Microwave-assisted synthesis of 5-aminopyrazol-4-yl ketones and the p38MAPK inhibitor RO3201195 for study in Werner syndrome cells

Article abstract of DOI:10.1016/j.bmcl.2008.05.037

5-Aminopyrazol-4-yl ketones are prepared rapidly and efficiently using microwave dielectric heating from β-ketonitriles by treatment with N,N′-diphenylformamidine followed by heterocyclocondensation by irradiation with a hydrazine. The inhibitory activity of RO3201195 prepared by this methodology was confirmed in hTERT-immortalized HCA2 and WS dermal fibroblasts at 200 nM concentration, both by ELISA and immunoblot assay, and displays excellent kinase selectivity for p38α MAPK over the related stress-activated kinase JNK.

Full text of DOI:10.1016/j.bmcl.2008.05.037

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3745–3748
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Microwave-assisted synthesis of 5-aminopyrazol-4-yl ketones and the
p38MAPK inhibitor RO3201195 for study in Werner syndrome cells
b,
a
a
b
Mark C. Bagley a, , Terence Davis , Matthew C. Dix , Paola G. S. Murziani , Michal J. Rokicki ,
*
*
David Kipling b,
*
a School of Chemistry, Main Building, Cardiff University, Park Place, Cardiff CF10 3AT, UK
b Department of Pathology, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
5-Aminopyrazol-4-yl ketones are prepared rapidly and efficiently using microwave dielectric heating
from b-ketonitriles by treatment with N,N0-diphenylformamidine followed by heterocyclocondensation
by irradiation with a hydrazine. The inhibitory activity of RO3201195 prepared by this methodology
was confirmed in hTERT-immortalized HCA2 and WS dermal fibroblasts at 200 nM concentration, both
by ELISA and immunoblot assay, and displays excellent kinase selectivity for p38a MAPK over the related
stress-activated kinase JNK.
Received 6 March 2008
Revised 9 May 2008
Accepted 10 May 2008
Available online 16 May 2008
Keywords:
p38 MAPK
Ó 2008 Elsevier Ltd. All rights reserved.
Werner syndrome
RO3201195
Microwaves
Heterocycles
Werner syndrome (WS) is a rare genetic disorder that is used as
a model disease to investigate normal human ageing and results
from mutation in the WRN gene.1 WS individuals show premature
onset of many clinical features of old age, early susceptibility to
several major age-related diseases and a shortened median life
expectancy (47 years) as a consequence of malignancy and myo-
cardial infarction. This accelerated in vivo ageing is associated with
premature senescence of WS cells in vitro that may underlie sev-
eral features of the rapid ageing seen in WS.2,3 Normal human cells
are capable of only a finite number of divisions in culture after
which is triggered a non-proliferative state known as senescence.
Whether cellular senescence plays a role in organismal ageing
has been hotly disputed,4 however, senescent cells are known to
accumulate during ageing, for example, in the skin,5 and evidence
is growing that supports the hypothesis that this finite prolifera-
tive capacity may be a causal agent of normal ageing processes.
Senescent cells display deleterious biochemical features, such as
a pro-inflammatory phenotype, suggesting a link between replica-
tive senescence and tissue degeneration.6 Normal and WS fibro-
blasts use telomere erosion as a cell division ‘counter’,7 but
recently it has been shown that this telomere-driven senescence
synergizes with an additional telomere-independent mechanism
in WS.8 Unlike normal human fibroblasts, proliferating WS cells
contain high levels of phosphorylated p38a,8 a stress-activated
MAPK, suggesting that WS cells undergo stress-induced premature
senescence.8,9 If accelerated ageing in WS is related to p38 activa-
tion and to accelerated cell ageing, WS may provide a powerful
model to link cellular signalling events to the ageing of mitotic tis-
sues in vivo. In addition, blocking the p38a response through the
use of a chemical inhibitor has the potential to interfere with the
pathology of WS, and so may provide opportunities for therapeutic
intervention.
