The effect of RO3201195 and a pyrazolyl ketone P38 MAPK inhibitor library on the proliferation of Werner syndrome cells

Article abstract of DOI:10.1039/c5ob02229k

Microwave-assisted synthesis of the pyrazolyl ketone p38 MAPK inhibitor RO3201195 in 7 steps and 15% overall yield, and the comparison of its effect upon the proliferation of Werner Syndrome cells with a library of pyrazolyl ketones, strengthens the evidence that p38 MAPK inhibition plays a critical role in modulating premature cellular senescence in this progeroid syndrome and the reversal of accelerated ageing observed in vitro on treatment with SB203580.

Full text of DOI:10.1039/c5ob02229k

Organic &
Biomolecular Chemistry
View Article Online
View Journal
PAPER
The effect of RO3201195 and a pyrazolyl ketone
P38 MAPK inhibitor library on the proliferation of
Werner syndrome cells†
Cite this: DOI: 10.1039/c5ob02229k
Mark C. Bagley,*a Jessica E. Dwyer,a Mohammed Baashen,‡a Matthew C. Dix,b
Paola G. S. Murziani,b Michal J. Rokicki,c David Kiplingc and Terence Davis*c
Microwave-assisted synthesis of the pyrazolyl ketone p38 MAPK inhibitor RO3201195 in 7 steps and 15%
overall yield, and the comparison of its effect upon the proliferation of Werner Syndrome cells with a
library of pyrazolyl ketones, strengthens the evidence that p38 MAPK inhibition plays a critical role in
modulating premature cellular senescence in this progeroid syndrome and the reversal of accelerated
ageing observed in vitro on treatment with SB203580.
Received 28th October 2015,
Accepted 11th November 2015
DOI: 10.1039/c5ob02229k
www.rsc.org/obc
of many of the clinical features of old age, show early suscepti-
bility to a number of major age-related diseases and have an
Introduction
P38α is one isoform of the mitogen-activated protein kinase abbreviated median life expectancy (47 years). Consequently,
(MAPK) intracellular enzymes which are central to the WS is widely used as a model disease to investigate the mecha-
regulation of cytokine biosynthesis and inflammatory cell sig- nisms underlying normal human ageing. Associated with
nalling.1,2 The reduction of pro-inflammatory cytokine levels these aged features, fibroblasts from WS individuals have a
offers a means for the treatment of inflammatory disorders much-reduced replicative capacity when compared with fibro-
such as rheumatoid arthritis and atherosclerosis.3,4 In blasts from normal individuals, and have a stressed, aged,
addition to inflammatory disorders, p38α is known to play a morphology and high levels of activated p38α.13–15 This accel-
role in the resistance of various cancers to chemotherapy, erated cellular senescence is thought to underlie whole body
cancer proliferation and metastasis.5,6 This makes p38α a com- ageing in this syndrome. As part of our interest in mechanisms
pelling therapeutic target for the design of safe and efficacious of premature cell senescence, we showed that administering
inhibitors suitable for clinical investigation.2,7 Following the the p38α inhibitor SB203580 to WS cells rescues all of the fea-
discovery that pyridinylimidazole p38α inhibitors such as tures of accelerated replicative decline.13 This observation
SB203580 mediate multiple cellular responses,1 including the suggested that the abbreviated life span of WS cells is due to
production of inflammatory cytokines, a wide variety of struc- stress-induced growth arrest mediated by p38α MAPK, which
turally-distinct chemotypes8,9 have been discovered to inhibit we speculate is transduced from the frequently stalled DNA
this enzyme with notable differences in binding motif.10 Many replication forks observed in these cells.16 It also offers a
of these have been used in stage II or III clinical trials.11
Werner syndrome (WS) is a rare autosomal recessive dis- means, intervene in a premature ageing syndrome.16
order.12 The mutated gene (WRN) encodes for a RecQ helicase
However, it has been shown that SB203580 is not specific
means to clinically regulate this process and, by therapeutic
involved in DNA replication, recombination and repair. Indi- for p38α, but also inhibits the related N-terminal c-Jun kinases
viduals living with the syndrome display the premature onset (JNKs) that may play a role in cellular senescence.17–19 Thus
more work is needed using inhibitors that do not target the
JNKs to verify the role played by p38α in WS cells. In addition,
aDepartment of Chemistry, School of Life Sciences, University of Sussex, Falmer,
SB203580 is not suitable for possible in vivo use.11
Brighton, East Sussex, BN1 9QJ, UK. E-mail: m.c.bagley@sussex.ac.uk
bSchool of Chemistry, Main Building, Cardiff University, Park Place, Cardiff,
This manuscript describes a rapid route to one such inhibi-
tor, RO3201195 (1),20 the study of this inhibitor in WS cells,
and its comparison with the canonical p38α inhibitor
SB203580 to investigate the role of p38α MAPK signal trans-
duction in replicative senescence. This pyrazolyl ketone p38α
MAPK inhibitor progressed to clinical trials, is orally bio-
available, exhibits high kinase selectivity, and inhibited the
CF10 3AT, UK
cInstitute of Cancer and Genetics, Cardiff University School of Medicine, Heath Park,
Cardiff, CF14 4XN, UK
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5ob02229k
‡Current address: Department of Chemistry, College of Science and Humanities,
Shaqra University, Kingdom of Saudi Arabia.
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
Scheme 1 Synthesis of 3-methoxybenzoylacetonitrile (6) by Heck
coupling (top) or Claisen ester condensation (bottom). Reagents and
conditions: (i) see Table 1; (ii) cHCl–MeOH (1 : 4), microwaves, 110 °C,
30 min; (iii) MeOH, H2SO4, reflux, 2.5 h (90%); or MeOH, H2SO4, micro-
waves, 110 °C, 10 min (94%); (iv) see Table 2; HCl (aq).
Fig. 1 P38 MAPK inhibitors SB203580 and RO3201195 (1), a pyrazolyl
ketone library 4 and disconnective approach to 1.
acrylates,27 cinnamates,28 crotonates29 and alkoxypropenes,30
there are very few examples of β-alkoxyacrylonitriles in these
and related processes. Enol ethers are less reactive than
electron-poor alkenes in Heck transformations and so these
slow reactions often require high catalyst loadings, more reac-
tive aryl iodides as substrates and the use of certain additives,
particularly when performed under solid–liquid phase transfer
conditions.31 Furthermore, α,β-disubstituted alkenes may
suffer from steric constraints and so, although direct, this
approach for the synthesis of benzoylacetonitriles was
untested. Ortar et al.32 did describe the arylation of an α-meth-
oxyacrylate back in 1987 using aryl iodides, palladium acetate
(3 mol%), NaHCO3 base and tetrabutylammonium chloride as
additive. Using Jeffery’s conditions for Heck-type processes as
a starting point,33 methods related to the conditions for aryla-
tion of acrylonitrile with aryl iodides34 were investigated in the
Heck reaction of 3-iodoanisole and 3-methoxyacrylonitrile (5).
