2-carboxamide series of p38α inhibitors, discovered through the application of virtual screening, is presented. Following evaluation of activity, selectivity and developability properties of commercially available analogues, a synthesis program enabled rapid assessment of the series' suitability for further lead optimisation studies.

Full text of DOI:10.1016/j.bmcl.2010.05.011

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3936–3940
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The discovery and initial optimisation of pyrrole-2-carboxamides as inhibitors
of p38
a
MAP kinase
b,
a
a
b,
Kenneth Down a, , Paul Bamborough , Catherine Alder , Amanda Campbell , John A. Christopher
,
*
*
Maria Gerelle a,à, Steve Ludbrook b, Dave Mallett a, Geoff Mellor b, David D. Miller a,§, Rosannah Pearson b,
Keith Ray a,–, Yemisi Solanke a, Don Somers b
a Respiratory Centre of Excellence in Drug Discovery, GlaxoSmithKline R&D, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
b Molecular Discovery Research, 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:
A novel pyrrole-2-carboxamide series of p38
a inhibitors, discovered through the application of virtual
Received 29 March 2010
Revised 5 May 2010
Accepted 5 May 2010
Available online 10 May 2010
screening, is presented. Following evaluation of activity, selectivity and developability properties of com-
mercially available analogues, a synthesis program enabled rapid assessment of the series’ suitability for
further lead optimisation studies.
Ó 2010 Elsevier Ltd. All rights reserved.
Keyword:
p38a MAP kinase
The p38
a
mitogen-activated protein kinase was first identified
less, at least seven phase II clinical trials of small-molecule p38a
in human monocytes as the target for a class of cytokine suppres-
inhibitors are currently in progress or have recently completed
for other indications, including pain, multiple myeloma, major
depressive disorder, acute respiratory distress syndrome, chronic
obstructive pulmonary disorder and acute cardiovascular dis-
sive anti-inflammatory compounds.1 From its central position in
the cell signalling pathway, p38
pro-inflammatory cytokines including IL-1, IL-6 and TNF-
itors of p38 suppress the production of cytokines in vitro and
a
regulates the expression of many
a. Inhib-
a
eases.8 Therefore, selective and structurally distinct p38
a inhibi-
have anti-inflammatory activity in vivo in models of rheumatoid
tors remain of interest. For instance, Pfizer reported ongoing
development of a triazolopyridine series, and we recently pre-
sented the discovery of Losmapimod, a biaryl amide (Fig. 1).9,10
Kinase inhibitors frequently inhibit multiple protein kinases.11 It
has been our experience that rational, focused screening against a
single kinase frequently yields hits, but these can often be more
arthritis (RA) and other diseases.2
Several compounds have progressed into clinical studies for RA,
but so far their success has been limited.3 Toxicity was the primary
reason for failure, often because of liver, gastro-intestinal (GI) or
neurological toxicity.4,5 It is possible that these may be related to
off-target effects, perhaps due to inhibition of other kinases, partic-
ularly for early examples where wider kinase selectivity was less
thoroughly measured.3 Results from two trials have been pub-
lished recently. Pamapimod led to liver enzyme elevation and ad-
verse events including infection, rash, dizziness and GI problems.6
The highly selective VX702 was comparatively well tolerated and
showed transient effects on biomarkers in the first few weeks of
treatment, but disappointingly this was not sustained.7 Neverthe-
F
VX702
Pamapimod
F
O
O
F
N
NH2
HO
O
F
F
N
N
N
Me
N
O
F
N
NH2
OH
F
F
H
F
Me
N
O
* Corresponding authors.
O
E-mail address: kenneth.d.down@gsk.com (K. Down).
Present address: Heptares Therapeutics Ltd, BioPark, Welwyn Garden City,
Hertfordshire AL7 3AX, UK.
Cl
N
N
N
H
O
Cl
N
à
HN
Present address: Chemistry Research Laboratory, University of Oxford, Oxford
Merck
dihydroquinazolinone
OX1 3TA, UK.
N
H
Losmapimod
§
Present address: University College London, Gower Street, London WC1E 6BT, UK.
–
Present address: Ray Bioscience Consulting Ltd, Malvern Cottage, Greenlands
Lane, Prestwood, Bucks HP16 9QU, UK.
