Imino-tetrahydro-benzothiazole derivatives as p53 inhibitors: Discovery of a highly potent in vivo inhibitor and its action mechanism

Article abstract of DOI:10.1021/jm060318n

Several neurological disorders manifest symptoms that result from the degeneration and death of specific neurons. p53 is an important modulator of cell death, and its inhibition could be a therapeutic approach to several neuropathologies. Here, we report

Full text of DOI:10.1021/jm060318n

J. Med. Chem. 2006, 49, 3645-3652
3645
Imino-tetrahydro-benzothiazole Derivatives as p53 Inhibitors: Discovery of a Highly Potent in
Vivo Inhibitor and Its Action Mechanism
Nicolas Pietrancosta,†,‡ Anice Moumen,† Rosanna Dono,† Paul Lingor,§ Veronique Planchamp,§ Fabienne Lamballe,†
Mathias Ba¨hr,§ Jean-Louis Kraus,*,†,‡,| and Flavio Maina*,†,|
DeVelopmental Biology Institute of Marseille (UniVersite´. de la Mediterrane´e), Inserm UMR623 (CNRS - INSERM) Campus de Luminy-Case
907, Marseille Cedex 09, France, Laboratoire de Chimie Biomole´culaire (UniVersite´. de la Mediterrane´e), Campus de Luminy-Case 907,
Marseille Cedex 09, France, and Department of Neurology, UniVersity of Go¨ttingen, Waldweg 33, 37075 Go¨ttingen, Germany
ReceiVed March 20, 2006
Several neurological disorders manifest symptoms that result from the degeneration and death of specific
neurons. p53 is an important modulator of cell death, and its inhibition could be a therapeutic approach to
several neuropathologies. Here, we report the design, synthesis, and biological evaluation of novel p53
inhibitors based on the imino-tetrahydrobenzothiazole scaffold. By performing studies on their mechanism
of action, we find that cyclic analogue 4b and its open precursor 2b are more potent than pifithrin-R (PFT-
R), which is known to block p53 pro-apoptotic activity in vitro and in vivo without acting on other pro-
apoptotic pathways. Using spectroscopic methods, we also demonstrate that open form 2b is more stable
than 4b in biological media. Compound 2b is converted into its corresponding active cyclic form through
an intramolecular dehydration process and was found two log values more active in vivo than PFT-R.
Thus, 2b can be considered as a new prodrug prototype that prevents in vivo p53-triggered cell death in
several neuropathologies and possibly reduces cancer therapy side effects.
Introduction
Neurodegenerative disorders, such as stroke, Alzheimer’s, and
Parkinson’s disease, manifest symptoms of degeneration and
progressive loss of specific neuron populations.1-3 Whereas
different neuronal subpopulations are affected in each of these
pathologies, the biochemical and cellular events leading to
neuronal dysfunction and cell death share a common final
cascade, which involves increased oxidative stress, the disruption
of calcium homeostasis, and the activation of an intrinsic
Figure 1. Structure of Pifithrin-R (PFT-r) and the general structure
of related analogues.
apoptotic program.3,4 The activation of the tumor suppressor
gene p53 plays a crucial role in regulating the death of neurons
in vitro, following apoptotic stimuli involving molecules such
Moreover, PFT-R protects cultured hippocampal neurons
as glutamate or DNA-damaging agents.5-8 Studies of p53-
against death induced by DNA-damaging agents.17,18 PFT-R
deficient mice have confirmed the essential role of p53 in
neuronal apoptosis caused by ischemic and excitotoxic insults.6,9
In vivo evidence of p53 implication in neuronal death has also
been reported in stroke,10,11 traumatic brain injury,12 and
Alzheimer’s disease.13 The biological function of p53 is largely
mediated through the transactivation of specific target genes,
such as p21, Bax, and PUMA.14 Moreover, p53 promotes
apoptosis by preventing the expression of survival genes, by
interference with the activity of specific transcription factors,
or by regulating the mitochondrial translocation of Bax.15
The involvement of p53 in neuronal death raises the pos-
sibility that p53 inhibitors might prove effective in suppressing
the degenerative processes in neurodegenerative disorders. The
antiparasitic compound, PFT-R (Figure 1) has been identified
for its ability to protect cells against radiation, excitotoxic, and
amyloid-induced neuronal death.16
prevents cell death by inhibiting p53 transcriptional activity,
mitochondrial damage, and caspase activation, whereas it has
no effects on p53 protein levels and phosphorylation.17 Its
specificity to block p53 has been proven by its failure to prevent
p53-independent cell death.17 Recent in vivo studies further
emphasized the potential of PFT-R in preventing neuronal death
and delaying neurodegeneration.17-19 On the basis of these
observations, the identification of more potent chemical com-
pounds able to efficiently inactivate p53 activity and an
understanding of their action mechanism are promising contri-
butions to neurodegenerative disease progression. Some com-
pounds based on the PFT-R scaffold and their preliminary
biological activities have already been published by our group,20
and some other compounds described by Bachechath have been
reported as inhibitors of apoptosis in Lymphocytes.21 In this
article, we report the complete synthesis of all of the p53
inhibitors, their unique specific neuroprotective activity through
p53 inhibition, and their action mechanism in vitro and in vivo.