The use of Pd(OAc)2 (10 mol%) as catalyst in MeCN–H2O (3 : 1)
(Table 1) under microwave-assisted heating at 130 °C for 1.5 h
(entry 4) with K2CO3 as base and tetrabutylammonium
bromide (TBAB) as additive gave, following aqueous work up,
the α-aryl enol ether 7 as a mixture of (E)- and (Z)-diastereo-
isomers, in a 2 : 1 ratio, as determined by 1H NMR spectro-
scopic analysis. The diastereoisomers could be separated by
column chromatography, albeit in variable and poor yield. Low
conversions and poor diastereocontrol has been observed
before in Heck reactions of β-alkoxyacrylonitriles25 but, in our
case, separation would not be required if the mixture could be
converted directly to the target benzoylacetonitrile 6.
production of the cytokines IL-6 and TNFα upon lipopolysac-
charide (LPS) challenge in rats.20 Thus, RO3201195 could be a
valuable chemical tool in dissecting the accelerated ageing
pathophysiology seen in WS cells and its rescue in vitro using
small molecule inhibitors of this stress signalling pathway.13,21
Our rapid approach to the inhibitor employs microwave
irradiation to accelerate a number of steps, preparing the
central pyrazole motif through cyclocondensation of hydrazine
3 and a suitably protected benzoylnitrile 2, derivatized by
homologation prior to heterocycle formation (Fig. 1). This
approach had been successful for the synthesis of a pyrazolyl
ketone library (4)22 and so should be suitable for rapid pro-
duction of the target inhibitor under microwave-assisted
conditions.
Results and discussion
The synthesis of a suitably functionalized β-ketonitrile deriva-
tive 2, as a precursor for pyrazole formation, was first investi-
gated using the Pd-catalyzed Heck reaction23,24 of
3-iodoanisole and 3-methoxyacrylonitrile (5). This approach
had the advantage that, potentially, it could access 3-methoxy-
benzoylacetonitrile (6) directly from commercially-available
materials, upon acid hydrolysis of the coupled product, methyl
vinyl ether 7 (Scheme 1, top), providing a very rapid route into
the pyrazole skeleton. Heck reactions of α- or β-substituted
enol ethers are known and have been shown to provide
efficient access to 1-arylpropanone derivatives.25 However,
although there are numerous examples of Heck reactions of
α,β-disubstituted alkenes such as methylacrylates,26 3-ethoxy-
Surprisingly the hydrolysis of the enol ether diastereoiso-
meric mixture 7 required very forcing conditions. Simple treat-
ment of the Heck reaction mixture with acid under ambient
conditions failed to provide β-ketonitrile 6. The hydrolysis of
related alkoxyacrylonitriles were notable, by their absence,
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
Table 1 Investigating conditions for Heck reaction of 3-iodoanisole
and 3-methoxyacrylonitrile (5) to give benzoylacetonitrile 6
Table 2 Investigating conditions for base-mediated Claisen con-
densation of methyl benzoate 9 and acetonitrile to give benzoylaceto-
nitrile 6
Yieldb
Additive Conditionsa
(%)
Entry
Base
Conditions
Yielda (%)
Entry Base
1
2
3
4
5
6
7
K2CO3
NaHCO3 TBAB
K2CO3
K2CO3
NaHCO3 TBAB
NaHCO3 TBAB
TBAB
Conductive heating, 90 °C, 24 h 36
1
2
NaOEt
NaOMe
NaH
NaH
NaH
LDA
LDA
MeCN, RT; reflux, 24 h
MeCN, RT; reflux, 24 h
THF, reflux, 1 h
THF, microwaves, 150 °C, 1 h
PhMe, 90 °C, 16 h
THF, −78 °C, 1.5 h
THF, −50 °C, 3 h
9
9
15
35
34
18
70
Conductive heating, 90 °C, 24 h 34
TBAB
TBAB
Microwaves,c 130 °C, 1 h
Microwaves,c 130 °C, 1.5 h
Microwaves,c 130 °C, 1.5 h
Microwaves,c 140 °C, 1 h
Microwaves,c 150 °C, 1.5 h
26
20
37
19
31
3
4b
5
6
7
K2CO3
DPPP
a Isolated yield of benzoylacetonitrile 6. b Reaction of 3-iodoanisole
(1 equiv.), 3-methoxyacrylonitrile (5) (4 equiv.), Pd(OAc)2 (10 mol%),
base (2 equiv.) and additive (10 mol%) in MeCN–H2O (3 : 1) under the
given conditions, followed by aqueous work up, microwave irradiation
in cHCl–MeOH (1 : 4) at 110 °C (hold time) for 30 min (by moderation
of initial power, 150 W) and aqueous work up, followed by purification
by column chromatography on silica. c Microwave irradiation in a
sealed tube at the given temperature, as measured by the instrument’s
in-built IR sensor, for the given time (hold time) by moderation of the
initial power (150 W).
a Isolated yield of benzoylacetonitrile 6 from methyl benzoate 9 after
purification by column chromatography on silica. b Microwave
irradiation in a sealed tube at the given temperature, as measured by
the instrument’s in-built IR sensor, for the given time (hold time) by
moderation of the initial power.
A well-established method35 for Claisen condensation using
sodium ethoxide or sodium methoxide as base was frustrated
by the almost immediate formation of a homogenous gelati-
nous mass and so a range of conditions were investigated
(Table 2). Heating this mixture in acetonitrile at reflux for 24 h
gave only a very low yield (9% in both cases) of the benzoylace-
tonitrile 6 (entries 1 and 2) and significant return of unreacted
ester and so alternative methods were employed. Carrying out
the reaction using NaH in THF at reflux for 1.5 h gave an
immediate improvement in efficiency and provided the con-
densation product 6 in 15% yield (entry 3), 35% yield if heated
at 150 °C for 1 h in a sealed tube under microwave irradiation
(entry 4). Changing the solvent to toluene and heating the
mixture at 90 °C for 16 h gave a similar yield (entry 5, 34%).
However, the use of freshly-prepared LDA as base at −50 °C in
THF (entry 7) gave the Claisen condensation product 6 in good
yield (70%), whereas at lower temperatures was inefficient
(entry 6). Given the high yield and speed of the microwave-
assisted esterification, this route now represented the quickest
and most efficient means to access this key intermediate.
Considering our previous success in the use of microwave
irradiation in cyclocondensation reactions for the preparation
of pyrazoles,17,36,37 the aminopyrazole core motif of BIRB
796,38 and pyrazolyl ketone libraries,22,39 benzoylacetonitrile 6
was homologated in a microwave-assisted Knoevenagel con-
densation using N,N′-diphenylformamidine (10) in xylenes at
180 °C for 20 min to give enaminone 11 in good yield (74%)
(Scheme 2). Cyclocondensation by microwave irradiation with
the hydrochloride salt of 4-fluorophenylhydrazine (3) in the
presence of Et3N in EtOH at 140 °C for 1 h gave 5-aminopyra-
zole 12 in superior yield (86%) to a range of other methods,39
after purification by column chromatography. Protodemethyl-
ation using BBr3 in CH2Cl2 (1 M) at RT overnight gave the
corresponding phenol 13 in quantitative yield, which was
reacted in a SN2 displacement reaction with ketal-protected
tosyl glycerate 14 under basic conditions. The reaction in DMF
in the presence of K2CO3 at 80 °C for 24 h gave only a very low
yield of product 15 (9%)39 but switching to DMSO and carrying
from the study of Doucet and Santelli following Heck reaction
of 3-ethoxyacrylonitrile, catalyzed by a tetraphosphane/palla-
dium complex.25 However, by resorting to more forcing con-
ditions, gratifyingly, microwave irradiation of the isolated
diastereoisomeric mixture 7 in cHCl–MeOH (1 : 4) at 110 °C for
30 min finally provided benzoylacetonitrile 6 after purification
by column chromatography but only in modest isolated yield
over the two steps (Table 1, entry 1, 36%).
Efforts to improve the overall yield of this process by chan-
ging the base from K2CO3 to NaHCO3, in accordance with
Heck methods for the synthesis of cinnamonitriles,34 carrying
out the hydrolysis in cHCl–MeOH (1 : 4) under microwave
irradiation as before (Table 1, entry 2), gave no improvement
in yield (34%). Similarly, carrying out the Heck reaction in a
sealed tube under microwave irradiation using K2CO3 at
130 °C for 1 h (entry 3) or 1.5 h (entry 4) was poorly efficient.