Figure 1. Structures of literature p38a inhibitors mentioned in the text.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.011

K. Down et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3936–3940
3937
effective against kinases other than the intended target.10b There-
fore, we have adopted an approach in which focused screening is di-
rected towards the protein kinase family rather than individual
targets.
Table 1
Data for initial compounds 1–613
H
N
O
R1
R2
N
Databases of commercially available compounds are used
extensively in virtual screening and are a valuable resource for lead
discovery. A database of millions of commercially available com-
pounds offered by various suppliers has been assembled by GSK
for this purpose. A search of this database was carried out using
a 3D pharmacophore query intended to be generally applicable
to kinases.12 Selected pharmacophore hits were purchased and
H
O
R2
R1
p38a pIC50
14
Compound
1
2-Methyl
6.6
6.1
O
S
S
2
3
2,6-Dichloro
4-Methoxy
screened against a panel of protein kinases in duplicate at 10 lM
compound concentration. Several examples of the pyrrole series,
<4.6
which is the subject of the rest of this report, showed sufficient
p38a
activity for progression to IC50 measurement.13
MeO
N
Compound 1 was the most potent from this set, with a pIC50 of
6.6 (mean, n = 9).14 Because of this encouraging activity, and to ex-
plore the mechanistic activity in a cellular environment, compound
1 was tested in a human lung fibroblast assay measuring the inhi-
bition of phosphorylation of Heat Shock Protein 27 (Hsp27), a
downstream marker on the p38 pathway. It showed good activity
with a pIC50 of 6.2 (mean, n = 6).15
4
2-Methyl
6.5
5
6
2,6-Dichloro
2-Methyl
5.7
5.0
N
Values are means of at least three experiments.
H
N
O
4
nevertheless showed interpretable trends, some of which are
exemplified in Table 1. Lipophilic ortho-substitution was preferred
on the R1 aryl ring (1 and 2), consistent with the geometry of the
ketone to phenyl linker observed in the crystal structure. However,
anything other than F or H in the para position resulted in greatly
reduced potency (3). At the amide R2 position, benzyl and benzyl-
like substituents were the most potent, with a reasonable tolerance
evident for substitution on the benzyl group, including at the ortho
position (4). The benzyl group could also be replaced by small alkyl
groups with only a small reduction in potency (5). More polar
amine groups were less favoured however, with substituted sec-
ondary amines being the least potent (6), indicating a preference
for a lipophilic group in the outer lipophilic subpocket of the site.
Despite the large number of purchased compounds screened,
O
2
N
H
Me
O
1
Once the structural integrity of the initial hits had been confirmed
by NMR, a ꢀ1.9 Å resolution crystal structure of 1 in complex with
p38a
was obtained (Fig. 2).16 The phenyl ring on the ketone at the
pyrrole 4-position occupies the inner lipophilic pocket adjacent to
the gatekeeper residue Thr106. The pyrrole NH donates a hydro-
gen-bond to the inner hinge acceptor backbone carbonyl of
His107. In addition, a hydrogen-bond to the hinge at Met109 is
formed from the carbonyl of the amide at the pyrrole 2-position.
The amide NH in the 2-position and the ketone carbonyl in the 4-
position interact with water molecules evident in the crystal struc-
ture. The methyl furan fills the outer lipophilic pocket near Ala111.
A further 120 available analogues were selected from the exter-
none were found showing better p38a potency than 1. To build
confidence in the series, 1 was profiled further.
The kinase selectivity of the series was excellent when screened
against a wider kinase panel. For example, 1 had a pIC50 < 5.5
against every member of an in-house panel of 51 kinases, and
nal suppliers compound database and screened against p38a.
Whilst compounds allowing direct pairwise comparisons could
not always be obtained, the SAR around the 120 tested compounds
showed no significant activity when screened at 10 lM against
the Dundee University panel of 18 kinases.17 In keeping with other
compounds from this series, some p38b activity was present
(pIC50 = 5.2), although for the series as a whole IC50s were generally
10-fold lower than p38a. Furthermore, against a panel of 227 ki-
nases at Ambit Biosciences, the only hits were p38a, p38b, JNK1
and JNK3.18 However, JNK binding was weak, consistent with the
low inhibition observed in internal assays measuring the catalytic
activity of JNK1 (pIC50 < 5).