* Corresponding author. Tel: +33 491 82 9141. Fax: +33 491 82 9416.
E-mail:
kraus@ibdm.univ-mrs.fr
(J.-L.K.).
E-mail:
maina@
ibdm.univ-mrs.fr (F.M.).
Chemistry
† Developmental Biology Institute of Marseille (Universite´. de la
Mediterrane´e).
We sought to develop novel p53 inhibitors able to efficiently
block p53 pro-apoptotic activity both in vitro and in vivo.
Although the similarity property principle states that structurally
‡ Laboratoire de Chimie Biomole´culaire (Universite´. de la Mediterrane´e).
§ University of Go¨ttingen.
| These authors contributed equally to this work.
10.1021/jm060318n CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/24/2006

3646 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Pietrancosta et al.
Scheme 1a
a (i) Thiourea, I2, 110 °C, 12 h; (ii) saturated Na2CO3, H2O; 1a (52%), 1b (47%), 1c (37%), for 2 steps; (iii) 4-R2-C6H4-C(O)-CH(R1)Br, toluene, rt, 48
h; PFT-r (54%), 2a (61%), 2b (55%), 2c (80%), 2d (41%), 2e (47%), 2f (40%); (iv) MeOH, rt; quantitative for 4b and 4e; (v) biological medium, 37 °C;
quantitative for 4, 4b, 4e; (vi) NH2-OH, 2b, CH2Cl2, 3 h; 2g (35%).
similar compounds are expected to have similar activities,22
small structural modifications can nevertheless produce either
a slight modulation or a dramatic loss of activity. Therefore,
considering the PFT-R scaffold as a privileged structure
according to Evans’ definition,23 we introduced chemical
modifications to generate a series of novel PFT-R analogues
(Scheme 1).
The synthesis of 2-aminothiazole hydroiodide salt precursors
was carried out according to the method described by King et
al.24 The solvent free reaction was performed by heating various
cyclic ketones (cyclohexanone, 2-decalone, and cycloheptanone)
with thiourea in the presence of iodine. Basic treatment of the
hydroiodide salt allowed the isolation of the corresponding free
aminothiazoles 3a-c, which could be N-alkylated at the
endocyclic N3-position by selected R-bromoacetophenones at
room temperature in toluene. The resulting N3-substituted
2-iminothiazole derivatives PFT-r and 2a-g were isolated in
yields ranging from 40 to 70% after purification by crystalliza-
tion.
oxime derivative 2g were tested for their ability to block p53-
induced death.
Results and Discussion
Cell-Based Assay in Vitro Evaluation. The compounds were
submitted first to an in vitro cell-based assay as a primary
screening. As the read-out system for p53 inhibitor screening,
a survival assay was performed using primary cultures of E15.5
cortical neurons exposed to DNA-damaging agents (PFT-R
being considered as a p53 reference inhibitor). Before screening
the new analogues, we first verified that anticancer drugs
etoposide or camptothecin induced the death of primary cortical
neuron cultures in a time- and concentration-dependent manner
as shown in Figure 2, and then, we ensured that cortical neuron-
induced death was p53-dependent. For this purpose, primary
cultures of p53-/- cortical neurons were submitted to etoposide
treatment, and as expected (Figure 2), no death was observed
after the etoposide treatment of these p53-mutated cortical
neurons.
Cyclic analogues were obtained from the open form simply
by stirring nitro PFT-r analogues 2b and e in protic solvents
We next performed an assay to discover whether the new
synthesized PFT-r analogues (2a-g, 4b, 4e) prevented cortical
neuron death induced by etoposide (Figure 3).
