Synthesis and in vivo activity of MK2 and MK2 substrate-selective p38αMAPK inhibitors in Werner syndrome cells

Article abstract of DOI:10.1016/j.bmcl.2007.10.036

A benzopyranopyridine inhibitor of mitogen-activated protein kinase-activated protein kinase 2 (MK2) is prepared rapidly and efficiently in one step using microwave dielectric heating, whereas a substrate-selective p38 MAPK inhibitor was prepared using co

Full text of DOI:10.1016/j.bmcl.2007.10.036

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6832–6835
Synthesis and in vivo activity of MK2 and MK2
substrate-selective p38aMAPK inhibitors in Werner syndrome cells
b,
b
Terence Davis,a,* Mark C. Bagley, Matthew C. Dix,
*
Paola G. S. Murziani,b Michal J. Rokicki,a Caroline S. Widdowson,b
Jameel M. Zayed,b Marcus A. Bachlera and David Kiplinga,*
aDepartment of Pathology, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK
bSchool of Chemistry, Main Building, Cardiff University, Park Place, Cardiff CF10 3AT, UK
Received 17 August 2007; revised 5 October 2007; accepted 6 October 2007
Available online 17 October 2007
Abstract—A benzopyranopyridine inhibitor of mitogen-activated protein kinase-activated protein kinase 2 (MK2) is prepared rap-
idly and efficiently in one step using microwave dielectric heating, whereas a substrate-selective p38 MAPK inhibitor was prepared
using conventional heating techniques. The former had MK2 inhibitory activity above 2.5 lM concentration, whereas the latter
showed no MK2 inhibition at 10 lM. However, rather than rescuing the reduced cellular growth rate and aged morphology of
hTERT-immortalised WS dermal fibroblasts, both induce a state resembling stress-induced cellular senescence, suggesting that these
inhibitors may have limited therapeutic use.
Ó 2007 Elsevier Ltd. All rights reserved.
Werner syndrome (WS) is a genetic disorder resulting
from mutation in the RecQ helicase-encoding WRN
gene.1 WS individuals show premature onset of many
clinical features of old age, show early susceptibility to
a number of major age-related diseases and have a
greatly abbreviated median life expectancy (47 years)
as a consequence of malignancy and myocardial infarc-
tion.1 WS is widely used as a model disease to investigate
normal human ageing processes.2 Several observations
suggest that accelerated cellular senescence may underlie
several features of accelerated ageing in WS, as cells
from a dividing tissue (e.g., skin fibroblasts) that shows
premature in vivo ageing also show accelerated in vitro
ageing,3,4 whereas cells from a dividing tissue (e.g., T
cells) that does not manifest premature ageing in vivo
do not.5 Whether cellular senescence plays a role in
organismal ageing has been hotly disputed;6 however,
recent observations demonstrate that senescent fibro-
blasts accumulate in the skin during ageing.7 Normal
and WS fibroblasts use telomere erosion as a cell divi-
sion ‘counter’ and senesce with short telomeres.8 How-
ever, unlike normal fibroblasts, WS fibroblasts show
activation of the stress-associated MAPK p38a (herein
p38), and studies using the p38 inhibitor SB203580 res-
cued the features of accelerated replicative decline and
allowed the WS cells to attain a normal lifespan.4 These
data suggest that WS cells show premature senescence
via activation of a ‘stress signal’, possibly due to the
stalled DNA replication forks that are characteristic of
cells deficient in the WRNp helicase,9 leading to a phe-
nomenon known as replication stress.10 This replication
stress synergises with telomere erosion to limit WS cell
lifespan. If accelerated ageing in WS is related to p38
activation and to accelerated cell ageing, then WS pro-
vides a powerful model to link cellular signalling events
to the ageing of mitotic tissues in vivo, and also may
provide opportunities for therapeutic intervention.