The microwave-assisted reaction using NaHCO3 at 130 °C for
1.5 h (entry 5) did give a modest yield of product (37%),
similar to the longer conductive heating processes, but this
was not improved further by raising the temperature of reac-
tion to 140 °C (entry 6) or to 150 °C in the presence of an
alternative additive DPPP (10 mol%) (entry 7). Although the
route had been successful for the preparation of ketonitrile 6,
the need to isolate enol ether 7, in order to carry out the hydro-
lysis under high-temperature conditions, and the modest yield
for the overall process (entry 5 gave the best yield, at 37%)
meant that an alternative route had to be sought.
Considering the original approach to RO3201195 (1) by
Roche,20 efforts were made to access ketonitrile 6 by a more
traditional Claisen ester approach. Fischer esterification of
3-methoxybenzoic acid (8) in methanol using sulfuric acid as
catalyst at reflux for 3 h gave methyl ester 9 in good yield
(90%). This was improved further by carrying out the process
in a sealed tube under microwave irradiation at 110 °C for
10 min to give methyl ester 9 in 94% yield (Scheme 1, bottom).
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
Scheme 2 Microwave-assisted synthesis of RO3201195. Reagents and
conditions: (i) xylenes, microwaves, 180 °C, 20 min; (ii) Et3N, EtOH,
140 °C, 1 h; (iii) BBr3in CH2Cl2 (1 M), 0 °C-RT, 18 h; (iv) K2CO3, DMSO,
100 °C, 40 h; (v) TsOH, MeOH–H2O, 50 °C, 18 h.
out the SN2 displacement at a slightly higher temperature
(100 °C) for prolonged period (40 h) gave phenyl ether 15 in
improved yield (42%) after purification by column chromato-
graphy, followed by recrystallization. Submitting the product
of tosylate displacement 15 to ketal deprotection directly, prior
to recrystallization, under acid-catalyzed aqueous conditions at
50 °C for 18 h gave RO3201195 (1) in 37% yield over the two
steps, after purification by column chromatography and recrys-
tallization. The target inhibitor exhibited spectroscopic data
that matched literature values.22,39 It was prepared in 15%
overall yield in 7 separate steps from commercially-available
material, three of them employing microwave irradiation, to
provide rapid access to material suitable for examination in
WS cells.
Fig. 2 Effects of p38α inhibitors on Werner syndrome cells. Effects of
SB203580 (a) and RO3201195 (b) on the proliferation rate of WStert cells,
with 100% representing the DMSO control. Titration curves for inhibition
of p38α activity for SB203580 (c) and RO3201195 (d) measured as the
ratio of phosphorylated HSP27 to total HSP27 (measured by ELISA). For
(a) to (d) DMSO are control cells, ‘An’ are cells treated with anisomycin
to activate p38α, 10 to 50 000 are cells treated with p38 inhibitors at the
indicated concentration. (e) Plot of % p38α activity versus % increase in
WStert cell proliferation rate for both SB203580 and RO3201195. (f) Pro-
liferation of Werner syndrome AG05229 primary fibroblasts in the pres-
ence of SB203580 (2.5 µM) or RO3201195 (10 µM) measured as
population doublings versus days. (g) Plot of % increase in proliferation
rate of WStert cells against % p38α activity using pyrazolyl ketone library
4. See Table 3 for compound identities. (h) Immunoblot showing that
neither SB203580 nor RO3201195 inhibit JNK at the optimal concen-
trations for their effects on cellular proliferation rates. C = DMSO
control, A = cells treated with anisomycin to activate p38α and JNK, and
the other three lanes show the effect of the given inhibitor concen-
trations in anisomycin-treated cells.
Due to limited supplies of primary WS fibroblasts, initial
experiments to determine the effects of novel kinase inhibitors
on cellular proliferation were performed using WStert cells (see
materials and methods for a description of these cells).
Despite being immortal, these telomerized cells retain the
slow proliferation rate typical of primary WS cells and show a
similar response to SB203580 treatment.14 Thus, these cells
can be used to test the effects of modulators of the p38-signal-
ling pathway prior to their use on primary cells.
WStert cells were grown in standard medium supplemented
with SB203580 at a final concentration ranging from 10 nM to
50 µM (Fig. 2a). SB203580 inhibited p38α even at the lowest 30 nM as measured by us, and others.40,41 SB203580 treatment
doses used of 10 nM with maximum inhibition seen at 1.0 µM resulted in an increased proliferation rate of the WStert cells
and above (Fig. 2c), and has an IC50 for p38α of approximately compared to DMSO-treated control cells even at the lowest con-
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
Table 3 Pyrazolyl ketone library 4 studied in WStert cellsa
centration used, with the effect on proliferation rate increasing
steadily with increasing SB203580 concentration and reaching
a maximum between 2.5 µM and 10 µM. When the level of
p38α activity was plotted against the effects upon proliferation
rate a sigmoid curve was found (Fig. 2e). At 2.5 µM SB203580
resulted in an increase in proliferation rate of approximately
22% compared to DMSO controls (Fig. 2a and e).
RO3201195 has an IC50 of approximately 190 nM with
maximal inhibition between 2.5 and 10 µM.39 In this work,
RO3201195 inhibited p38α at a minimum concentration of
25 nM, with the maximal effect seen at 10.0 µM (Fig. 2d).
RO3201195 had an increasing effect on the proliferation rate
of WStert cells up to 10.0 µM concentration, thereafter becom-
ing inhibitory (Fig. 2b). As seen for SB203580, when the level
of p38α activity was plotted against the effects of RO3201195
upon proliferation rate a sigmoid curve was found (Fig. 2e):
indeed the curve was essentially identical with the curve
obtained using SB203580. The maximal increase seen in
the proliferation rate of WStert cells was 19% at 10.0 µM
(Fig. 2b and e).
The above data show that the optimal concentration of
these inhibitors for the maximal effects on WStert cells are
2.5 µM for SB203580 and 10.0 µM for RO3201195. The inhibi-
tors completely inhibited p38α but did not inhibit total JNK
activity in WStert cells at these concentrations as measured by
analyzing the phosphorylation of their downstream targets
HSP27 and c-Jun by immunoblot (Fig. 2h). WStert cells are
stimulated with anisomycin that activates p38α and JNKs as
shown by the increased presence of the band in the A lane
of the p-HSP27 panel, and the apperance of the doublet in the
p-c-Jun and c-Jun panels (Fig. 2h). RO3201195 at 10.0 µM
and SB203580 at 2.5 µM concentration reduced the p-HSP27
band to the level seen in the unstimulated control
(C lane), but had no effect on the doublet seen in p-c-Jun and
c-Jun.
We next used the p38α inhibitors on the primary Werner
syndrome fibroblast strain WAG05229 that was close to replica-
tive senescence to determine their effects on cellular senes-
cence. For SB203580 a concentration of 2.5 µM was used,
whereas for RO3201195 10.0 µM concentration was employed,
as these were the lowest concentrations that resulted in
maximal proliferation rate and 100% p38α inhibition (see
Fig. 2). The cells were re-fed with DMEM containing the inhibi-
tors as described previously.15 DMSO-treated fibroblasts proli-
ferated very slowly and only achieved a single population
doubling in the 88 days of the experiment (Fig. 2f). Inhibitor
treated cells, however, had a much elevated proliferation rate,
and managed approximately 7.5 population doublings prior to
4
R1
R2
A
Bb
C
D
E
F
H
I
J
OMe
OMe
H
H
H
H
H
OMe
OMe
H
4-Iodophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Bromophenyl
2,6-Dichlorophenyl
2,4-Difluorophenyl
Phenyl
4-Tolyl
Methyl
4-Chloromethyl
4-Chloromethyl
K
L
OMe
a See ref. 22 for preparation. b Corresponds with compound 12 from
Scheme 2.