i
(EtOH, PrOH). The corresponding tricyclic nitro derivatives
4b and e resulting from a dehydration process were isolated in
quantitative yields and fully characterized. Some of these
derivatives have already been reported.20,21 The ability of PFT-r
analogues to undergo this dehydration process depends on the
electron-donating or electron-withdrawing properties of the
phenyl ring substituents. Compounds bearing withdrawing
substituents such as a nitro group on the phenyl ring, for
example, 2b and e, were found to be more sensitive to the
dehydration process (few hours at room temperature) than
analogues bearing electron-donating substituents, which needed
a longer time to cyclize (2a, c, d, f) (The cyclization process
will be discussed in more detail below.) The new opened PFT
analogues (2a-f), cyclic PFT analogues (4a-g) as well as
As can be seen in Figure 3, tricyclic analogue 4b (ED50 )
30nM) is one order magnitude more active than PFT-r in
protecting cortical neurons exposed to etoposide. In contrast,
the replacement of the cyclohexene ring by larger cycles (e.g.,
cycloheptene or octahydro-naphthalene: 2f and c, respectively)
abolished inhibitory effects, showing that ring size is determina-
tive of activity. Moreover, the introduction of different substit-
uents on the aromatic ring (2a and e) modulate their inhibitory
activities. Interestingly, 2b, the corresponding open form of the
cyclic analogue 4b, shows a p53 inhibitory activity similar to
that of PFT-r, whereas oxime 2g was found inactive in
preventing p53-induced death. This later observation is of
interest because oxime 2g cannot be cyclized.

Mechanism of Action in ViVo of NoVel p53 Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3647
Figure 2. Survival of wild-type (+/+) and p53-/- mutant cells exposed to etoposide, evaluated in a time- and dose-dependent manner. Cortical
neuron cultures without etoposide treatment are set to 100%. p < 0.001 (*).
Figure 3. Percentage of survival effect of p53 inhibitors. None of the new derivatives were toxic at 10 µM. Cortical neuron cultures without
etoposide treatment are set to 100%. Plots show means of at least six independent experiments, each performed in triplicate, with standard errors
(sem). p < 0.01 (*) and p < 0.001 (**).
At this point, we selected analogue 2b (open form) and its
corresponding cyclic form 4b as the most representative
analogues for mechanistic biochemistry studies and in vivo
activities.
Action Mechanism Studies. Specificity of 4b Inhibitory
Activity. This primary screening allowed us to identify 4b as
the most efficient inhibitor of p53-triggered neuronal death in
vitro. We first investigated whether 4b would also affect p53-
independent cell death. Knowing that the overexpression of a
cytoplasmic fragment of the Met receptor tyrosine kinase
(p40Met) in cortical neurons induces apoptosis25 in a p53-
independent manner, we took advantage of this experimental
Figure 4. p53-independent death of cortical neurons was not prevented
by 4b treatment. Mouse embryonic cortical neurons were electroporated
system to test the action of 4b and PFT-r (reference compound)
on other pro-apoptotic pathways. Interestingly, 4b and PFT-r
with either an empty vector (-) or with the pro-apoptotic fragment of
Met tyrosine kinase receptor (p40Met) and then treated with either
PFT-r or 4b at indicated concentrations. Forty hours after transfection,
TUNEL was performed, and the percentage of TUNEL-positive cells
was determined. Results are the mean values of two independent
experiments.
did not prevent cortical neuronal death induced by p40Met
,
showing the remarkable specificity in the inhibitory action of
4b on p53 (Figure 4). In contrast to published results, which
have shown that some PFT analogues prevent apoptosis in
lymphocytes through both dependent and independent p53
pathways,21 we found that the tested analogues were clearly p53-
dependent inhibitors in our cortical neuron apoptosis assay.
We found that etoposide treatment triggered p53 phospho-
26
rylation on the S15 residue and the up-regulation of the p53
transcriptional target p21/WAF126,27 in wild-type cortical neu-
rons but not in p53-/-mutant cells. Similar to PFT-r,26
analogues 4b and 2b did not prevent p53 phosphorylation on
the S15 residue (data not shown). We therefore used p21/WAF1
increased protein levels as a read-out to evaluate the effects of
p53 Posttranscriptional Activity of 4b. We next biochemi-
cally assayed the ability of compounds 2b, 4b, and PFT-r to
antagonize p53 by testing p53 phosphorylation and the induction
of its downstream target, p21/WAF1.

3648 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Pietrancosta et al.
Figure 5. Time course experiment to assay the abilities of 4b (300
nM), 2b (3 µM) and PFT-r (3 µM) to prevent p21/WAF1-increased
protein levels.
the studied compounds on p53 transcriptional activity. Com-
pounds 4b and 2b prevented p53-triggered increase in protein
levels of p21/WAF1 similar to that in PFT-r (Figure 5),
indicating that these analogues behaved as p53 posttranscrip-
tional activity inhibitors.16
Performing time course as well as concentration-dependent
experiments, we found that lower concentrations of 4b (30 nM)
were sufficient to prevent the increase of p21/WAF1 levels
compared to those in PFT-r and 2b, which required higher
concentrations (300 nM) to induce the same effect (data not
shown). Altogether, these results indicate that 4b efficiently
blocks p53-triggered cell death and p21/WAF1 expression in
cortical neurons exposed to etoposide at concentrations one order
magnitude lower than that in PFT-r.