However, p38 regulates many cellular processes making
p38 inhibitors problematical for therapeutic use due to
the possibilities of deleterious side effects.11
Many young WS cells are enlarged with prominent F-
actin stress fibres and resemble aged normal cells.4 The
p38 pathway is involved in stress fibre production via
activation of MK2 that then phosphorylates HSP27.12
In addition, recent data suggest that MK2 acts as a
checkpoint kinase that can lead to cell-cycle arrest.13
Moreover, MK2 activity up-regulates the expression of
Keywords: Werner syndrome; Inhibitors; Heterocycles; Microwaves;
Inflammation; p38 MAP kinases; Senescence.
*
Corresponding authors. Tel.: +44 29 2087 4029; fax: +44 29 2087
4030 (M.C.B.); e-mail: Bagleymc@cardiff.ac.uk
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.036

T. Davis et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6832–6835
6833
inflammatory pathways,14 and WS is associated with
O
NH2
N
inflammatory diseases.15 These data suggest the possibil-
ity that MK2 may be involved in the phenotypic charac-
teristics seen in WS. If so, then therapeutic targeting of
MK2 may help surmount complications in the use of
p38 inhibitors, as the number of pathways inhibited will
be reduced. The recent disclosure of small molecule
inhibitors of MK2 makes this approach possible.16,17
We synthesized two different MK2 inhibitors (see
Schemes 1 and 2). The first biphenyl 1 is a substrate spe-
cific inhibitor that actively prevents the activation of
MK2 by p38, while leaving p38 activation of other tar-
gets alone (e.g., ATF2).16 The second, diaminocyano-
pyridine 2, is an ATP-competitive inhibitor of MK2
that is effective in attenuating TNFa production in cel-
lular assays with established in vivo activity.17
HO
HO
HO
HO
CN
NH2
i,ii
H
90%
OH
O
6
2·TFA
Scheme 2. Synthesis of MK2 inhibitor 2. Reagents and conditions: (i)
malonitrile dimer, EtOH–AcOH (1:1), microwaves, 140 °C (initial
power 120 W, which was moderated to maintain a constant reaction
temperature), 1 h; (ii) Et3SiH, TFA, 0 °C, 4 h.
MK2 inhibitory activity was assessed by the ability to
prevent the anisomycin-induced phosphorylation of
HSP27 in human hTERT-immortalised HCA2 cells
using an ELISA system as previously described.20 In
addition, the inhibitors were tested for their ability to
modify the growth rate and morphology of immorta-
lised HCA2 and WS cells using daily replaced drug sup-
plemented growth medium as previously described.4
Finally, their effects on p38 activation were assessed by
immunoblots.20 For inhibitor 2, the same results were
found using either the free base or the TFA salt.
Our route to the substrate-selective inhibitor 1 involved
the Friedel–Crafts acylation of fluorobiphenyl 3 using
succinic anhydride/AlCl3, followed by Clemmensen
reduction of ketoacid 4 to give carboxylic acid 5, suitable
for amide formation with 4-aminophenol (Scheme 1).
Benzotriazol-1-yloxytripyrrolidino-phosphonium hexa-
fluorophosphate (pyBOP)-mediated coupling gave the
inhibitor 1,18 albeit it in very poor (15%) yield. This
unoptimized process, undoubtedly a consequence of
low aniline reactivity, did provide sufficient quantity on
small scale (0.2 mmol) for biological evaluation.
Inhibitor 1 prevents the ability of p38 to activate MK2
using an in vitro kinase assay with a Kai pp of 330 nM,
by binding in the vicinity of the p38 active site causing
subtle structural perturbations that result in suboptimal
positioning of substrates and cofactors.16 However,
inhibitor 1 at 10 lM has, at best, only a small effect
on the anisomycin-induced phosphorylation of HSP27
in HCA2 cells (Fig. 1a), or in WS cells (Fig. 1c).