Finally, we used a pyrazolyl ketone library 4 (Table 3) of
compounds related to RO320119522 for their effects on the pro-
liferation of WStert cells (Fig. 2g). These were used at a concen-
tration of 1.5 µM that resulted in a varied degree of p38α
inhibition from approximately 5% to greater than 95% (Fig. 2g
and ref. 22). When the effect on the proliferation rate of WStert
cells by this pyrazolyl ketone library were plotted against the %
p38α activity (as assessed by ELISA22), a linear response was
seen (Fig. 2g), providing further support that these effects on
cellular proliferation are due to p38α inhibition.
The data presented in this paper showing that
RO3201195 has essentially identical effects on the proliferation
of WS cells as SB203580, and that the proliferation rate is
dependent on p38 activity, provide very strong support that the
prevention of premature senescence seen in WS fibroblasts by
SB203580 reported previously13 is via p38α signalling. If pre-
mature cellular senescence does underlie the accelerated
ageing seen in WS individuals, these data also provide further
support that the use of p38 inhibitors in vivo may alleviate
this accelerated ageing.16 Finally, RO3201195 is shown to be a
useful tool for the dissection of signalling pathways in human
cells, as it is efficacious, relatively potent and is more kinase
selective than SB203580.20 In addition, it has shown thera-
peutic efficacy in vivo in rats in regulating cytokine production
upon LPS challenge,20 so may prove useful for future in vivo
use.
Experimental
Materials and methods
growth arrest (Fig. 2f). There was essentially no difference Commercially available reagents were used without further
whichever inhibitor was used. Thus, as the effects of these purification; solvents were dried by standard procedures. Light
inhibitors on WS cells are essentially identical and yet they petroleum refers to the fraction with bp 40–60 °C and ether
show very different kinase specificity profiles at the concen- refers to diethyl ether. Unless otherwise stated, reactions were
trations used (the only common denominator being p38), the performed under an atmosphere of air. Flash chromatography
conclusion is that the effects on proliferation seen in both was carried out using Merck Kieselgel 60 H silica or Matrex
WStert cells and primary WS fibroblasts result from the inhi- silica 60. Analytical thin layer chromatography was carried out
bition of p38α.
using aluminium-backed plates coated with Merck Kieselgel
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
60 GF254 that were visualised under UV light (at 254 and/or (0.82 g, 70%) as a yellow solid, mp 86.3–87.4 °C (MeOH) (lit.,42
360 nm). Microwave irradiation experiments were performed mp 87–88 °C) (Found [ES]: 198.0523. C10H9NO2Na [MNa]
in a sealed Pyrex tube using a self-tunable CEM Discover or requires 198.0525); IR (neat) νmax/cm−1 2948 (CH str.), 2252
CEM Explorer focused monomodal microwave synthesizer at (CuN str.), 1694 (CvO str.), 1580 (CC), 1451 (CH), 1258 (C–O),
1
the given temperature using the instrument’s in-built IR tem- 1012 (C–O); H NMR (500 MHz, CDCl3) δH/ppm 7.49–7.41 (3H,
perature measuring device, by varying the irradiation power 2-, 5- and 6-CH), 7.21 (1H, d, J = 8, 4-CH), 4.07 (2H, s, CH2),
(initial power given in parentheses).
3.88 (3H, s, Me); 13C NMR (126 MHz, CDCl3) δC/ppm 186.9 (C),
Fully characterized compounds were chromatographically 160.2 (3-C), 135.7 (1-C), 130.1 (5-CH), 121.2 (4-CH), 120.9
homogeneous. Melting points were determined on a Kofler (6-CH), 113.6 (CN), 112.8 (2-CH), 55.6 (Me), 29.4 (CH2); m/z (EI)
hot stage apparatus or Stanford Research Systems Optimelt 175 (M•+, 36%), 135 ([M − CH2CN]+, 100).
and are uncorrected. Specific rotations were measured at the
3-Methoxybenzoylacetonitrile (6) by Claisen ester conden-
indicated temperature (in °C) using a ADP440 polarimeter sation using NaH as base (Table 2, entry 5). NaH (60% dis-
(Bellingham + Stanley) at the sodium D line and are given in persion in mineral oil; 0.49 g, 12.0 mmol) was added portion-
deg cm3 g−1 dm−1 with concentration c in 10−2 g cm−3. Infra- wise to a stirred solution of dry MeCN (0.65 mL, 20.3 mmol) in
red spectra were recorded in the range 4000–600 cm−1 on a dry toluene (5 mL) at 0 °C under Ar, and the mixture was
Perkin-Elmer 1600 series FTIR spectrometer using an ATR warmed to room temperature and stirred for 2 h. The reaction
probe or using KBr disks or as a nujol mull for solid samples mixture was then cooled to 0 °C before dropwise addition of a
and thin films between NaCl plates for liquid samples and are solution of methyl 3-methoxybenzoate (9) (1.05 g, 6.3 mmol)
reported in cm−1. NMR spectra were recorded using a Varian in dry toluene (5 mL). Once complete, the solution was
VNMRS instrument operating at 400 or 500 MHz or a Bruker allowed to warm to room temperature and stirred at 90 °C for
Avance III 400 MHz for 1H spectra and 101 or 126 MHz for 13
C
16 h. After cooling, the reaction mixture was filtered and the
spectra; J values were recorded in Hz and multiplicities were filtrate was extracted with water (20 mL). The isolated solids
expressed by the usual conventions. Low resolution mass were then dissolved in the aqueous extract, acidified to pH 3–4
spectra were determined using a Waters Q-TOF Ultima using using aqueous HCl solution (1 M), extracted with EtOAc (3 ×
electrospray positive ionization, A Waters LCT premier XE 20 mL), dried (Na2SO4) and evaporated in vacuo. Purification
using atmospheric pressure chemical ionization (APcI), an by column chromatography on silica gel (dry load), gradient
Agilent 6130 single quadrupole with an APcI/electrospray dual eluting with hexanes to EtOAc–hexanes (2 : 3), and recrystalliza-
source, a Fisons Instrument VG Autospec using electron tion (EtOAc–hexanes) gave the title compound (0.36 g, 34%) as
ionization at 70 eV (low resolution) or a ThermoQuest Finni- a colourless solid, mp 87.3–88.2 °C (EtOAc) (lit.,42 mp
gan LCQ DUO electrospray, unless otherwise stated. TOFMS 87–88 °C), with identical spectroscopic properties.
refers to time-of-flight mass spectrometry, ES refers to electro-
3-Methoxybenzoylacetonitrile (6) by Heck coupling, followed
spray ionization, CI refers to chemical ionization (ammonia), by enol ether hydrolysis (Table 1). 3-Methoxyacrylonitrile (5)
FTMS refers to Fourier transform mass spectrometry, NSI (0.28 g, 3.40 mmol) was added to a stirred solution of 3-iodo-
refers to nano-electrospray ionization and EI refers to electron anisole (0.20 g, 0.86 mmol), base (1.71 mmol), tetrabutyl-
ionization. A number of high resolution mass spectra were ammonium bromide (27 mg, 86 µmol) and Pd(OAc)2 (19 mg,
obtained courtesy of the EPSRC Mass Spectrometry Service at 86 µmol) in water–MeCN (3 : 1) (3 mL) under Ar and the
University College of Wales, Swansea, UK using the ionization mixture was heated at 90 °C for 20 h (entries 1 and 2) or irra-
methods specified.