Evaluation in Vivo. We next examined whether 2b and 4b
could suppress p53-dependent neuronal death in vivo. For these
studies, we analyzed the survival of retinal ganglion cells
(RGCs) in the paradigm of axotomy-induced apoptotic cell
death. The phosphorylation of p53 on the S15 residue occurs at
day 1 after optic nerve transection with a peak at day 4 in the
RGC layer and returns to control levels at day 7 post axotomy.
The p53 phosphorylation correlates with a protracted up-
regulation of p21/WAF1 expression with a peak at day 7 after
optic nerve axotomy (data not shown). These results show that
p53 plays an important role in neuronal death in this model.
We assayed the survival of RGCs after the intravitreal treatment
with 2b, 4b, and PFT-r. Both 4b and 2b inhibitors were well
tolerated in rats after intraocular injections, as has been
previously reported for PFT-r after injection in mice.28 The
intraocular administration of PFT-r resulted in the increased
survival of RGCs at a concentration of 6 µM (131 ( 27%) but
not at 0.06 µM (98 ( 15%), relative to that of the vehicle control
(100 ( 18%) (Figure 6).
Remarkably, 2b already showed significant protective effects
on RGCs at 0.06 µM (150% ( 5). In contrast, 4b was not
effective in vivo, even at 6 µM. These studies clearly show
that cyclic analogue 4b, the most active compound in vitro, does
not show any activity in vivo. In contrast, the corresponding
open compound 2b is as efficient as PFT-r in preventing cell
death in vitro but two log values more active in vivo. To explain
these in vivo observations, we then performed stability and
degradation studies of compounds 2b and 4b.
Stability and Degradation Studies. In contrast to ketone
analogue 2b, which could be cyclized into its correspond-
ing analogue 4b (Scheme 2), oxime analogue 2g, known as
a stable protecting group in various conditions (acido-
basic media, aqueous, or different organic solvents),29 can-
not be cyclized. Recall that 2g was found to be inactive
in preventing p53-induced death in vitro (Figure 3), indicat-
ing that the ability of open compounds to undergo the cycliza-
tion process is an essential feature for inhibitory activity (Figure
3).
Figure 6. Upper panel: representative areas of flat mounted middle
retinal diameters after intraocular injections of DMSO, 2b (0.06 µM),
and PFT-r (0.06 µM) showing RGC after staining with FluoroGold.
Bar ) 40 µm. Lower panel: increased RGC survival after treatment
with p53 inhibitors. RGC numbers in DMSO injected animals are set
to zero. Intraocular injection of PFT-r slightly increased survival of
RGC at the concentration of 6 µM but not at 0.06 µM. p53 inhibition
only partially restored RGC levels, indicating that RGC degeneration
is also controlled by p53-independent pathways. Compound 2b showed
significant protective effects on RGC survival at 0.06 µM, whereas
PFT-r was only active at 6µM, and compound 4b was not effective
in vivo. RGC counts are given at the inner retinal diameters. All data
is given as mean ( sem. p < 0.01 (**); p < 0.001 (***).
media. We also found by analytical chromatography that the
degradation process of 2b was negligible. Indeed, 2b was only
converted into its corresponding cyclic derivative 4b (t1/2 ) 8
h ( 0.5) when incubated in biological conditions (data not
shown). Consistent with our data, the same cyclization process
has been recently reported for PFT-r.21,30 Interestingly, the
longer preincubation time of 2b in biological media significantly
increased its inhibitory activity.
As expected, oxime derivative 2g was found to be very stable
after incubation in the same biological media for more than 2
days. In contrast, cyclic compound 4b was no longer detected
after 24 h of incubation, and its half-life (t1/2 ) 6 h ( 0.5) was
higher than that of PFT-r (t1/2 ) 4 h ( 0.5).
It should be emphasized that we never observed the retro-
conversion of cyclic compound 4b into its open analogue 2b,
confirming that the observed dehydration is an irreversible
process. These results show that open form 2b should be
transformed, in situ, into its cyclic form 4b to become active.
Thus, 2b can be viewed as a prodrug of 4b. Open form 2b,
allows the generation over the time of the cyclic active form
4b. A stationary state occurred, during which a sufficient
concentration of 4b remained present to allow an efficient p53
inhibition in vivo.