SB203580, a p38 inhibitor, prevents HSP27 phosphory-
lation in WS cells under similar conditions (Fig. 1c). As
the reported inhibition of MK2 at 10 lM is an in vitro
assay with purified proteins,16 it is possible that we find
little inhibition of MK2 due to the cellular context. Such
effects have been seen for other kinase inhibitors.22
Inhibitor 1 and SB203580 have only a small effect on
the anisomycin-induced activation of p38 in WS cells
as expected, as these inhibitors act to affect p38 activity,
not its activation. Continuous treatment of WS cells
with inhibitor 1 above 200 nM resulted in complete ces-
sation of growth (Fig. 1b).
The synthesis of inhibitor 2 from salicylaldehyde 6 by con-
densation with malonitrile dimer in EtOH–AcOH was
first investigated under traditional conductive heating
methods at reflux for 21 h, followed by evaporation, addi-
tion of trifluoroacetic acid (TFA) and reduction with tri-
ethylsilane. However, under these conditions,17 the TFA
salt of 2 did not precipitate and only unreacted starting
material was obtained so recourse was made to more forc-
ing conditions. Microwave irradiation at 140 °C for 1 h in
a sealed tube using a single-mode microwave synthesizer
(CEM DiscoverTM), followed by triethylsilane reduction
in TFA, precipitated the TFA salt of 219 in 90% yield
(Scheme 2), which could be liberated as the free base by
treatment with ethanolic ammonia.
In contrast, inhibitor 2 prevents the anisomycin-in-
duced phosphorylation of HSP27 above 2.5 lM in
HCA2 cells, with the maximal inhibition achieved at
25 lM (Fig. 2a). The IC50 seems to be between 2.5
and 10 lM, higher than the reported IC50 of
0.92 lM to attenuate LPS-induced TNFa production
in U937 cells.17 However, this agrees with data show-
ing that inhibitor 2 prevents the arsenite-induced
phosphorylation of HSP27 in mouse embryonic fibro-
blasts and HeLa cells at 15 lM.21 Inhibitor 2 at
25 lM has no effect on the activation of p38 in con-
trol HCA2 cells, but may accentuate p38 activation
in anisomycin-treated cells (Fig. 2b). Additionally,
inhibitor 2 does not prevent the p38 phosphorylation
of MK2, whereas SB203580 does. Similar effects were
seen in WS cells (Fig. 3a). Continuous treatment of
HCA2 and WS cells with inhibitor 2 at concentrations
above 2.5 lM caused a complete cessation of growth
(Fig. 3c).
O
OH
i
X
47%
F
F
3
4 X = O
ii, 57%
5 X = H2
OH
iii
15%
O
F
N
H
1
Scheme 1. Synthesis of MK2 substrate-selective p38 inhibitor 1.
Reagents and conditions: (i) succinic anhydride, AlCl3, ClCH2
CH2Cl, 24 h; (ii) Zn, HgCl2, HCl (aq), 5 min; then 4, toluene,
H2O, reflux, 30 h; (iii) PyBOP, dry CH2Cl2, 0 °C; 4-aminophenol,
Et3N, rt, 24 h.

6834
T. Davis et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6832–6835
Figure 1. (a) Effects of inhibitor 1 on MK2 activity in HCA2 cells
assessed by ELISA. White bars indicate total HSP27, dark grey bars p-
HSP27 and black bars the p-HSP27/HSP27 ratio. D are DMSO-
treated cells, An are anisomycin-treated cells, 10.0 are cells pre-treated
with 10 lM inhibitor 1 followed by anisomycin treatment. (b) Effects
of inhibitor 1 on the growth rate of WS cells: *zero growth rate.
Growth rates are measured as population doublings (PD) per day with
cells grown in standard EMEM in tissue culture flasks: PD = log(Nf/
Ni)/log2 where Nf is the number of cells counted and Ni is the number
of cells seeded. (c) Immunoblot showing effect of inhibitor 1 (10 lM)
on the anisomycin-induced activity of the p38 pathway in WS cells.