In our previous studies, we tested this hypothesis to dramatic
effect in hTERT-immortalized WS dermal fibroblasts and in pri-
mary WS cells. When these cells were treated with the prototypical
p38 inhibitor SB203580, all of the features of accelerated replica-
tive decline were rescued, including growth rate and cell morphol-
ogy,8 indicating that the abbreviated life span of WS cells could be
due to a stress-induced growth arrest mediated by p38a MAPK.9
Subsequently we showed that alternative p38 inhibitors with im-
proved kinase selectivity, including BIRB 79610 and VX-745,11
could be prepared using microwave-assisted methods and re-
ported their activity in WS cells. However BIRB 796 also inhibits
the related stress-activated JNK kinases,12 as does SB203580, and
as young WS cells show elevated levels of phosphorylated JNK1a1
and JNK2a1 isoforms of JNK (our unpublished data), facile access to
another chemotype without JNK inhibitory activity that is orally
bioavailable is required to corroborate our findings and thence pro-
gress towards a clinical study. The 5-aminopyrazol-4-yl ketone
p38 inhibitor RO3201195 (Fig. 1)13 fits this profile, combining good
cellular activity, pharmacokinetic properties, metabolic stability
* Corresponding authors.
E-mail address: Bagleymc@cardiff.ac.uk (M.C. Bagley).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.037

3746
M. C. Bagley et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3745–3748
tive heating and microwave irradiation in a sealed vessel at 100–
140 °C in the presence or absence of Et3N (Table 1).16
O
O
N
It was apparent from the microwave irradiation experiments
that pyrazolyl ketone formation was, for the most part, rapid and
high-yielding. For hydrazine hydrochlorides, the addition of Et3N
to the reaction mixture improved the efficiency significantly. From
pyrazolyl ketone 4b, further elaboration to RO3201195 involved
deprotection of the phenolic ether by boron tribromide-mediated
protodemethylation, followed by O-alkylation of 5 with (S)-O-iso-
propylideneglycerol tosylate to give (R)-ketal 6 which could be
readily deprotected under acid-catalyzed aqueous conditions to
give the (S)-diol RO320119517 (Scheme 2) in accordance with
Roche’s original strategy.13 Although the yield for these final steps
was not high, it did provide sufficient compound for study in WS
cells.
N
O
N
N
H
N
H
BIRB 796
O
heteroannulation
O
N
H2N
O
-alkylation
HO
N
The ability of RO3201195 to inhibit p38a and JNK was tested in
human hTERT-immortalized HCA2 cells by ELISA as described pre-
viously.11,18 Kinase activity is detected using antibodies specific for
phosphorylated (S73)HSP27 and antibodies that detect total levels
of HSP27, the degree of activation being measured as the ratio of
phospho-protein/total protein. In this system p38 activation by
anisomycin activates MK2 that then phosphorylates HSP27. For
JNK the phosphorylation status of the JNK substrate c-jun is mon-
itored. In addition, the effect of RO3201195 on the p38 signalling
pathway in WS cells was assessed by immunoblots.11,18
HO
F
RO3201195
Figure 1. The p38 MAPK inhibitors BIRB 796 and RO3201195, the latter showing
our disconnective strategy.
and bioavailability with an excellent selectivity profile as deter-
mined in a panel of 105 kinases.
That RO3201195 can inhibit the p38a-signalling pathway is
shown in Figure 2a and c. In control cells, low p38a activity is indi-
cated by low p-HSP27 levels and a low p-HSP27/HSP27 ratio
(DMSO columns). Anisomycin treatment activates p38a that in-
creases p-HSP27 levels and the p-HSP27/HSP27 ratio (An columns).
