The neuroprotective action of JNK3 inhibitors based on the 6,7-dihydro-5H-pyrrolo[1,2-a]imidazole scaffold

Article abstract of DOI:10.1016/j.bmcl.2005.07.076

Imidazole-based structures of p38 inhibitors served as a starting point for the design of JNK3 inhibitors. Construction of a 6,7-dihydro-5H-pyrrolo[1,2-a] imidazole scaffold led to the synthesis of the (S)-enantiomers, which exhibited p38/JNK3 IC₅₀ ratio of up to 10 and were up to 20 times more potent inhibitors of JNK3 than the relevant (R)-enantiomers. The JNK3 inhibitory potency correlated well with inhibition of c-Jun phosphorylation and neuroprotective properties of the compounds in low K⁺-induced cell death of rat cerebellar granule neurones.

Full text of DOI:10.1016/j.bmcl.2005.07.076

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4666–4670
The neuroprotective action of JNK3 inhibitors based on the
6,7-dihydro-5H-pyrrolo[1,2-a]imidazole scaffold
Piotr P. Graczyk,a,* Afzal Khan,a Gurpreet S. Bhatia,a Vanessa Palmer,a Darren Medland,a
Hirotoshi Numata,a Hitoshi Oinuma,a Jacqueline Catchick,a Angela Dunne,a Moira Ellis,a
Caroline Smales,a Jonathan Whitfield,a Stephen J. Neame,a Bina Shah,a Daniel Wilton,a
Louise Morgan,a Toshal Patel,a Raymond Chung,a Howard Desmond,a
James M. Staddon,a Nobuaki Satob and Atsushi Inoueb
aEisai London Research Laboratories, University College London, Bernard Katz Building, Gower Street, London WC1E 6BT, UK
bTsukuba Research Laboratories, Eisai Co., Ltd, Tsukuba-shi, Ibaraki 300-2635, Japan
Received 9 June 2005; revised 26 July 2005; accepted 29 July 2005
Available online 8 September 2005
Abstract—Imidazole-based structures of p38 inhibitors served as a starting point for the design of JNK3 inhibitors. Construction
of a 6,7-dihydro-5H-pyrrolo[1,2-a]imidazole scaffold led to the synthesis of the (S)-enantiomers, which exhibited p38/JNK3 IC50
ratio of up to 10 and were up to 20 times more potent inhibitors of JNK3 than the relevant (R)-enantiomers. The JNK3 inhibitory
potency correlated well with inhibition of c-Jun phosphorylation and neuroprotective properties of the compounds in low
K+-induced cell death of rat cerebellar granule neurones.
Ó 2005 Elsevier Ltd. All rights reserved.
Contribution of apoptotic mechanisms to neurodegen-
erative diseases, such as AlzheimerÕs,1 HuntingtonÕs,2
and ParkinsonÕs diseases,3 as well as amyotrophic lat-
eral sclerosis (ALS),4 is becoming increasingly appreci-
ated.5 Therefore, the JNK pathway, which controls
apoptosis in various cell death paradigms, has devel-
oped into an attractive drug target for neurodegenera-
tive disorders.6,7 Inhibitors of this pathway, such as
SP6001258 and TAT-fused peptide inhibitor of JNK
substrate binding,9 exhibited neuroprotective proper-
ties in preclinical studies, and CEP-1347, an inhibitor
of the JNK pathway, had advanced to Phase III for
ParkinsonÕs disease.10
of hippocampal CA3 neurones of JNK3 knockout
mice to kainic-acid-induced injury.12 These mice are
also protected against cerebral ischemia-hypoxia13
and partially against toxicity due to MPTP, which
serves as a model of ParkinsonÕs disease.14 Important-
ly, the JNK3 knockout animals exhibit normal pheno-
type.12 Furthermore, mice in which the endogenous
substrate of JNK, c-Jun, is replaced by a mutant
Jun allele, which cannot be phosphorylated by JNK,
are viable and fertile, and resistant to neuronal apop-
tosis induced by kainic acid.15
At the initial stages of the JNK3 program at Eisai, we
decided to obtain a JNK3 inhibitor by modification of
known structure acting in an ATP-competitive manner,
similar to the approach adopted by others.7 As the imid-
azole-based p38 inhibitor SB203580 had been recog-
nized to inhibit JNK3a1 with a submicromolar IC50
value,16 we decided to use a more structurally attractive
p38 inhibitor, SB227931 (p38 IC50 0.046 lM),17 as a
starting point for our initial drug design, hoping that
it, too, would inhibit JNK3. To verify this assumption,
we synthesized 1, which showed inhibition of JNK3
with an IC50 value of 0.13 lM.
Although this pathway can be inhibited at multiple
steps, we focused on c-Jun N-terminal kinase 3
(JNK3) due to its relatively selective expression in
brain tissue11 and an expected better drug safety pro-
file. Validity of JNK3 as a target for neurodegenera-
tive disorders has been supported by the resistance
*
Corresponding author. Tel.: +44 2074131147; fax: +44
2074131121; e-mail: piotr_graczyk@eisai.net
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.076