diated at 130 °C or 140 °C for 1–1.5 h in a pressure-rated glass
vial (10 mL) using a CEM Discover microwave synthesizer
(entries 3–6) by moderating the initial power (150 W). After
Synthetic procedures
3-Methoxybenzoylacetonitrile (6) by Claisen ester conden- cooling, the solution was partitioned between water and EtOAc
sation using LDA as base (Table 2, entry 6). n-BuLi (2.5 M in and the aqueous layer was further extracted with EtOAc (2 ×
hexanes; 5.3 mL, 13.2 mmol) was added dropwise to a stirred 10 mL). The organic extracts were combined, washed with
solution of DIPA (1.9 mL, 13.6 mmol) in dry THF (2 mL) at brine (20 mL), dried (Na2SO4) and evaporated in vacuo. The
0 °C under Ar, and the mixture was stirred at 0 °C for 10 min. crude residue was dissolved in MeOH (2 mL) in air and con-
In a separate flask, a solution of methyl 3-methoxybenzoate (9) centrated HCl was added (10.2 M; 0.5 mL). The mixture was
(1.1 g, 6.6 mmol) and dry acetonitrile (0.5 mL, 9.5 mmol) in irradiated at 110 °C for 1 h in a pressure-rated glass vial
dry THF (5 mL) was cooled to −50 °C. The cold LDA solution (10 mL) using a CEM Discover microwave synthesiser by mod-
was added dropwise and the reaction mixture was stirred at erating the initial power (150 W). After cooling in a flow of
−50 °C for 3 h. Saturated aqueous NH4Cl solution (10 mL) was compressed air, the solution was partitioned between water
then added. The solution was allowed to warm to room tem- and EtOAc, and the aqueous layer was further extracted with
perature, extracted with EtOAc (3 × 20 mL), washed with EtOAc (2 × 10 mL). The organic extracts were combined,
aqueous HCl solution (1 M; 20 mL) and brine (20 mL), dried washed with brine (20 mL), dried and evaporated in vacuo.
(MgSO4) and evaporated in vacuo. Purification by column Purification by column chromatography on silica gel (dry
chromatography on silica gel (dry load), gradient eluting with load), gradient eluting with hexanes to EtOAc–hexanes (2 : 3),
hexanes to EtOAc–hexanes (2 : 3), gave the title compound gave the title compound as a yellow solid, mp 87.0–87.6 °C
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
(MeOH) (lit.,42 mp 87–88 °C), with identical spectroscopic benzoic acid (8) (1.48 g, 9.7 mmol) in MeOH (10 mL) and the
properties. mixture was irradiated at 110 °C for 10 min in a pressure-rated
(E)- and (Z)-3-Methoxy-3-(3-methoxyphenyl)prop-2-enenitrile glass tube (35 mL) using a CEM Discover microwave synthe-
(7) by Heck coupling. 3-Methoxyacrylonitrile (5) (0.72 g, sizer by moderating the initial power (200 W). After cooling in
8.7 mmol) was added to a stirred solution of potassium a flow of compressed air, the solvent was evaporated in vacuo
carbonate (0.63 g, 4.5 mmol), tetrabutylammonium bromide and the residue was partitioned between water and Et2O. The
(80 mg, 0.24 mmol), 3-iodoanisole (0.47 g, 2.0 mmol) and ethereal solution was washed with saturated aqueous NaHCO3
palladium acetate (50 mg, 0.20 mmol) in water–MeCN (4 : 1) solution, dried (Na2SO4) and evaporated in vacuo to give the
(12 mL) under Ar and the solution was stirred at 90 °C for title compound (1.52 g, 94%) as a colourless oil, with identical
24 h. After cooling, the solution was partitioned between water physical and spectroscopic properties.
and EtOAc and the aqueous layer was further extracted with
2-(3-Methoxybenzoyl)-3-(phenylamino)acrylonitrile (11). A
EtOAc (2 × 20 mL). The organic extracts were combined, solution of 3-methoxybenzoylacetonitrile (6) (0.20 g, 1.1 mmol)
washed with brine (20 mL), dried (Na2SO4) and evaporated in and N,N′-diphenylformamidine (10) (0.24 g, 1.2 mmol) in dry
vacuo. Purification by column chromatography on silica gel xylenes (7.5 mL) was irradiated at 180 °C under Ar for 20 min
(dry load), gradient eluting with hexanes to EtOAc–hexanes in a pressure-rated glass tube (35 mL) using a CEM Discover
(15 : 85), gave the title compounds (0.26 g, 68%; Z : E (1 : 2)) as microwave synthesizer by moderating the initial power (200 W).
yellow oils [Rf 0.5 (Z)-7 and 0.3 (E)-7 in EtOAc–light petroleum After cooling in a stream of compressed air, the solution
(1 : 4)] (Found [ES]: 212.0679. C11H11NO2Na [MNa] requires was diluted with hexanes to give a precipitate. Isolation by
212.0682); IR (neat) νmax/cm−1 3069 (C–H str.), 2950 (C–H str.), gravity filtration gave the title compound (0.23 g, 74%) as a col-
2198 (CuN str.), 1605 (CvC str.), 1579 (C–C str.), 1437 (C–H ourless solid, mp 107.4–108.8 °C (lit.,22 mp 105 °C) (Found
bend), 1215 (C–O str.), 1117 (C–O str.), 1047 (C–O str.); [ES]: 279.1126. C17H15N2O2 [MH] requires 279.1128); IR (neat)
1H NMR (500 MHz, CDCl3) δH/ppm [(Z)-7] 7.31 (1H, t, J = 8, νmax/cm−1 3062 (C–H str.), 2921 (C–H str.), 2204 (CuN str.),
5′-CH), 7.11 (1H, d, J = 8, 6′-CH), 7.06 (1H, s, 2′-CH), 7.00 (1H, 1634 (CvO str.), 1596 (N–H bend), 1571 (C–C str.), 1392 (C–C
d, J = 8, 3′-CH), 4.96 (1H, s, 2-CH), 4.24 (3H, s, 3-OMe), 3.84 str.), 1313 (C–N str.), 1225 (C–O str.), 1044 (C–O str.), 990 (C–H
(3H, s, 3′-OMe); δH/ppm [(E)-7] 7.40–7.33 (2H, 5′,6′-CH), 7.30 bend), 867 (N–H wag); 1H NMR (500 MHz, CDCl3) δH/ppm
(1H, s, 2′-CH), 7.03 (1H, d, J = 7, 4′-CH), 4.65 (1H, s, 2-CH), 12.76 (1H, d, J = 12, NH), 8.06 (1H, d, J = 12, 3-CH), 7.57 (1H,
3.85 (6H, app s, 3- and 3′-OMe); 13C NMR (126 MHz, CDCl3) d, J = 8, 6′-CH), 7.46 (3H, m, 2′-, 3″, and 5″-CH), 7.40 (1H, t, J =
δC/ppm [(Z)-7] 170.9 (3-C), 159.7 (3′-C), 135.3 (1′-C), 129.7 (5′- 8, 5′-CH), 7.30 (1H, m, 4″-CH), 7.23 (2H, d, J = 8, 2″- and
CH), 118.9 (6′-CH), 117.3 (CN), 116.7 (4′-CH), 112.2 (2′-CH), 6″-CH), 7.11 (1H, d, J = 8, 6′-CH), 3.89 (3H, s, Me); 13C NMR
71.2 (2-CH), 59.6 (3-OMe), 55.4 (3′-OMe); δC/ppm [(E)-7] 173.0 (126 MHz, CDCl3) δC/ppm 192.1 (C), 159.6 (3′-C), 154.1 (3-CH),
(3-C), 159.4 (3′-C), 134.1 (1′-C), 129.5 (5′-CH), 120.3 (6′-CH), 139.2 (1′-C), 138.1 (1″-C), 130.2 (3″, 5″-CH), 129.4 (5′-CH), 126.6
118.4 (CN), 117.4 (4′-CH), 112.9 (2′-CH), 69.9 (2-CH), 56.8 (4″-CH), 120.5 (6′-CH), 120.3 (1-CN), 118.9 (4′-CH), 117.9
(3-OMe), 55.4 (3′-OMe); m/z (EI) 189 (M•+, 100%), 132 (29), (2″, 6″-CH), 112.7 (2′-CH), 83.5 (2-C), 55.5 (Me); m/z (EI): 278
69 (39).