We next analyzed the stability of 4b when incubated in
different experimental conditions: in ethanol/H2O solution or
in horse serum for 1 or 24 h. Compound 4b showed a
characteristic absorption at λmax ) 323 nm in ethanol/H2O
solution. After 24 h of incubation in horse serum, a total
disappearance of this peak was observed, suggesting that no
more free 4b was present in the incubation media. In contrast,
a new absorption peack appeared at λmax ) 398 nm, indicating
that new products were formed. Because the incubation biologi-
Compounds 2b, 4b, and oxime 2g stabilities were next
assayed in different media: ethanol/H2O, serum, or neurobasal
culture media. Using an NMR technique, we found that 2b was
converted into 4b when incubated in chemical and/or biological

Mechanism of Action in ViVo of NoVel p53 Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3649
Scheme 2a
a (i) NH2-OH, 2b, CH2Cl2, 3 h; 2g (35%).
without purification. Tetrahydrofuran (THF) was distilled over
sodium benzophenone ketyl immediately prior to use. Methylene
dichloride (CH2Cl2) was distilled over P2O5 just prior to use.
Toluene and dimethylformamide (DMF) were of anhydrous quality
from commercial suppliers (Aldrich, Acros, Carlo Erba Reagents).
Nuclear magnetic resonance spectra were recorded at 250 MHz
cal media contained several reactive moieties (free amino acids
and proteins), we hypothesized that biological species could
react with 4b to form new adducts.
The disappearance of 4b in the biological condition could
explain the observed loss of in vivo activity. It could also be
possible that during the time taken (4 days) because of low
solubility a deposition of active compound 4b in biological
conditions could occur in vivo.
1
for H and 62.9 MHz for 13C on a Bru¨ker AC-250 spectrometer.
Chemical shifts are expressed as δ units (part per million) downfield
from TMS (tetramethylsilane). Electro-spray mass spectral analysis
and LC-MS analysis were obtained on a Waters Micromass ZMD
spectrometer for the ES-MS analysis by direct injection of the
sample solubilized in acetonitrile. The LC-MS analysis was carried
out by using a Waters model 2690 pump and a Waters C18
Symmetry column with a two-mobile phase system (0.1% formic
acid in water and 0.1% formic acid in acetonitrile). Microanalyses
were carried out by Service Central d’Analyses du CNRS (Venai-
son, France) and were within 0.4% of the theoretical values.
Analytical thin layer chromatographies (TLC) and preparative thin
layers chromatographies (PLC) were performed using silica gel
plates 0.2 mm thick and 1 mm thick, respectively (60F254 Merck).
Preparative flash column chromatographies were carried out on
silica gel (230-240 mesh, G60 Merck). The melting points were
not determined because of the amorphous character of our peptides
and derivatives.
Synthesis of 2-Aminothiazole Hydroiodide Salts Derivatives.
General Procedure I. A mixture of ketone (20.0 mmol, 1.0 equiv),
thiourea (40.0 mmol, 2.0 equiv) and iodine (20.0 mmol, 1.0 equiv)
was stirred at 110 °C for 12 h. The reaction mixture was cooled to
room temperature. Hot water (20 mL) was then added to solubilize
the crude material, and the resulting solution was stirred for 30
min. The aqueous solution was washed with diethyl ether and then
neutralized by the addition of solid NaHCO3. The pale yellow
crystals were collected by filtration and identified without any
further purification as the expected compound.
Conclusion
The presented mechanistic studies as well as the stability
studies support the hypothesis that cyclic compound 4b could
be the active form generated over time from its corresponding
open precursor 2b, which acts as a prodrug precursor. Indeed,
longer 2b preincubation time in the biological assay enhanced
its in vitro inhibitory activity. Moreover, the noncyclizable
oxime analogue 2g, inactive in blocking p53-induced death,
confirms the requirement of cyclic analogues for p53 inhibition.
Open form 2b being more stable than its cyclic analogue 4b,
slowly released this later and remained active until its reaction
with the alkylating species present in biological media. Thus,
the increased availability of 2b over time is likely to ensure a
prolonged p53 inhibition after injection in vivo, which is an
essential feature for possible therapeutic application. These
results support the finding that a subtle balance between the
open prodrug precursor and the cyclic active form of the imino-
tetrahydrobenzothiazole analogues regulates their inhibitory
activity in vivo. Issues of potency and efficacy of this scaffold
as p53 inhibitors have to take into account this slow kinetic of
intramolecular cyclization to design analogues that could be
attractive drugs for in vivo therapeutic perspectives.
The identification of open analogue 2b, a two log values more
active p53 inhibitor than reference p53 inhibitor PFT-r in vivo,
and its action mechanism studies strengthen the interest to
develop novel p53 inhibitors based on the imino-tetrahydro-
benzothiazole scaffold with reduced cyclization kinetics. Further
studies are needed to address their true therapeutic potential.
This strategy may be applicable not only to block p53-triggered
death but may result in more effective treatments when
combined with the trophic support provided by survival signals.
2-Amino-4,5,6,7-tetrahydrobenzothiazole Hydroiodide Salt
(1a). The title compound was obtained by condensation of
cyclohexanone with thiourea according to general procedure I.