P. P. Graczyk et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4666–4670
4667
Preparation of (S)-4 and (S)-5, which are representative
examples of the series, is given in Scheme 1. Synthesis of
amine 8, from a commercially available pyrrolidinone 6,
was performed by modification of the methodology
developed by Leutenegger et al.19
Amine 8 was reacted with bromoketone 10, which was
readily available by bromination of ketone 9 with
CuBr2.20 Formation of the imidazole ring occurred
during reflux of an ethanolic solution of salt 11 in the
presence of catalytic amount of hydrobromic acid.
Treatment of the product with TBAF afforded alcohol
12, which was then protected under phase-transfer con-
ditions to form the MPM ether 13. Stannylation of 13,
followed by Stille coupling with 4-iodo-2-methylthiopyr-
imidine, gave di(hetero)aryl-substituted imidazole 14.
Substitution of the methylthio group with the n-propyla-
mino was achieved by oxidation of sulfur to the relevant
sulfoxide and reacting it at room temperature with neat
propylamine (liquid ammonia in a sealed tube in the
case of (S)-3 and (R)-3). The MPM group in (S)-4 could
be removed under acidic conditions to provide (S)-5.
To increase inhibitory potency, we decided to modify
structure 1 by reducing conformational flexibility of
the side chain. This could be achieved by introducing
an additional ring (n = 1, 2) linking imidazole C(2) with
the side chain, as shown in structure 2. We also expected
that formation of a chiral center in 2 would enable
further control of selectivity, particularly against p38,
as this kinase contributes to synaptic plasticity within
the CNS.18
Furthermore, the target structure would possess several
points for derivatization, which may be utilized to opti-
mize potency and ADME properties of the compounds.
Herein, we describe the synthesis of three enantiomeric
pairs 3–5, their biological properties and rationalization
of the results based on an in silico approach.
Scheme 1. Reagents and conditions: (a) (i) TBDPSCl, imidazole, DMF,
rt, 2 h; (ii) LawessonÕs reagent, THF, rt, 2 h, 81%; (b) (i) MeI, CH2Cl2, rt,
5 h; (ii) NH4Cl, MeOH, 50 °C, 3.5 h; (iii) NaOH, (87%); (c) CuBr2,
AcOEt, reflux, 5 h, 73%; (d) AcOEt, rt, 16 h; (e) (i) cat HBr/AcOH, 33%
aq EtOH, reflux, 5.5 h, (ii) TBAF, THF, 0 °C—rt, 1 h, 62% (overall for
steps d and e); (f) p-methoxybenzyl chloride, 50% aq NaOH, n-
Bu4NHSO4 (cat), rt, 12 h, 99%; (g) (i) n-BuLi, THF, ꢀ78 °C, 30 min;
(ii) Bu3SnI (neat); (iii) 4-iodo-2-methylthiopyrimidine, DMF, Pd(PPh3)4
(cat), 80 °C, 4 days, 48%; (h) (i) H2O2, CH3COOH, 48 h; (ii) n-PrNH2
(neat), rt, 36%; (j) (i) 6 N HCl(aq), MeOH, 125 °C, 1 h; (ii) NaOH, 78%.