Methyl 3-methoxybenzoate (9) using conductive heating. A
(M•+, 49%), 277 ([M − H]+, 100), 135 (52).
[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-3-methoxyphenyl
catalytic amount of concentrated H2SO4 (0.01 mL, 0.20 mmol) ketone (12). A mixture of 2-(3-methoxybenzoyl)-3-(phenyl-
was added to a stirred solution of 3-methoxybenzoic acid (8) amino)acrylonitrile (11) (0.13 g, 0.46 mmol), 4-fluorophenylhy-
(1.48 g, 9.7 mmol) in MeOH (10 mL) and the mixture was drazine hydrochlororide (3·HCl) (73 mg, 0.47 mmol) and
stirred at reflux for 3 h. After cooling to room temperature, the triethylamine (20 µL, 140 µmol) in ethanol (3 mL) was irra-
solvent was evaporated in vacuo and the residue was parti- diated at 140 °C for 1 h in a pressure-rated glass vial (10 mL)
tioned between water and Et2O. The ethereal solution was using a CEM Discover microwave synthesizer by moderating
washed with saturated aqueous NaHCO3 solution, dried the initial power (150 W). After cooling in a stream of com-
(Na2SO4) and evaporated in vacuo to give the title compound pressed air, the solvent was evaporated in vacuo. Purification
(1.45 g, 90%) as a colourless oil (Found [ES]: 189.0520. by column chromatography on silica gel (dry load), gradient
C9H10O3Na [MNa] requires 189.0522); IR (neat) νmax/cm−1 2953 eluting with hexanes to EtOAc–hexanes (3 : 7), gave the title
(C–H str.), 1718 (CvO str.), 1601 (CC str.), 1276 (C–O str.), compound (0.11 g, 86%) as a pink solid, mp 130.1–132.0 °C
1
1221 (C–O str.); H NMR (500 MHz, CDCl3) δH/ppm 7.64 (1H, (Found [ES]: 312.1138. C17H15FN3O2 [MH] requires 312.1143);
d, J = 8, 6-CH), 7.57 (1H, br. s, 2-CH), 7.35 (1H, t, J = 8, 5-CH), IR (neat) νmax/cm−1 3318 (N–H str.), 2925 (C–H str.), 1610
7.11 (1H, dd, J = 8, 2, 4-CH), 3.92 (3H, s, CO2Me), 3.86 (3H, s, (CvO str.), 1595 (N–H bend), 1539 (C–C str.), 1286 (C–N str.),
OMe); 13C NMR (126 MHz, CDCl3) δC/ppm 166.9 (C), 159.6 1223 (C–O str.), 1051 (C–O str.), 838 (N–H wag); 1H NMR
(3-C), 131.5 (1-C), 129.3 (5-CH), 122.0 (6-CH), 199.5 (4-CH), (400 MHz, CDCl3) δH/ppm 7.81 (1H, s, 3-CH), 7.56 (2H, m, 2′,
144.0 (2-CH), 55.4 (OMe), 52.1 (CO2Me); m/z (EI): 166 6′-CH), 7.42 (2H, m, 5″, 6″-CH), 7.35 (1H, s, 2″-CH), 7.25 (2H,
(M•+, 14%), 135 ([M − OMe]+, 39), 93 (100).
m, 3′, 5′-CH), 7.11 (1H, m, 4″-CH), 6.04 (2H, bs, exch. D2O,
Methyl 3-methoxybenzoate (9) using microwave irradiation. NH2), 3.89 (3H, s, Me); 13C NMR (101 MHz, CDCl3) δC/ppm
1
A
catalytic amount of concentrated H2SO4 (0.01 mL, 189.4 (C), 162.2 (d, JCF = 250, 4′-CF), 159.8 (3″-C), 150.6 (5-C),
4
0.20 mmol) was added to a stirred solution of 3-methoxy- 142.0 (3-CH), 141.1 (1″-C), 133.3 (d, JCF = 4, 1′-C), 129.5
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
3
(5″-CH), 126.1 (d, JCF = 9, 2′, 6′-CH), 120.7 (6″-CH), 117.8 (4″- 1222 (C–F str.), 1053 (C–O str.), 839 (NH2 wag.); 1H NMR
2
CH), 116.9 (d, JCF = 23, 3′, 5′-CH), 113.0 (2″-CH), 104.8 (4-C), (500 MHz, d6-DMSO) δH/ppm 7.80 (1H, s, 3H), 7.61 (2H, m, 2′,
55.4 (OMe); m/z (APcI) 312 (MH+, 100%).
6′-CH), 7.47–7.37 (3H, 3′, 4′- and 5″-CH), 7.33 (1H, d, J = 7,
[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-3-hydroxyphenyl 6″-CH), 7.25 (1H, m, 2″-CH), 7.14 (3H, 4″-CH and NH2), 4.95
ketone (13). Boron tribromide solution (1 M in CH2Cl2; (1H, bs, CHOH), 4.65 (1H, bs, CH2OH), 4.09 (1H, m, OCHH),
1.7 mL, 1.70 mmol) was added dropwise to a solution of 3.95 (1H, m, OCHH), 3.82 (1H, m, CHOH), 3.47 (2H, d, J = 5,
[5-amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-3-methoxyphenyl CH2OH); 13C NMR (126 MHz, d6-DMSO) δC/ppm 187.5 (C),
1
ketone (12) (0.10 g, 0.34 mmol) in dry CH2Cl2 (1 mL) at 0 °C 161.1 (d, JCF = 244, 4′-C), 158.7 (3″-C), 151.2 (5-C), 141.4
4
under Ar. The mixture was allowed to warm to room tempera- (3-CH), 140.9 (1″-C), 133.7 (d, JCF = 3, 1′-C), 129.6 (5″-CH),
ture and stirred for 20 h. Water (15 mL) was then added cau- 126.4 (d, 3JCF = 9, 2′, 6′-CH), 120.1 (6″-CH), 117.8 (4″-CH), 116.3
2
tiously and the mixture was extracted with EtOAc (3 × 15 mL). (d, JCF = 23, 3′, 5′-CH), 113.3 (2″-CH), 103.5 (4-C), 69.9
The organic extracts were combined, washed with brine, dried (CHOH), 69.8 (CH2O), 62.6 (CH2OH); m/z (EI) 371 (M•+, 10%),
(MgSO4) and evaporated in vacuo to give the title compound 325 (33), 160 (100).