Yield: 57%. Mp ) 185-187 °C. 1H NMR (DMSO-d6) δ
2.50-2.48 (m, 4 H), 1.78-1.75 (m, 4 H). MS-ES m/z 283
(M + H)+.
2-Amino-5,6,7,8-tetrahydro-4H-cycloheptathiazole Hydro-
iodide Salt (1b). The title compound was obtained by condensation
of cycloheptanone with thiourea according to general procedure I.
1
Yield: 52%. Mp ) 110 °C. H NMR (DMSO-d6) δ 8.2 (s, 2 H),
2.65-2.50 (m, 4 H), 1.74 (m, 6 H). MS-ES m/z 297 (M + H)+.
Experimental Section
Chemistry. Unless otherwise noted, starting materials and
2-Amino-4,4a,5,6,7,8,8a,9-octahydro-naphtho[2,3-d]thiazole
reagents were obtained from commercial suppliers and were used
Hydroiodide Salt (1c). The title compound was obtained by

3650 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Pietrancosta et al.
condensation of decalone with thiourea according to general
1-(4-Nitrophenyl)-2-(4,4a,5,6,7,8,8a,9-octahydro-2-imino-3(2H)-
naphthothiazolyl)ethanone Hydrobromide Salt (2e). The title
compound was obtained according to general procedure III. Yield:
1
procedure I. Yield: 55%. Mp ) 58 °C. H NMR (DMSO-d6) δ
6.40 (s, 2 H), 1.20-2.50 (m, 14 H). (M + H+))336. MS-ES m/z
337 (M + H)+.
47%. Mp ) 239-240 °C (MeOH/EtOAc). H NMR (250 MHz,
1
DMSO-d6) δ 9.71 (s, 1 H), 8.34 (d, 2 H, J ) 8.9 Hz), 8.18 (d, 2 H,
J ) 8.9 Hz), 5.80 (s, 2 H), 2.61-1.20 (m, 14 H). ES/MS m/z 372
(M + H)+.
1-(4-Methylphenyl)-2-(5,6,7,8-tetrahydro-2-imino-4H-cyclo-
heptathiazolyl)ethanone Hydrobromide Salt (2f). The title
compound was obtained according to general procedure III. Yield:
61%. Mp ) 175 °C. (EtOH/EtOAc). 1H NMR (250 MHz, DMSO-
d6) δ 9.60 (s, 1 H), 7.95 (d, 2 H, J ) 8.2 Hz), 7.45 (d, 2 H, J ) 8.2
Hz), 5.22 (s, 2 H), 2.65-2.50 (m, 7 H), 1.74 (m, 6 H). ES/MS m/z
301 (M + H)+.
Synthesis of 2-Aminothiazole Derivatives. General Procedure
II. The previous 2-aminothiazole hydroiodide salt derivative was
dissolved in a hot saturated aqueous solution of Na2CO3 (20 mL).
After cooling, the aqueous solution was extracted with CH2Cl2 (3
× 20 mL). The organic layer was dried over anhydrous MgSO4,
filtered, and concentrated under reduced pressure. The desired
compound was purified by flash chromatography to yield the desired
compound as an oil, which was solidified by trituration in diethyl
ether.
2-Amino-4,5,6,7-tetrahydrobenzothiazole (3a). The title com-
pound was obtained according to general procedure II. Yield: 92%.
2-(4-Nitro-phenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-ben-
zothiazolyl)ethanone Oxime, Hydrobromide Salt (2g). A mixture
of NH2-OH (10 mmol, 1 equiv) and 2b (10 mmol, 1 equiv) was
stirred for 3 h in CH2Cl2 (70 mL) at room temperature. The title
compound was obtained after filtration and recristalization in
1
Rf 0.35 (toluene/EtOAc 3:1). Mp ) 38 °C. H NMR (DMSO-d6)
δ 6.40 (s, 2 H), 1.20-2.50 (m, 14 H). MS-ES m/z 155 (M + H)+.
2-Amino-4,4a,5,6,7,8,8a,9-octahydro-naphtho[2,3-d]thiazole
(3b). The title compound was obtained according to general
procedure II. Yield: 87%. Rf 0.30 (toluene/EtOAc 3:1). Mp ) 36
1
AcOEt/MeOH (3:1). Yield: 35%. Mp ) 235 °C. H NMR (250
1
MHz, DMSO-d6) δ 9.65 (s, 1 H), 8.18 (d, 2 H, J ) 8.9 Hz), 7.93
(d, 2 H, J ) 8.9 Hz), 6.10 (s, 2 H), 2.56-2.37 (m, 4 H), 2.12 (br,
1 H), 1.74 (m, 4 H). ES/MS m/z 413 (M + H)+.
°C. H NMR (DMSO-d6) δ 5.3 (s, 2 H), 1.20-2.50 (m, 14 H).