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P. P. Graczyk et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4666–4670
Table 1. Inhibitory activities (IC50) of enantiomeric pairs 3–5 and the relevant p38/JNK3 selectivity ratios
a
JNK3 IC50 (nM)
a
p38 IC50 (nM)
Compound
p38/JNK3 IC50
(S)-3
(R)-3
(S)-4
(R)-4
(S)-5
(R)-5
61.2 ( 12)
270 ( 47)
10.9 ( 3.6)
233 ( 46)
2.5 ( 0.1)
3.0 ( 0.1)
295 ( 27)
116 ( 17)
54 ( 4)
4.8
0.43
5.0
217 ( 26)
28 ( 2)
31 ( 2)
0.93
11
10
a Values are means of three experiments, standard error is given in parentheses. ATP concentration in both enzyme assays was 1.0 lM.
None of the synthetic stages was accompanied by
racemization, which was verified by determination of
the optical purity of final compounds and key
intermediates.21
Docking simulations suggest that (R)-3 and (R)-4 fit the
ATP-binding site of p38 better than the ATP-binding
site of JNK3 because Val196 in JNK3 is replaced with
a smaller Ala157 in p38. Furthermore, the (R)-enantio-
mers may participate in larger hydrophobic interactions
with the C-terminal domain of p38. This may explain
why (R)-3 inhibits p38 more strongly than JNK3,
although no such difference can be seen for (R)-4. Inter-
estingly, the (S)-enantiomers inhibit p38 lesser than
JNK3 because the methoxybenzyl group is located at
the ribose binding site of p38 where it undergoes desta-
bilizing interactions with Tyr35. The above differences
are reflected in the selectivities, for example, p38/JNK3
IC50 4.8 for (S)-3 and p38/JNK3 IC50 0.43 for (R)-3.
Inhibitory activities of compounds 3–5 against JNK3
and p38 are given in Table 1.
The (S)-enantiomers of MPM ethers 3 and 4 are more
potent JNK3 inhibitors than their (R) counterparts. In
particular, the JNK3 IC50 value for (S)-4 is about 20
times lower than that for (R)-4. This observation can
be rationalized by docking (S)-4 and (R)-4 within the
JNK3 ATP-binding site using published coordinates
for the protein.22
Due to similar cell membrane permeability, enantiomer-
ic compounds with different selectivity profiles offer a
very useful tool to study the importance of alternative
signaling pathways for biological phenomena in cell sys-
tems. As the relative contribution of JNK- and p38-de-
pendent pathways in different cell death mechanisms is
still a matter of debate,23 we examined the effect of the
three enantiomeric pairs 3–5 on neuroprotection in cer-
ebellar granule neurones (CGN). In this model, incuba-
tion of cells in the presence of 25 mM KCl prevents cell
death. Reducing the KCl concentration to 5 mM induc-
es c-Jun phosphorylation and apoptosis.24
Compound (S)-4 fits the ATP-binding site of JNK3 very
well (Fig. 1A) and participates in several binding inter-
actions identified by Scapin et al.22 for analogous com-
pounds. Furthermore, the methoxybenzyl group in
(S)-4 binds to the glycine-rich loop in the N-terminal
domain of JNK3. In contrast, the methoxybenzyl group
in (R)-4 is shifted toward the C-terminal domain
(Fig. 1B). Some of the hydrophobic interactions are lost
and as a consequence (R)-4 would exhibit a weaker
JNK3 inhibition than (S)-4.
In agreement with this explanation, enantiomeric alco-
hols (S)-5 and (R)-5, in which the methoxybenzyl group
is absent, display virtually the same inhibitory potencies
against JNK3. A very low IC50 value for both enantio-
mers can be attributed to the possible hydrogen bond
formation between the hydroxyl group of both enantio-
mers and enzyme Asn152 and/or Ser193. As these
hydrogen bonds can also be formed with p38, there is
virtually no difference in selectivity against p38 between
isomeric alcohols (p38/JNK3 IC50 11 vs. 10 for (S)-5 and
(R)-5, respectively).
The transcription factor c-Jun is suggested to be a JNK
substrate involved in neuronal death.25 Therefore, effica-
cy in c-Jun phosphorylation inhibition should follow the
JNK3 inhibition profile.26 As can be seen in Figure 2,
Figure 2. Inhibition of c-Jun phosphorylation by enantiomeric pairs
3–5 at concentrations of 10 and 1 lM. Phospho-c-Jun band intensity in
immunoblot is expressed as a percentage of the signal at low K+ in the
absence of drug. Hi, 25 mM KCl; Lo, 5 mM KCl. The data are average
of four experiments; error bars represent standard deviation.
Figure 1. View of (A) (S)-4 and (B) (R)-4 within the JNK3 ATP-
binding site (arrows point to the methoxybenzyl group in both
molecules).