RO3201195 pre-treatment increasingly inhibits the anisomycin-in-
duced activity of p38a, as indicated by the decreasing p-HSP27 lev-
els and the p-HSP27/HSP27 ratio. Maximal inhibition is achieved
between 2.5 and 10.0 lM RO3201195. The IC50 in this system is
approximately 180–200 nM, similar to the reported IC50 for
RO3201195 inhibition of p38a induced TNFa or IL-1b production
in peripheral blood mononucleocytes of 500 nM and 400 nM,
Our synthetic approach to RO3201195 (Fig. 1) utilized a linear
strategy: the 5-amino-4-benzoylpyrazole motif would be assem-
bled by regiospecific heterocyclocondensation of the corresponding
hydrazine and a b-anilino-a-benzoylacrylonitrile formed by Knoe-
venagel-type condensation. O-Alkylation installs the propyloxy
side chain to improve the pharmacokinetic properties as described
by Roche.13 We have shown that related heterocyclocondensation
reactions are accelerated dramatically by microwave dielectric
heating and used this in the synthesis of polysubstituted pyra-
zoles14 and the 5-aminopyrazole core of BIRB 796.10 For the synthe-
sis of RO3201195, two benzoylacetonitriles, 2a and 2b, the latter
readily available from 1 by reaction with acetonitrile under basic
conditions,15 were reacted with N,N0-diphenylformamidine to give
cyclocondensation precursors 3a,b (Scheme 1). Although the Knoe-
venagel condensation proceeded in good yield for benzoylaceto-
nitriles 2a and 2b under traditional conductive heating,
microwave irradiation at 180 °C afforded 3b more efficiently after
only 20 min. A variety of conditions were investigated for heterocy-
clocondensation with a range of hydrazines, present as the free base
or hydrochloride salt, including a traditional reflux under conduc-
Table 1
Microwave-assisted conditions for the synthesis of 4a–n
Entry
4
R1
R2
Yield%a
C
A
B
D
E
1b
2b
3
a
b
c
d
e
f
g
h
i
j
k
l
m
n
H
Ph
41
37
75
43
66
37
95
78
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
4-Fluorophenyl
4-Nitrophenyl
4-Methoxyphenyl
tert-Bu
p-Tolyl
Me
36
41
86c
4b
5b
6b
7
71
82
58
36
17
53
71
67
9
O
O
CO2Me
46
77
78
CN
CN
i
ii or
iii
8
9
H
Pentafluorophenyl
2,4-Difluorophenyl
2-Fluorophenyl
4-Bromophenyl
Pentafluorophenyl
2,5-Dichlorophenyl
ref. 15
10b
11b
12b
13
14
74
72
63
>98
90
92
NHPh
OMe
R1
R1
18
1
2a R1 = H;
3a R1 = H (ii 95%; iii 70%);
89
95
2b R1 = OMe
3b R1 = OMe (ii 85%; iii 90%)
H
a
O
Isolated yield of 4 from one of the methods A–E. Method A: EtOH, reflux, 3 h
(5 h for entry 2). Method B: microwaves, EtOH, 100 °C, 40 min (entry 1) or 120 °C,
20 min (entry 2) or 1 h. Method C: microwaves, EtOH, Et3N, 100 °C (entries 1 and 2)
or 120 °C (entries 10–12), 1 h. Method D: microwaves, EtOH, 140 °C, 20 min (entry
2) to 1 h (otherwise). Method E: microwaves, EtOH, Et3N, 140 °C, 1 h. ‘Microwaves’
indicates microwave irradiation in a 10 mL PyrexTM tube using a CEM DiscoverTM
single-mode instrument at the specified temperature by moderating the initial
magnetron power (100 W).
see Table 1
R2NHNH2
N
N
H2N
R1
R2
4a–n
b
The corresponding hydrazine hydrochloride was used.
On scale up (124 mg of 3b), irradiation at 110 °C for 1 h followed by 140 °C for
Scheme 1. Synthesis of pyrazol-4-yl ketones 4a–n. Reagents and conditions: (i)
MeCN, NaOEt, 80 °C (Ref. 15); (ii) PhN@CHNHPh, dry xylenes, reflux, 1.5–2 h; (iii)
PhN@CHNHPh, xylenes, microwaves, 180 °C (initial power: 3a, 100 W; 3b, 160 W),
20 min.
c
40 min avoided untoward pressure build up and gave 4b (95 mg, 68%) suitable for
elaboration to RO3201195.