(0.10 g, quant.) as a brown solid, mp 192.2–194.9 °C (Found
[ES]: 298.0984. C16H13FN3O2 [MH] requires 298.0986); IR (neat) Cells and cell growth
νmax/cm−1 3320 (N–H str.), 3204 (br. O–H), 3072 (C–H str.),
Human adult dermal primary Werner syndrome AG05229 and
1610 (CvO str.), 1592 (N–H bend), 1538 (C–C str.), 1310 (C–N
AG03141 fibroblasts were obtained from the Coriell Cell Re-
str.), 1126 (C–O str.), 841 (N–H wag); 1H NMR (500 MHz,
positories (Camden, NJ, USA). WStert cells are AG03141 fibro-
CD3OD) δH/ppm 7.78 (1H, s, 3-CH), 7.59 (2H, m, 2′, 6′-CH),
blasts that have been immortalised by the ectopic expression
7.37–7.29 (3H, 3′, 5′- and 5″-CH), 7.23 (1H, d, J = 7, 6″-CH),
of human enzyme telomerase and have been described
7.18 (1H, s, 2″-CH), 7.00 (1H, dd, J = 7, 1, 4″-CH); 13C NMR
previously.14
1
(100 MHz, CD3OD) δC/ppm 191.6 (C), 164.1 (d, JCF = 247, 4′-
Cells were grown in DMEM growth medium as previously
CF), 159.2 (3″-C), 153.4 (5-C), 143.6 (3-CH), 142.7 (1″-C), 135.0
described.15 Cell proliferation rates for the WStert cells were
4
3
(d, JCF = 3, 1′-C), 131.1 (5″-CH), 128.5 (d, JCF = 9, 2′, 6′-CH),
measured as cumulated population doublings (CPDs) divided
by the number of days of the experiment concerned and
expressed as a percentage of the proliferation rate of control
cells (with the control being 100%). The number of CPDs
achieved at each passage of the cells was calculated according
to the formula: PDs = log(Nt/No)/log2, where Nt is number of
cells counted and No is number of cells seeded. For primary
cells the CPDs were plotted versus days in culture. For asses-
sing the effects of the various kinase inhibitors the culture
medium was supplemented with the inhibitor dissolved in
DMSO, with the medium being replaced daily. For controls an
equivalent volume of the inhibitor solvent (DMSO) was added
to the medium.
3
120.6 (6″-CH), 120.1 (4″-CH), 118.0 (d, JCF = 24, 3′, 5′-CH),
116.0 (2″-CH), 105.7 (4-C); m/z (EI) 297 (M•+, 91%), 296
([M − H]+, 100), 204 (31).
5-Amino-1-(4-fluorophenyl)-4-{3-[2(S)-3-dihydroxypropoxy]-
benzoyl}pyrazole (RO3201195) (1). (S)-2,2-Dimethyl-1,3-dioxo-
lan-4-ylmethyl p-toluenesulfonate (14) (0.60 mL, 2.5 mmol)
and anhydrous K2CO3 (0.59 g, 4.25 mmol) were added to a
solution of [5-amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-3-
hydroxyphenyl ketone (13) (0.48 g, 1.61 mmol) in dry DMSO
(15 mL) under argon and the mixture was heated at 100 °C for
40 h. After cooling, the solution was diluted with water
(25 mL) and extracted with EtOAc (3 × 20 mL). The organic
extracts were combined, washed with brine, dried (MgSO4) and
evaporated in vacuo. Purification by column chromatography
on silica gel (dry load), gradient eluting with light petroleum
to EtOAc–light petroleum (2 : 3), gave [5-amino-1-(4-fluoro-
phenyl)-1H-pyrazol-4-yl] {3-[(R)-2,2-dimethyl-1,3-dioxolan-4-yl)-
methoxy]phenyl} ketone (15), which was dissolved in MeOH–
water (4 : 1) (4 mL). p-Toluene sulfonic acid monohydrate
(40 mg, 0.21 mmol) was added and the solution was heated at
50 °C for 18 h. After cooling, the solvent was evaporated in
vacuo. The residue was dissolved in EtOAc, washed with satu-
rated aqueous NaHCO3 solution, dried (MgSO4) and evapor-
ated in vacuo. Purification by column chromatography on
silica gel (dry load), gradient eluting with light petroleum to
EtOAc, followed by recrystallization (EtOAc–hexanes) gave the
title compound (0.22 g, 37%) as a colourless solid, mp
154.1–154.6 °C (EtOAc) (Found [ES]: 372.1353. C19H19FN3O4
[MH] requires 372.1354); [α]2D1 −28 (c 0.2, EtOAc); [α]D22 + 6.5
P38 inhibitor assay
The ability of SB203580 and RO3201195 to inhibit the p38α
signalling pathway was tested in human immortalised HCA2
cells using an ELISA system (obtained from Cell Signalling,
NEB, UK) as previously described.39 Kinase activity was
detected using antibodies specific for HSP27 and its phos-
phorylated form, the degree of activation being measured as
the ratio of phospho-protein/total protein (Fig. 2d and e). In
this system activation of p38α by anisomycin activates the
kinase MK2 that then phosphorylates the small heat shock
protein HSP27. As MK2 is the major HSP27 kinase,43 the
activity of p38α can be assessed by the phosphorylation status
of HSP27.
Immunoblot assays for stress kinase activity in WS cells
(c 0.6, MeOH); IR (neat) νmax/cm−1 3440 (N–H str.), 3330 (O–H The ability of SB203580 and RO3201195 to inhibit the p38α
str.), 3229 (O–H str.), 2896 (C–H str.), 1633 (CvO str.), 1597 and JNK signalling pathways in WStert cells was tested by
(NH2 str.), 1540 (CvN str.), 1498 (C–C str.), 1292 (C–O str.), immunoblot detection of the phosphorylated versions of their
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
downstream targets HSP27 (pHSP27) and c-Jun (p-c-Jun) as EPSRC Mass Spectrometry Service at the University of Wales,
previously described.15,44
Swansea UK for mass spectra.
Conclusions
Notes and references
This manuscript describes the chemical synthesis and biologi-
cal evaluation of a p38α MAPK inhibitor, RO3201195 (1), in
WS cells. Two complementary routes for the chemical syn-
1 J. C. Lee, J. T. Laydon, P. C. McDonnell, T. F. Gallagher,
S. Kumar, D. Green, D. McNulty, M. J. Blumenthal,
J. R. Heys, S. W. Landvatter, et al., Nature, 1994, 372, 739.
2 G. L. Schieven, Curr. Top. Med. Chem., 2005, 5, 921.
3 E. Salgado, J. R. Maneiro, L. Carmona and J. J. Gomez-
Reino, Ann. Rheum. Dis., 2014, 73, 871.
4 M. Back and G. K. Hansson, Nat. Rev. Cardiol., 2015, 12,
199–211.
5 V. Grossi, A. Peserico, T. Tezil and C. Simone, World
J. Gastroenterol., 2014, 20, 9744.
thesis of
a key intermediate, benzoylacetonitrile 6, are
described, both of them involving the use of microwave dielec-
tric heating for rapid reaction kinetics. Of these two routes, a
classical approach based upon a Claisen ester condensation
was found to be the most efficient. The benzoylacetonitrile
intermediate 6 was transformed by Knoevenagel condensation,
followed by cyclocondensation with a hydrazine, both under
microwave irradiation, to give the core pyrazole motif. Phenox-
ide alkylation by SN2 displacement of tosylate, followed by
ketal deprotection, gave the target inhibitor in a sequence of
7 steps and 15% overall yield.
6 K. Yumoto, M. R. Eber, J. E. Berry, R. S. Taichman and
Y. Shiozawa, Clin. Cancer Res., 2014, 20, 3384.
7 C. Dominguez, D. A. Powers and N. Tamayo, Curr. Opin.
Drug Discovery Dev., 2005, 8, 421.