P. P. Graczyk et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4666–4670
4669
compound (S)-3 almost completely suppressed the phos-
phorylation of c-Jun at a concentration of 10 lM, while
its enantiomer, (R)-3, a weaker JNK3 inhibitor (IC50
270 nM), was ineffective at this dose. A much more
potent JNK3 inhibitor, (S)-4 (IC50 10.9 nM), showed
clear effect, even at 1 lM concentration (Fig. 2), while
its enantiomer was less active. Alcohols (S)-5 and (R)-
5, both very potent JNK3 inhibitors (IC50 of about
3 nM), exhibited a virtually complete suppression of
c-Jun phosphorylation in the range of studied concen-
trations (Fig. 2). Therefore, the degree of inhibition of
c-Jun phosphorylation in this system reflects the JNK3
inhibitory potency. Furthermore, the effects on c-Jun
phosphorylation for (S)-3 and (R)-3 are opposite to
what could be anticipated based on p38 inhibition
(IC50 295 and 116 nM, respectively).
To determine whether inhibition of JNK3 and c-Jun
phosphorylation correlate with neuroprotection, we fol-
lowed the survival of cerebellar granule neurones by
MTT27 or lactate dehydrogenase (LDH)28 colorimetric
assays. The results for enantiomeric pairs 3–5 are given
in Figure 3 as a percentage of survival at three drug
concentrations: 0.1, 1, and 10 lM.
Compounds (S)-3 and (R)-3 had no effect on survival up
to a concentration of 1 lM, regardless of the assay used
(Fig. 3A). At a concentration of 10 lM, the neuropro-
tective properties of the (S)-enantiomer were significant-
ly stronger than those of (R)-3, as expected based on a
lower JNK3 IC50 value for (S)-3 (61.2 nM vs. 270 nM,
Table 1) and in agreement with a stronger inhibition
of c-Jun phosphorylation by (S)-3 (Fig. 2). Since (S)-3
is a weaker p38 inhibitor than (R)-3 (IC50 295 and
116 nM, respectively), the survival effects indicate that
p38-dependent pathway is unimportant in this cell death
paradigm, in agreement with Cao et al.23
Figure 3. The effect of enantiomeric pairs 3–5 on the survival of
cerebellar granule neurons, as measured by MTT and LDH assays (in
triplicate, error bars represent standard deviation). Death was induced
by withdrawal of K+. Complete survival (100% of mean values)
corresponds to the assay reading at high K+ concentration, while 0%
survival (cell death) is the reading at low K+. (A) (S)-3 and (R)-3; (B)
(S)-4 and (R)-4; (C) (S)-5 and (R)-5.
Both enantiomers of 4 exhibited clear dose-dependent
effects on survival. Neuroprotective properties of (S)-4
and (R)-4 correlate very well with their JNK3 IC50 val-
ues. Just as inhibitory potency of (S)-4 is stronger than
that of (S)-3 (10.9 nM vs. 61.2 nM, respectively), so a
clear protective effect of (S)-4 could already be seen at
a concentration of 1 lM (Fig. 3B). The effects of (R)-4
were weaker (JNK3 IC50 233 nM).
the JNK pathway inhibition. The results of additional
SAR studies on this template will be reported in due
course.
Alcohols (S)-5 and (R)-5, which do not differ in terms of
JNK3 inhibitory activity (IC50 values of 2.5 and 3.0 nM,
respectively), showed survival profiles that were very
similar. Due to their high inhibitory potency against
JNK3, the compounds showed an almost complete
neuroprotection at a concentration of 1 lM.
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20
phase: (S)-3: ee > 99%; ½aꢁD ꢀ271.3 (c 0.91, MeOH); (R)-
20
3: ee > 99%; ½aꢁD +269.9 (c 0.41, MeOH); (S)-4: ee > 99%;
20
20
20
½aꢁD ꢀ237.6 (c 0.87, MeOH); (R)-4: ee 99%; ½aꢁD +247.6
20
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(m, 1H), 2.71–2.89 (m, 2H), 2.97–3.07 (m, 1H), 3.35 (q,
J = 6.6 Hz, 2H), 3.51 (dd, J = 9.5, 5.2 Hz, 1H), 3.66 (dd,
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1H), 4.32 (d, J = 11.7 Hz, 1H), 5.11 (bs, 2H), 6.36 (d,
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4H), 7.54 (dd, J = 8.7, 5.5 Hz, 2H), 7.98 (d, J = 5.2 Hz,
1H); HRMS (CI) calcd for C28H30FN5O2, 488.2462
(M+1); found: 488.2467. (S)-5: 1H NMR (400 MHz,
CDCl3) d [ppm] 0.98 (t, J = 7.4 Hz, 3H), 1.62 (sextet,
J = 7.3 Hz, 2H), 2.34–2.46 (m, 1H), 2.67–2.86 (m, 2H),
2.99–3.09 (m, 1H), 3.33 (q, J = 6.6 Hz, 2H), 3.72 (dd,
J = 11.4, 6.7 Hz, 1H), 3.87 (dd, J = 11.4, 3.6 Hz, 1H), 4.88
(bs, 1H), 5.19 (bs, 1H), 6.34 (d, J = 5.3 Hz, 1H), 7.01(t,
J = 8.7 Hz, 2H), 7.47 (dd, J = 8.7, 5.5 Hz, 2H), 7.99 (d,
J = 4.4 Hz, 1H); HRMS (CI) calcd for C20H22FN5O,
368.1887 (M+1); found: 368.1874.
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