The wider developability properties were promising. Cyto-
chrome P450 inhibition was low, for instance 1 had an IC50 of
15.8
ers tested.19
In vivo rat PK was measured, showing the compound had mod-
lM against the 2C9 isoform and was less potent against oth-
erate oral bioavailability (F% = 18), with high clearance (56 ml/min/
kg), short half-life (0.16 h) and low VDISS (0.93 l/kg).20
With these encouraging data in hand a synthesis program was
initiated to further explore the series. The array was prepared
using chemistry shown in Scheme 1. 2-Trichloroacetylpyrrole
was subjected to a Friedel–Crafts acylation with the appropriate
acid chloride, formed from the carboxylic acid with thionyl chlo-
ride, to give the corresponding ketone. This was reacted directly
Figure 2. Crystal structure of 1 (green, with surface shaded) in complex with p38
(orange). Hydrogen-bonds to the inhibitor are shown as magenta dotted lines.
Water molecules are represented by red crosses.
a

3938
K. Down et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3936–3940
H
N
H
H
N
b
c
N
O
O
O
R1
R1
R1
+
Cl
Cl
Cl
Cl
Cl
HO
Cl
O
O
Cl
O
O
a
d
a
H
N
H
N
e
O
O
R1
R1
R1
HO
R2
N
Cl
H
O
O
Scheme 1. Reagents and conditions: (a) DMA, SOCl2, rt; (b) AlCl3, chloroform, reflux; (c) NaOH (aq), rt; (d) primary and secondary amines, DMF, rt; (e) anilines, DMA, rt.
with primary and benzyl amines in DMF to give the desired
amides. Anilines failed to react using this chemistry and so a differ-
ent route was developed via the carboxylic acid, which could be
rapidly obtained by treating the trichloroacetyl group with aque-
ous sodium hydroxide. This failed to react using common carbox-
ylic acid coupling conditions, but in situ formation of the acid
chloride using thionyl chloride, followed by addition of the anilines
gave the desired amides.
SAR knowledge from other p38
porated into the array design. Figure 3 shows an overlay between
the p38 crystal structures of pyrrole-2-carboxamide 1 (green)
a compound classes was incor-
a
and a Merck dihydroquinazolinone (magenta).21 The back-pocket
binding aryl group at the 4-position of the pyrrole superimposes
well on the C-5 aryl group of the dihydroquinazolinone (circled
area). In the dihydroquinazolinone series, aryl groups (in particular
2,4-difluorophenyl, 2-chlorophenyl and 2-chloro-4-fluorophenyl)
were preferred C-5 substituents. These groups were included as
4-aryl substituents in the pyrrole series. Heterocycles and fused
bicyclic systems were also prepared.
The dihydroquinazolinone series has no analogous position to
the pyrrole 2-carboxamide substituent. Figure 4 shows, however,
how this carboxamide forms the hinge hydrogen-bond to Met109
in a similar way to the amide in the biaryl amide series (circled
area).10 In that series, aryl and benzyl substituted amide com-
pounds gave the greatest enzyme potency, whilst alkyl substitu-
ents gave better all-round properties.10c Therefore anilines,
benzylamines and small primary alkyl groups were used as pyrrole
2-position substituents. The X-ray structure showed that this sub-
stituent was partially exposed to solvent, so water solubilising
groups were also included.
Figure 4. Overlaid X-ray structures of p38a (orange) complexed with 1 (green) and
a biphenyl amide (magenta).
10c
Data for selected compounds are summarised in Table 2. Chang-
ing the amide substituent of 1 to a 2,6-difluorobenzylamine (7)
gave a marginal increase in potency but led to reduced activity in
the pHsp27 assay. The addition of a 4-fluoro substituent to the aryl
group was tolerated (8), giving similar potency to compound 7 in
both assays. Changing the 2-methyl to a 2-fluoro (9) gave a 10-fold
potency increase over compound 1, but despite this had lower po-
tency in the pHsp27 assay.
Reasoning that the lower cellular activity might be related to
solubility, a solubilising 4-(N-methylpiperazinyl)benzylamine sub-
stituent was introduced into the pyrrole 2-amide position (10).