SB203580 (2.5 lM) is used as a control. p-HSP27 and p-p38 are the
phosphorylated forms of HSP27 and p38.
Figure 3. (a) Effects of inhibitor 2 (25 lM) and SB203580 (2.5 lM) on
the activation of MK2 and HSP27 in WS cells. (b) Effects of inhibitor 2
(25 lM) on HSP27 phosphorylation in WS cells over 6 d. (c) Growth
rate of WS and HCA2 cells in the presence of increasing concentra-
tions of inhibitor 2: *zero growth rate. (d) WS cells stained with
phalloidin-FITC to visualize F-actin. Bar = 50 lm. (e) Effects of 25 lM
inhibitor 2 on activation of cofilin.
seen in primary WS cells, in that many are enlarged with
prominent F-actin stress fibres (Fig. 3d). These fibres are
associated with elevated p-HSP27 levels that are reduced
by SB203580 (Fig. 1c) and inhibitor 2 (Fig. 3b). Treat-
ment with inhibitor 2 at 10 lM or above accentuated
this phenotype, with more cells having increased levels
of stress fibres, indeed they now resembled senescent
cells (Fig. 3d). Similar effects are seen with inhibitor 1
(not shown). Inhibitor 2 shows similar effects on
HCA2 cells (not shown).
The growth arrest caused by these inhibitors is unlikely
to be related to MK2 inhibition, as it appears to occur at
inhibitor 2 levels insufficient to inhibit MK2 by more
than 50%, and inhibitor 1 shows no MK2 inhibitory
activity. Likewise, the increased levels of stress fibres
are unlikely to result from MK2 activity, as inhibitor 2
at 25 lM results in decreased p-HSP27 levels in WS cells
that are maintained over 6 days (Fig. 3b). These inhibi-
tors may be targeting a second kinase, although as these
inhibitors work in very different ways, the probability
that they would hit the same off-target seems small.
One possible off-target may be the stress kinase JNK;
however, we have shown that these inhibitors have no
effects on JNK in a cell-based assay (not shown). Alter-
natively, these effects may simply be due to cell toxicity.
If so, then unlike other cell toxic chemicals such as arse-
nite, their effects are not transduced via the p38- or
JNK-signalling pathways. A further possibility is that
these inhibitors may increase F-actin stress fibre produc-
tion by activating the lim kinase (LIMK) pathway
Figure 2. (a) ELISA results for the effects of inhibitor 2 base on MK2
activity in HCA2 cells. The key is as for Figure 1a. 1.0–50.0 are cells
pre-treated with inhibitor 2 followed by anisomycin treatment. (b)
Effects of inhibitor 2 (25 lM) and SB203580 (2.5 lM) on the activation
of p38 and MK2. For MK2 the upper band is phosphorylated MK2
(p-MK2).
The WS cells used in this work have been immortalised
using human telomerase expression;4 however, these
cells retain the slow growth and stressed morphology

T. Davis et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6832–6835
6835
9. Rodriguez–Lopez, A. M.; Jackson, D. A.; Iborra, F.; Cox,
L. S. Aging Cell 2002, 1, 30.
10. Pichierri, P.; Franchitto, A. Bioessays 2004, 26, 306.
11. Goldstein, D. M.; Gabriel, T. Curr. Top. Med. Chem.
2005, 5, 1017.
12. Shi, Y.; Kotlyarov, A.; Laabeta, K.; Gruber, A. D.; Butt,
E.; Marcus, K.; Meyer, H. E.; Friedrich, A.; Volk, H. D.;
Gaestel, M. Mol. Cell. Biol. 2003, 23, 7732.