M. C. Bagley et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3745–3748
3747
O
O
N
O
N
H2N
ii
N
N
H2N
R'O
R'O
RO
F
F
4b R = Me
5 R = H
6 R' = CMe2
RO3201195 R' = H
i
iii
Scheme 2. Synthesis of RO3201195 from pyrazol-4-yl ketone 4b. Reagents and
conditions: (i) BBr3, CH2Cl2, 0 °C; RT, 2.5 h; H2O (59%); (ii) (S)- -a,b-isopropylid-
eneglycerol-c-tosylate, K2CO3, DMF, 80 °C, 24 h (9%); (iii) p-TsOH, MeOH, H2O,
50 °C, 18 h (48%). RT, room temperature.
Figure 3. Effects of RO3201195 treatment on the activity of p38a in hTERT-immor-
talized WS cells. Lane 1, WS cells; lane 2, WS cells + anisomycin (A); lanes 3–5 WS
cells + A + 2.5, 10.0 and 25.0 lM RO3201195; lane 6, WS cells + A + 2.5 lM
SB203580. p-p38 and p-HSP27 are the phosphorylated forms of p38 and HSP27.
With MK2 two bands are seen (arrows): the upper is activated (phosphorylated) MK2.
L
low level of activated p38 as assessed by the presence of activated
MK2 (upper band in lane 1 of Fig. 3) and low levels of p-HSP27.
Anisomycin treatment greatly increases the activation of p38a
causing an increase in activated MK2 and p-HSP27 levels (lane
2). RO3201195 pre-treatment inhibits the anisomycin-induced
activity of p38a, as indicated by the much-reduced levels of acti-
vated MK2 and p-HSP27 (lanes 3–5). As the level of activated
MK2 is reduced in the RO3201195-treated cells compared to the
control (compare lanes 1 and 5), RO3201195 not only inhibits
the anisomycin-induced activity of p38a, but also the p38a activity
that is believed to be caused by genomic stress in the WS cells.
In conclusion, the p38a MAPK inhibitor RO3201195 and other
phenyl pyrazol-4-yl ketones can be prepared rapidly and efficiently
from b-(anilino)-a-benzoylacrylonitriles by microwave irradiation
with the corresponding hydrazine in ethanolic solvent. Further
elaboration provided RO3201195, the inhibitory activity of which
was confirmed in HCA2 and WS cells by ELISA and immunoblot as-
say, showing excellent selectivity for p38a MAPK over JNK. Given
this selectivity profile, RO3201195 would appear to be ideal for
further study to rescue premature senescence and the accelerated
ageing of WS cells in culture. These studies are now underway in
our laboratories and will be reported in due course.
Acknowledgements
Figure 2. ELISA results for the effect of RO3201195 on p38a activity (a and c) and
on JNK activity (b and d). For the upper panels, total protein is indicated by the
white bars, and the phosphorylated protein by the dark grey bars. In the lower
panel the ratio of phosphorylated protein to total protein is indicated by the black
bars. DMSO are cells with only DMSO treatment, An are cells treated with aniso-
mycin, and 0.010–50.000 are cells pre-treated with increasing concentrations of
RO3201195 followed by treatment with anisomycin. p-HSP27 and p-c-jun are the
phosphorylated forms of HSP27 and c-jun, respectively.
We thank the BBSRC (BB/D524140), EPSRC (GR/S25456; DTA
award to CSW) and SPARC (awards to T.D. and M.C.B.) for support
of this work and the EPSRC Mass Spectrometry Service at the Uni-
versity of Wales, Swansea UK for mass spectra.
References and notes
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respectively, and the IC50 for RO3201195 inhibition of p38a-in-
duced TNFa production in THP-1 cells of 250 nM.13 Anisomycin
treatment also activates JNK1 and JNK2 resulting in elevated levels
of p-c-jun and an increased p-c-jun/c-jun ratio (Fig. 2b and d).