MS-ES m/z 209 (M + H)+.
2-Amino-5,6,7,8-tetrahydro-4H-cycloheptathiazole (3c). The
title compound was obtained according to general procedure II.
Yield: 90%. Rf 0.35 (toluene/EtOAc 3:1). Mp ) 35 °C. 1H NMR
(DMSO-d6) δ 5.2 (s, 2 H), 2.65-2.50 (m, 4 H), 1.74 (m, 6 H).
MS-ES m/z 169 (M + H)+.
Preparation of 2-Iminothiazole Derivatives. General Proce-
dure IV. The corresponding hydrobromide salt was stirred in
MeOH at room temperature for 6 h or in a biological medium at
37 °C for 6 h. The medium was concentrated and extracted with
CH2Cl2 after evaporation, and the compound was purified by
chromatography.
Preparation of 2-Iminothiazole Derivatives. General Proce-
dure III. A mixture of the previous 2-aminothiazole derivative (1.0
mmol, 1.0 equiv) and R-bromomethyl ketone (1.0 mmol, 1.0 equiv)
were solubilized in anhydrous toluene (20 mL). The reaction
mixture was stirred at room temperature for several hours while
the desired compound precipitated as an hydrobromide salt. The
precipitate was then filtered from the reaction mixture, washed with
a small amount of toluene, and recrystallized from either MeOH/
EtOAc or EtOH/EtOAc.
1-(4-Methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-ben-
zothiazolyl)ethanone Hydrobromide Salt (1) (PFT-r). The title
compound was obtained according to general procedure III. Yield:
54%. Mp ) 256-257 °C. 1H NMR (250 MHz, DMSO-d6) δ 9.60
(s, 1 H), 7.95 (d, 2 H, J ) 8.2 Hz), 7.45 (d, 2 H, J ) 8.2 Hz), 5.70
(s, 2 H), 2.51-2.31 (m, 5 H), 1.83-1.79 (m, 2 H), 1.36 (br s, 1
H), 1.02 (d, 3 H, J ) 6.5 Hz). ES/MS m/z 287 (M + H)+.
1-(4-Methoxyphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-
benzothiazolyl)ethanone Hydrobromide Salt (2a). The title
compound was obtained according to general procedure III. Yield:
61%. Mp ) 205 °C (EtOH/EtOAc). 1H NMR (250 MHz, DMSO-
d6) δ 9.47 (s, 1 H), 8.03 (d, 2 H, J ) 8.9 Hz), 7.16 (d, 2 H, J ) 8.9
Hz), 5.67 (s, 2 H), 3.89 (s, 3 H), 2.55 (m, 2 H), 2.32 (m, 2 H), 1.73
(m, 4 H). ESM/MS m/z 303 (M + H)+.
1-(4-Nitrophenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-ben-
zothiazolyl)ethanone Hydrobromide Salt (2b). The title com-
pound was obtained according to general procedure III. Yield: 55%.
Mp ) 240 °C (MeOH/EtOAc). 1H NMR (250 MHz, DMSO-d6) δ
9.54 (s, 1 H), 8.46 (d, 2 H, J ) 8.9 Hz), 8.28 (d, 2 H, J ) 8.9 Hz),
5.80 (s, 2 H), 2.56-2.37 (m, 4 H), 1.74 (m, 4 H). ES/MS m/z 318
(M + H)+.
1-(4-Methylphenyl)-2-(4,4a,5,6,7,8,8a,9-octahydro-2-imino-
3(2H)-naphthothiazolyl)ethanone Hydrobromide Salt (2c). The
title compound was obtained according to general procedure III.
Yield: 80%. Mp ) 205 °C (MeOH/EtOAc). 1H NMR (250 MHz,
DMSO-d6) δ 9.71 (s, 1 H), 8.34 (d, 2 H, J ) 8.9 Hz), 8.18 (d, 2 H,
J ) 8.9 Hz), 5.80 (s, 2 H), 2.61-1.20 (m, 17 H). ES/MS m/z 341
(M + H)+.
2-(4-Methylphenyl)imidazo[2,1-b]-5,6,7,8-tetrahydrobenzothi-
azole (4) (PFT-â). The title compound was obtained according to
1
general procedure IV. H NMR (250 MHz, DMSO-d6) δ 7.95 (d,
2 H, J ) 8.2 Hz), 7.45 (d, 2 H, J ) 8.2 Hz), 7.3 (s, 1 H), 2.51-
2.31 (m, 5 H), 1.83-1.79 (m, 2 H), 1.36 (br s, 1 H), 1.02 (d, 3 H,
J ) 6.5 Hz). ES/MS m/z 269 (M + H)+.