13. Manke, I. A.; Nguyen, A.; Lim, D.; Stewart, M. Q.; Elia,
A. E.; Yaffe, M. B. Mol. Cell 2005, 17, 37.
resulting in phosphorylation of the actin depolymerising
protein cofilin.23 It has previously been shown that cofi-
lin is phosphorylated in young primary WS cells and
also in senescent WS and normal cells.4 Indeed, inhibi-
tor 2 treatment increases the level of phosphorylated
cofilin in HCA2 cells, although not in WS cells
(Fig. 3e); however, cofilin may already be maximally
activated in the WS cells. Finally, an ironic conclusion
would be that, as MK2 inhibition in WS cells does not
duplicate what is seen using p38 inhibitors,4 these data
may actually suggest that MK2 is not involved in the
WS phenotype, although this is speculative. The devel-
opment of new MK2 inhibitors with much lower IC50
values in cell-based systems would be needed to show
this. In conclusion, two MK2 inhibitors have been pre-
pared and tested for their effects on WS cells. The herein
reported effects of these inhibitors suggest that despite
the observed activity of inhibitor 2 in a 2-h rat LPS chal-
lenge assay (20 mpk, IP, 68% inhibition),17 they would
be unsuitable for long-term therapeutic use due to toxic-
ity issues and the possibility of deleterious side effects.
14. Dean, J. L. E.; Sully, G.; Clark, A. R.; Saklatvala, J. Cell
Signal. 2004, 16, 1113.
15. Davis, T.; Kipling, D. Rejuv. Res. 2006, 9, 402.
16. Davidson, W.; Frego, L.; Peet, G. W.; Kroe, R. R.;
Labadia, M. E.; Lukas, S. M.; Snow, R. J.; Jakes, S.;
Grygon, C. A.; Pargellis, C.; Werneberg, B. G. Biochem-
istry 2004, 43, 11658.
17. Anderson, D. R.; Hegde, S.; Reinhard, E.; Gomez, L.;
Vernier, W. F.; Lee, L.; Liu, S.; Sambandam, A.; Snider,
P. A.; Masih, L. Bioorg. Med. Chem. Lett. 2005, 15, 1587.
18. Inhibitor 1 was obtained as a colourless solid, mp 128–130
°C (found: MH+ 350.1548, C22H21FNO2 [MH+] requires
350.1551); IR (KBr) (m/cmÀ1) 3317, 2965, 2816, 1654,
1514, 1350, 1230, 1100, 880; 1H NMR (400 MHz, CDCl3)
(d/ppm) 7.44–7.40 (2H, m, ArH), 7.39–7.32 (1H, m, ArH),
7.30–7.18 (7H, ArH), 7.16–7.04 (2H, m, ArH), 6.92 (1H,
br s, NH), 6.73 (2H, d, J 8 Hz, ArH), 4.95 (1H, br s, OH),
2.70 (2H, t, J 7.3, CH2), 2.29 (2H, t, J 7.4, CH2), 2.04 (2H,
tt, J 7.3, 7.4, CH2); 13C NMR (100 MHz, CDCl3) (d/ppm)
170.8 (C), 159.7 (d, 1JC–F 247 Hz, C), 152.5 (C), 140.8 (C),
133.6 (C), 130.7 (C), 130.6 (d, 3JC–F 3.8 Hz, CH), 129.1 (d,
Acknowledgments
We thank the BBSRC (BB/D524140), EPSRC (GR/
S25456; DTA award to C.S.W.) and SPARC (awards
to T.D. and P.M.) for support of this work and the
EPSRC Mass Spectrometry Service at the University
of Wales, Swansea UK for mass spectra.
3
4JC–F 3.1 Hz, CH), 128.8 (d, JC–F 7.9 Hz, CH), 128.7
4
(CH), 128.6 (C), 124.4 (d, JC–F 3.7 Hz, CH), 122.1 (CH),
116.1 (d, 2JC–F 22.8 Hz, CH), 115.6 (CH), 36.5 (CH2), 34.7
(CH2), 26.8 (CH2); MS (APcI) m/z 350 (MH+, 100%).