This resulted in a compound with excellent enzyme activity, but
this did not translate into greater activity in the pHsp27 assay. This
lower potency in the pHsp27 assay was a consistent trend with the
benzylamine amides. However, when pinacolylamine was intro-
duced (11, racemic), there was no drop-off between the enzyme
and pHsp27 potency. This compares favourably with the most
cell-active benzylamine (12), which still suffers a significant reduc-
tion from its enzyme potency. Compound 11 also showed inhibi-
tion of TNF-a release from LPS-stimulated PBMC cells with
pIC50 = 5.6 (mean, n = 5) and from whole blood with pIC50 = 6.5
(mean, n = 3).22
Greatly reduced enzyme potency resulted when larger substit-
uents were added at the 4-position of the aryl group (e.g. 13). Sim-
ilarly, the benzothiophene substituent was very weakly active (14).
These results, along with compound 15, confirmed the earlier
hypothesis that the only 4-substituent small enough to be toler-
ated at this position was fluorine. Introducing polarity to the small
alkyl amide substituent (e.g. 16) resulted in reduced enzyme po-
tency, consistent with the position of this group in the outer lipo-
Figure 3. Overlaid X-ray structures of p38a (orange) complexed with 1 (green) and
philic pocket of p38a.
with a dihydroquinazolinone (magenta).

K. Down et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3936–3940
3939
Table 2
p38 inhibition and pHsp27 human lung fibroblast cellular mechanistic assay data for
Acknowledgements
a
compounds 7–17
The authors thank the Department of Screening and Compound
Profiling for the generation of kinase inhibition data, the Depart-
ment of Biological Reagents and Assay Development for protein
production and purification, Fiona Lucas for supporting the TNF-
H
N
H
N
O
O
e.g.11
R
N
H
N
H
Ar
O
O
a
release from LPS-stimulated PBMC cells and whole blood, Dave
F
Langley for coordinating compound acquisition activities, and Ni-
cole Hamblin and Simon Macdonald for helpful comments during
the preparation of the manuscript.
Compound
1
Ar
R
p38
a
pHsp27
6.2
6.6
7.0
O
F
F
F
References and notes
7
8
5.5
5.4
1. Lee, J.; Laydon, J.; McDonnell, P.; Gallgher, T.; Kumar, S.; Green, D.; McNulty, D.;
Blumenthal, M.; Heys, J.; Landvatter, S.; Strickler, J.; McLaughlin, M.; Siemens,
I.; Fisher, S.; Livi, G.; White, J.; Adams, J.; Young, P. Nature 1994, 372, 739.
2. Goldstein, D. M.; Gabriel, T. Curr. Top. Med. Chem. 2005, 5, 1017.
3. Goldstein, D. M.; Kuglstatter, A.; Lou, Y.; Soth, M. J. J. Med. Chem. 2010, 53, 2345.
4. Genovese, M. C. Arthritis Rheum. 2009, 60, 317.
F
F
F
F
6.6
5. Sweeney, S. E. Nat. Rev. Rheum. 2009, 5, 475.
6. Cohen, S. B.; Cheng, T. T.; Chindalore, V.; Damjanov, N.; Burgos-Vargas, R.;
DeLora, P.; Zimany, K.; Travers, H.; Caulfield, J. P. Arthritis Rheum. 2009, 60, 335.
7. Damjanov, N.; Kauffman, R. S.; Spencer-Green, G. T. Arthritis Rheum. 2009, 60,
1232.
F
F
9
7.6
7.9
5.8
5.7
8. Available from: http://clinicaltrials.gov, February 2010.
9. Jerome, K. D.; Rucker, P. V.; Xing, L.; Shieh, H. S.; Baldus, J. E.; Selness, S. R.;
Letavic, M. A.; Braganza, J. F.; McClure, K. F. Bioorg. Med. Chem. Lett. 2010, 20,
469.
10
10. (a) Walker, A.; Aston, N.; Bamborough, P.; Buckton, J.; Edwards, C.; Holmes, D.;
Jones, K.; Patel, V.; Smee, P.; Somers, D.; Vitulli, G. J. Med. Chem. 2009, 52, 6257;
(b) Angell, R. M.; Bamborough, P.; Cleasby, A.; Cockerill, S. G.; Jones, K. L.;
Mooney, C. J.; Somers, D. O.; Walker, A. L. Bioorg. Med. Chem. Lett. 2008, 18, 318;
(c) Angell, R. M.; Angell, T. D.; Bamborough, P.; Brown, D.; Brown, M.; Buckton,
J. B.; Cockerill, S. G.; Edwards, C. D.; Jones, K. L.; Longstaff, T.; Smee, P. A.; Smith,
K. J.; Somers, D. O.; Walker, A. L.; Willson, M. Bioorg. Med. Chem. Lett. 2008, 18,
324; (d) Angell, R. M.; Aston, N. M.; Bamborough, P.; Buckton, J. B.; Cockerill, S.