The effects of RO3201195 (1) on cellular proliferation were
first examined using WStert cells, where it was found that the
inhibitor elicited a very similar response to the prototypical
p38 MAPK inhibitor SB203580. The maximal increase seen in
8 D. J. Diller, T. H. Lin and A. Metzger, Curr. Top. Med.
Chem., 2005, 5, 953.
9 D. M. Goldstein and T. Gabriel, Curr. Top. Med. Chem.,
2005, 5, 1017.
the proliferation rate of WStert cells was 19% at 10.0 µM con- 10 S. T. Wrobleski and A. M. Doweyko, Curr. Top. Med. Chem.,
centration of RO3201195 (1). At this concentration, the inhibi-
2005, 5, 1005.
tor completely inhibited p38α but did not inhibit total JNK 11 J. Saklatvala, Curr. Opin. Pharmacol., 2004, 4, 372.
activity in WStert cells, as determined by immunoblot assay. 12 D. Kipling, T. Davis, E. L. Ostler and R. G. Faragher,
Following these findings, the cellular senescence of primary
Werner syndrome fibroblast strain WAG05229 was investigated 13 T. Davis, D. M. Baird, M. F. Haughton, C. J. Jones and
on treatment with SB203580 or RO3201195 (1). Despite the D. Kipling, J. Gerontol., Ser. A, 2005, 60, 1386.
fact that these inhibitors showed very different kinase speci- 14 T. Davis, M. F. Haughton, C. J. Jones and D. Kipling,
ficity profiles at the concentrations used, the effects on WS Ann. N. Y. Acad. Sci., 2006, 1067, 243.
Science, 2004, 305, 1426.
cells were essentially identical. Thus, we concluded that the 15 T. Davis and D. Kipling, Biogerontology, 2009, 10, 253.
effects on proliferation of treating WStert cells or primary WS 16 T. Davis and D. Kipling, Rejuvenation Res., 2006, 9, 402.
fibroblasts with SB203580 or RO3201195 (1) resulted from 17 M. C. Bagley, T. Davis, M. J. Rokicki, C. S. Widdowson and
inhibition of p38α. This was further supported by studying the
D. Kipling, Future Med. Chem., 2010, 2, 193.
effects of a pyrazolyl ketone library on WStert cells at a given 18 J. Bain, L. Plater, M. Elliott, N. Shpiro, C. J. Hastie,
concentration, which showed a linear relationship between the
H. McLauchlan, I. Klevernic, J. S. Arthur, D. R. Alessi and
proliferation rate and % p38α activity. Thus, we conclude
P. Cohen, Biochem. J., 2007, 408, 297.
RO3201195 (1) is a useful chemical tool for dissecting signal- 19 K. Godl and H. Daub, Cell Cycle, 2004, 3, 393.
ling pathways in human cells in order to understand the effect 20 D. M. Goldstein, T. Alfredson, J. Bertrand, M. F. Browner,
of p38 MAPK signalling upon cell proliferation in this human
progeroid syndrome.
K. Clifford, S. A. Dalrymple, J. Dunn, J. Freire-Moar,
S. Harris, S. S. Labadie, J. La Fargue, J. M. Lapierre,
S. Larrabee, F. Li, E. Papp, D. McWeeney, C. Ramesha,
R. Roberts, D. Rotstein, B. San Pablo, E. B. Sjogren,
O. Y. So, F. X. Talamas, W. Tao, A. Trejo, A. Villasenor,
M. Welch, T. Welch, P. Weller, P. E. Whiteley, K. Young and
S. Zipfel, J. Med. Chem., 2006, 49, 1562.
Acknowledgements
We thank the EPSRC-BBSRC-MRC sponsored network
SMS-Drug (EP/I037229/1; award to MCB), EPSRC (studentship 21 M. C. Bagley, T. Davis, P. G. S. Murziani, C. S. Widdowson
award to JED), Saudi Cultural Bureau (support for MB), BBSRC and D. Kipling, Pharmaceuticals, 2010, 3, 1842.
(BB/D524140/1; award to DK, MCB and TD), SPARC (awards to 22 M. C. Bagley, T. Davis, M. C. Dix, P. G. Murziani,
MCB and TD), ESRC under the New Dynamics of Ageing
Initiative (RES-356-25-0024; award to MCB, DK and TD) and
M. J. Rokicki and D. Kipling, Future Med. Chem., 2010, 2,
203.
the R M Phillips Trust (award to MCB) for support of this work 23 T. Mizoroki, K. Mori and A. Ozaki, Bull. Chem. Soc. Jpn.,
and Dr Alaa Abdul-Sada at the University of Sussex and the
1971, 44, 581.
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
24 R. F. Heck and J. P. Nolley, J. Org. Chem., 1972, 37, 2320.
36 M. C. Bagley, M. C. Lubinu and C. Mason, Synlett, 2007, 704.
25 A. Battace, M. Feuerstein, M. Lemhadri, T. Zair, H. Doucet 37 M. C. Bagley, M. Baashen, V. L. Paddock, D. Kipling and
and M. Santelli, Eur. J. Org. Chem., 2007, 3122. T. Davis, Tetrahedron, 2013, 68, 8429.
26 M. Beller and T. H. Riermeier, Tetrahedron Lett., 1996, 37, 38 M. C. Bagley, T. Davis, M. C. Dix, C. S. Widdowson and
6535. D. Kipling, Org. Biomol. Chem., 2006, 4, 4158.
27 T. Sakamoto, Y. Kondo, Y. Kashiwagi and H. Yamanaka, 39 M. C. Bagley, T. Davis, M. C. Dix, P. G. Murziani,
Heterocycles, 1988, 27, 257.
28 N. A. Cortese, C. B. Ziegler, B. J. Hrnjez and R. F. Heck,
J. Org. Chem., 1978, 43, 2952.
29 A. F. Littke and G. C. Fu, J. Am. Chem. Soc., 2001, 123,
6989.
30 C. M. Andersson, A. Hallberg and G. D. Daves, J. Org.
Chem., 1987, 52, 3529.
M. J. Rokicki and D. Kipling, Bioorg. Med. Chem. Lett.,
2008, 18, 3745.
40 S. P. Davies, H. Reddy, M. Caivano and P. Cohen, Biochem.
J., 2000, 351, 95.
41 K. Godl, J. Wissing, A. Kurtenbach, P. Habenberger,
S. Blencke, H. Gutbrod, K. Salassidis, M. Stein-Gerlach,
A. Missio, M. Cotten and H. Daub, Proc. Natl. Acad.
Sci. U. S. A., 2003, 100, 15434.
31 T. Jeffery, J. Chem. Soc., Chem. Commun., 1984, 1287.
32 S. Cacchi, P. G. Ciattini, E. Morera and G. Ortar, Tetra- 42 C. Ota, S. Kumata and S. Kawaguchi, EP 1544202A1, 2005,
hedron Lett., 1987, 28, 3039.
56–57.
33 T. Jeffery, Tetrahedron, 1996, 52, 10113.
34 H. Zhao, M.-Z. Cai and C.-Y. Peng, Synth. Commun., 2002,
32, 3419.
35 J. B. Dorsch and S. M. McElvain, J. Am. Chem. Soc., 1932,
54, 2960.
43 J. X. Shi, X. Su, J. Xu, W. Y. Zhang and Y. Shi,
Am. J. Physiol.: Lung Cell. Mol. Physiol., 2012, 302, L793.
44 T. Davis, M. C. Dix, M. J. Rokicki, A. J. C. Brook,
C. S. Widdowson, D. Kipling and M. C. Bagley, Chem. Cent.
J., 2011, 5, 83.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2015