19. Inhibitor 2ÆTFA was obtained as an orange solid, mp
>290 °C (found: MH+ 271.0824, C13H11N4O3 [MH+]
requires 271.0826); IR (KBr) (m/cmÀ1) 3480, 3397, 3359,
3258, 3223, 3112, 3067, 2926, 2859, 2230, 1675, 1652, 1559,
References and notes
1. (a) Martin, G. M.; Oshima, J.; Gray, M. D.; Poot, M.
J. Am. Geriatr. Soc. 1999, 47, 1136; (b) Yu, C.-E.; Oshima,
J.; Fu, Y.-H.; Wijsman, E. M.; Hisama, F.; Alisch, R.;
Matthews, S.; Nakura, J.; Miki, T.; Ouais, S.; Martin, G.
M.; Mulligan, J.; Schellenberg, G. D. Science 1996, 272,
258.
1
1521, 1505, 1457, 1444, 1394, 1324, 1287, 1158, 1132; H
NMR (400 MHz, d6-DMSO) (d/ppm) 9.85–8.00 (2H, br s,
2OH), 7.30–6.60 (2H, br s, NH2), 7.10 (2H, br s, NH2),
6.64 (1H, s, 6H), 6.44 (1H, s, 9H), 3.49 (2H, s, 5H); 13C
NMR (125 MHz, d6-DMSO) (d/ppm) (one
C not
2. Kipling, D.; Davis, T.; Ostler, E. L.; Faragher, R. G.
Science 2004, 305, 1426.
observed) 158.9 (C), 158.5 (C), 145.3 (C), 142.9 (C),
142.3 (C), 116.0 (C), 115.0 (CH), 108.7 (C), 104.0 (CH),
86.9 (C), 70.1 (C), 22.3 (CH2); MS (ES) m/z 271 (MH+,
100%).
3. Tollefsbol, T. O.; Cohen, H. J. Age 1984, 7, 75.
4. (a) Davis, T.; Baird, D. M.; Haughton, M. F.; Jones, C. J.;
Kipling, D. J. Gerontol. 2005, 60A, 1386; (b) Davis, T.;
Haughton, M. F.; Jones, C. J.; Kipling, D. Ann. N.Y.
Acad. Sci. 2006, 1067, 243.
5. James, S. E.; Faragher, R. G. A.; Burke, J. F.; Shall, S.;
Mayne, L. V. Mech. Ageing Dev. 2000, 121, 139.
6. Rubin, H. Nat. Biotechnol. 2002, 20, 675.
7. Herbig, U.; Ferreira, M.; Condel, L.; Carey, D.; Sedivy, J.
M. Science 2006, 311, 1257.
8. (a) Wright, W. E.; Shay, J. W. Curr. Opin. Genet. Dev.
2001, 11, 98; (b) Baird, D. M.; Davis, T.; Rowson, J.;
Jones, C. J.; Kipling, D. Hum. Mol. Genet. 2004, 13, 1515.
20. Bagley, M. C.; Davis, T.; Dix, M. C.; Widdowson, C. S.;
Kipling, D. Org. Biomol. Chem. 2006, 4, 4158.
21. Vertii, A.; Hakim, C.; Kotlarov, A.; Gaestel, M. J. Biol.
Chem. 2006, 281, 26966.
22. Trujillo, J. I.; Meyers, M. J.; Anderson, D. R.; Hegde, S.;
Mahoney, M. W.; Vernier, W. F.; Buchler, I. P.; Wu, K.
P.; Yang, S.; Hartmann, S. J.; Reitz, D. B. Bioorg. Med.
Chem. Lett. 2007, 17, 4657.
23. Pritchard, C. A.; Hayes, L.; Wojnowski, L.; Zimmer, A.;
Marais, R. M.; Norman, J. C. Mol. Cell. Biol. 2004, 24, 5937.