RO3201195 is reported to inhibit JNK2 in an in vitro assay with a
Kd of 5.0 lM;13 however, RO3201195 pre-treatment of HCA2 cells
does not prevent the anisomycin-induced phosphorylation of c-
jun up to 50 lM. As the ELISA measures both JNK1 and JNK2 activ-
ity, these data show that RO3201195 does not inhibit total cellular
JNK activity, and do not preclude the possibility that RO3201195
can inhibit either JNK1 or JNK2 alone.13
The ability of RO3201195 to inhibit the p38a-signalling path-
way in hTERT-immortalized AG03141 WS cells was tested by im-
muno-detection of activated versions of p38a, MK2 and HSP27
on Western blots as described previously11 (Fig. 3). In control WS
cells, phosphorylated p38 is not detected; however, there is a

3748
M. C. Bagley et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3745–3748
11. Bagley, M. C.; Davis, T.; Dix, M. C.; Rokicki, M.; Kipling, D. Bioorg. Med. Chem.
Lett. 2007, 17, 5107.
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Glimore, T.; Graham, A. G.; Hickey, E.; Klaus, B.; Madwed, J.; Moriak, M.; Moss,
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microwave power (100 W). After cooling in a stream of compressed air, the
mixture was evaporated in vacuo and purified by column chromatography on
silica, eluting with petroleum ether–EtOAc (6:4), to give 4b (19 mg, 86%).
17. 5-Amino-1-(4-fluorophenyl)-4-{3-[2(S),3-dihydro-xypropoxy]benzoyl}pyrazole
(RO3201195)[Rf 0.27 (CH2Cl2–MeOH, 9:1)] wasobtainedas a colourlesssolid, mp
75 °C (MeOH) (Found: MH+, 372.1354. C19H19N3O4F requires [MH+], 372.1352);
22
½aꢀD þ 4:8 (c 0.23, MeOH);IR(KBr)/cmꢁ1 mmax 3404, 2928, 1623, 1600, 1576, 1537,
1513, 1503, 1440, 1313, 1288, 1223, 1049, 932, 843, 767; 1H NMR (400 MHz,
DMSO-d6): dH 3.43–3.48 (2H, m, CH2OH), 3.78–3.85 (1H, m, CHOH), 3.90–3.96
(1H, m, OCHHCHOH), 4.05–4.11 (1H, m, OCHHCHOH), 4.72 (1H, t, J 5.7 Hz,
HOCH2), 5.02 (1H, d, J 4.8 Hz, HOCH), 7.13–7.20 (3H, NH2 and 40-ArH), 7.24 (1H, s,
20-ArH), 7.31–7.35 (1H, m, 50-ArH), 7.37–7.46 (3H), 7.58–7.64 (2H), 7.80 (1H, s, 3-
H); 13C NMR (100 MHz, DMSO-d6): dC 62.6 (CH2), 69.7 (CH2), 69.9 (CH), 103.4 (C),
14. Bagley, M. C.; Lubinu, M. C.; Mason, C. Synlett 2007, 704.
15. Dorsch, J. B.; McElvain, S. M. J. Am. Chem. Soc. 1932, 54, 2960.
2
3
113.2 (CH), 116.2 (d, JC–F 21.2 Hz, CH), 117.8 (CH), 120.1 (CH), 126.4 (d, JC–F
9.1 Hz, CH), 129.7 (CH), 133.7 (d, 4JC–F 3.0 Hz, C), 140.9 (C), 141.5 (CH), 151.2 (C),
158.7 (C), 161.1 (d, 1JC–F 245.9 Hz, C), 187.6 (C); m/z (APcI) 372 (MH+, 100%).
18. Davis, T.; Bagley, M. C.; Dix, M. C.; Murziani, P. G. S.; Rokicki, M. J.; Widdowson,
C. S.; Zayed, J. M.; Bachler, M. A.; Kipling, D. Bioorg. Med. Chem. Lett. 2007, 17,
6832.
16. In
a typical procedure, a mixture of 3-methoxy-2-benzoyl-3-phenyl-
aminoacrylonitrile (3b) (20 mg, 0.07 mmol) and p-fluorophenylhydrazine
hydrochlororide (12 mg, 0.07 mmol) and triethylamine (11 lL, 0.08 mmol) in
ethanol (0.8 mL) was irradiated in a sealed tube at 140 °C for 1 h using a CEM
DiscoverTM single-mode microwave synthesizer, by moderating the initial