G.; deBoeck, S. J.; Edwards, C. D.; Holmes, D. S.; Jones, K. L.; Laine, D. I.; Patel, S.;
Smee, P. A.; Smith, K. J.; Somers, D. O.; Walker, A. Bioorg. Med. Chem. Lett. 2008,
18, 4428; (e) Angell, R. M.; Angell, T. D.; Bamborough, P.; Bamford, M. J.; Chung,
C.; 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. Bioorg. Med.
Chem. Lett. 2008, 18, 4433.
N
N
F
F
11
12
6.7
7.3
6.8
6.1
HO
HO
HO
HO
F3C
F
13
14
15
<4.6
4.7
NT
NT
5.9
11. Paolini, G. V.; Shapland, R. H. B.; van Hoorn, W. P.; Mason, J. S.; Hopkins, A. L.
Nat. Biotechnol. 2006, 24, 805.
12. The pharmacophore was constructed manually from a crystal structure of a
non-selective kinase inhibitor (unpublished results) complexed with cyclin-
dependent kinase 2. Hydrogen-bonding and aromatic interactions between the
inhibitor and conserved residues of the ATP-binding site acids were included in
the pharmacophore query. The distance tolerances of the resulting model were
set loosely in order to be suitable for multiple protein kinases. Searching was
carried out using Catalyst (Accelrys Inc., www.accelrys.com). All molecules
identified from the external suppliers’ database in this way were passed
through proprietary in silico developability filters before being clustered and
examined visually in order to make a final selection.
S
F
F
F
7.5
F
OH
16
17
5.9
6.0
NT
13. Compounds 1–6 were sourced from Bionet (Key Organics Ltd,
www.keyorganics.net).
14. Recombinant full length human p38a was expressed in E. coli as an N-terminal
F
F
5.8a
6-His-tagged fusion protein and activated by incubation with MKK6 (Millipore,
Dundee, UK) in the presence of ATP and stored frozen in aliquots at À80 °C. Its
activity was assessed using a time-resolved fluorescence resonance energy
transfer (TR-FRET) assay. Briefly, p38
a (typically 0.1–0.2 nM final) diluted in
F
assay buffer (12.5 mM HEPES, pH 7.4, with 1 mM DTT) was added to wells of a
black, shallow 384-well plate (Greiner Bio-One, Stroudwater, Gluocester, UK)
containing various concentrations of compound or DMSO vehicle (less than 2%
v/v final). The reaction was initiated by the addition of biotinylated
pIC50 values are mean of at least three experiments.
a
n = 2 data only.
recombinant ATF2 (residues 19–96) substrate (0.4 nM final), ATP (125
final) and 5 mM MgCl2 final in 12.5 mM HEPES buffer as above to a total
volume of 6 l. The reaction was incubated for 120 min at room temperature
and then terminated by the addition of stop reagent (3 l) containing 60 mM
EDTA and detection reagents in buffer (40 mM HEPES, pH 7.4, 150 mM NaCl
and 0.3% w/v BSA). Detection reagents comprise antiphosphothreonine-
71ATF2 monoclonal antibody at 0.35 nM final concentration (Cell Signalling
Technology, Beverly Massachusetts, USA) labelled with W-1024 europium
chelate (Perkin-Elmer, Turku, Finland) and allophycocyanin-labelled
streptavidin at 258 nM final concentration (Prozyme, San Leandro, California,
lM
Anilides were also prepared at the pyrrole 2-amide position, but
were generally less potent than the benzylamines and alkylamines
(e.g. 17). This is consistent with docking studies that predict that
the inflexibility of the aniline results in a steric clash with the pro-
tein backbone around the Met109 residue.
l
l
In conclusion, we have presented a novel series for p38a discov-
ered through the application of 3D pharmacophore virtual screen-
ing. Initial exploration and SAR optimisation resulted in lead
compounds with good enzyme potency and excellent selectivity,
as well as mechanistic and functional cellular assay potency result-
ing in a series suitable for further lead optimisation.
USA). The reaction mixture (9 ll total volume) was further incubated for at
least 60 min at room temperature. The degree of phosphorylation of ATF2 was
measured using a suitable time-resolved fluorimeter such as a Rubystar (BMG,
Aylesbury, Buckinghamshire, UK) or Envision (Perkin-Elmer Ltd, Seer Green,
Beaconsfield, UK) as
a ratio of specific 665 nm energy transfer signal to
reference europium 620 nm signal.

3940
K. Down et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3936–3940
15. Hsp27 phosphorylation in human lung fibroblast cells was measured as
described in Ref. 10e.
19. CYP450 inhibition was carried out as described in Ref. 10d.
20. Pharmacokinetic parameters in male CD rats were determined following
intravenous (iv) and oral (po) administration at 1 mg/kg. Compound was
administered as a solution in DMSO/PEG200/sterile water (1:7:2 v/v/v), 1 ml/
kg (iv) and 4 ml/kg (po). Blood was collected over a 7-h time period. Plasma
16. An apo crystal of unphosphorylated human p38a (expressed, purified and
crystallized as previously described, Ref. 10b) was soaked with 1 mM 1 for 40 h
and cryoprotected as before. X-ray diffraction data were collected in-house on
a Rigaku-MSC MicroMax007 rotating anode generator with a MarResearch
Mar345 image plate detector mounted on a Mar-dtb. The data were processed
and the structure solved as in Ref. 10b. The final R-factor and R-free achieved
for the complex were 23.2% and 29.7%, respectively. Coordinates have been
deposited in the PDB as entry 3MPT. Figures were produced using Pymol
(DeLano, W.L., DeLano Scientific, Palo Alto CA, USA. http://www.pymol.org).
17. Available from: http://www.kinase-screen.mrc.ac.uk/.
18. Assays were carried out by Ambit Biosciences, San Diego. The ability of
compounds to compete with the binding of the human kinase (expressed as
fusion to T7 bacteriophage) to immobilized ATP-site probe ligands was
determined as previously described, see: Fabian, M. A.; Biggs, W. H., III;
Treiber, D. K.; Atteridge, C. E.; Azimioara, M. D.; Benedetti, M. G.; Carter, T. A.;
Ciceri, P.; Edeen, P. T.; Floyd, M.; Ford, J. M.; Galvin, M.; Gerlach, J. L.; Grotzfeld,
R. M.; Herrgard, S.; Insko, D. E.; Insko, M. A.; Lai, A. G.; Lélias, J.-M.; Mehta, S. A.;
Milanov, Z. V.; Velasco, A. M.; Wodicka, L. M.; Patel, H. K.; Zarrinkar, P. P.;
Lockhart, D. J. Nat. Biotechnol. 2005, 23, 329. Compounds were screened at
was prepared following centrifugation and compound extracted from 50
plasma using protein precipitation with acetonitrile. Samples were evaporated
under nitrogen and re-suspended in 200 of 20:80 acetonitrile/water.
ll
l
l
Analysis was performed using LC–MS/MS on the API365 with a 3 min fast
gradient comprising 0.1% formic acid in water and 0.1% formic acid in
acetonitrile (mobile phases), 30
Luna C18 column (50 Â 2.1 mm, 5
using a non-compartmental approach.
l
l injection volume, flow rate 0.8 ml/min and
lm). Pharmacokinetic data was generated
21. Stelmach, J. E.; Liu, L.; Patel, S. B.; Pivnichny, J. V.; Scapin, G.; Singh, S.; Hop, C.
E.; Wang, Z.; Cameron, P. M.; Nichols, E. A.; O’Keefe, S. J.; O’Neill, E. A.; Schmatz,
D. M.; Schwartz, C. D.; Thompson, C. M.; Zaller, D. M.; Doherty, J. B. Bioorg. Med.
Chem. Lett. 2003, 13, 277.
22. TNF-
a release from LPS-stimulated PBMC cells and whole blood was carried
out as previously described, see: Christopher, J. A.; Bamborough, P.; Alder, C.;
Campbell, A.; Cutler, G. J.; Down, K.; Hamadi, A.; Jolly, A. M.; Kerns, J. K.; Lucas,
F. S.; Mellor, G. W.; Miller, D. D.; Morse, M. A.; Pancholi, K. D.; Rumsey, W.;
Solanke, Y. E.; Williamson, R. J. Med. Chem. 2009, 52, 3098.
10
lM. Binding was only detected for p38a, p38b, JNK1 and JNK3 (% of binding
remaining relative to DMSO control was 0.7%, 17%, 12% and 34%, respectively).