2-(4-Nitrophenyl)imidazo[2,1-b]-5,6,7,8-tetrahydrobenzothia-
zole (4b). The title compound was obtained according to general
1
procedure IV. Yield: 55%. Mp ) 240 °C. H NMR (250 MHz,
DMSO-d6) δ 7.65 (d, 2 H, J ) 8.5 Hz), 7.45 (s, 1 H), 7.15 (d, 2 H,
J ) 8.5 Hz), 2.40-2.50 (m, 4 H), 1.80-1.95 (m, 4 H). ES/MS m/z
300 (M + H)+
2-(4-Nitrophenyl)imidazo[2,1-b]- 4,4a,5,6,7,8,8a,9-octahydro-
naphthothiazole (4e). The title compound was obtained according
1
to general procedure IV. Yield: 30%. Mp ) 211 °C. H NMR
(250 MHz, DMSO-d6) δ 7.65 (d, 2 H, J ) 8.5 Hz), 7.45 (s, 1 H),
7.15 (d, 2 H, J ) 8.5 Hz), 2.55-1.25 (m, 14 H). ES/MS m/z 354
(M + H)+.
Stability Assay. The stability of compounds was established
according to the following procedure. First, the compounds were
incubated in the same biological conditions used to evaluate the
inhibitory activity on cortical neurons (37 °C; neurobasal medium
+ B27 + glutamate). Then, the extraction of the biological medium
was performed with methylene chloride, and after solvent evapora-
tion, the residue was analyzed by NMR. The disappearance of the
Hb (5.4 ppm) proton in open form 2b as well as the appearance of
the Ha proton (7.3 ppm) in corresponding cyclic form 4b allowed
us to characterize both the structure and half-lives of the two
analogues during in situ conversion.
Antibodies and Reagents. The antibodies used were anti-tubulin
(Sigma), anti-phospho p53 (Cell Signaling), and anti-p21 (Santa
Cruz) and Cy-3 labeled anti-rabbit (Dianova, Hamburg, Germany).
Etoposide (Sigma) was used at the concentrations indicated in the
figure legends. No toxic effects were observed at the concentrations
used.
1-(4-Methoxyphenyl)-2-(4,4a,5,6,7,8,8a,9-octahydro-2-imino-
3(2H)-naphthothiazolyl)ethanone Hydrobromide Salt (2d). The
title compound was obtained according to general procedure III.
Cortical Neuron Culture and Mice. Cortical neuron cultures
were performed according to the following procedure. Neocortices
were removed from 15 day-old wild-type or p53-/- mutant mouse
embryos and digested with trypsin-EDTA for 15 min at 37 °C.
Cortical neurons were washed twice with a DMEM/F-12 medium
supplemented with 10% horse serum (DMEM/HS), once with a
neurobasal medium supplemented with a B27 nutrient (NB/B27,
1
Yield: 41%. Mp ) 190 °C (EtOH/EtOAc). H NMR (250 MHz,
DMSO-d6) δ 9.47 (s, 1 H), 8.03 (d, 2 H, J ) 8.9 Hz), 7.16 (d, 2 H,
J ) 8.9 Hz), 5.67 (s, 2 H), 3.89 (s, 3 H), 2.55-1.25 (m, 14 H).
ES/MS m/z 357 (M + H)+.

Mechanism of Action in ViVo of NoVel p53 Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3651
50:1) (all from Invitrogen), and were then dissociated with a fire-
polished glass Pasteur pipet. For biochemistry, the dissociated cells
were seeded on poly-DL-ornithine (poly-O, Sigma)-coated 6 cm
diameter dishes (Falcon) at a density of 6 × 106/dish in DMEM/
HS. Four hours later, the medium was replaced by NB/B27. The
cells were cultured for 3 days (3DIV). Electroporation of cortical
neurons was performed as previously described.25 All mice were
kept at the IBDM animal facilities, and all experiments were
performed in accordance with institutional guide lines.
containing the retina was fixed in 4% para-formaldehyde (Sigma)
for 1 h. The retinas were then extracted and flat mounted in
glycerol-PBS (1:1) on glass slides. The number of FG-positive
RGCs was determined by fluorescence microscopy (Zeiss-Axioplan,
Oberkochen, Germany) using a UV filter (365/420 nm). Three fields
of 62 500 µm2 were counted in each retinal quadrant at eccentricities
of 1/6 (I, inner), 3/6 (M, middle), and 5/6 (O, outer) of the retinal
radius. The RGC counts were performed by two different investiga-
tors according to a blind protocol.
Survival Assays. Compounds were assessed for their ability to
protect cortical neurons against etoposide (1.25 and 2.5 µM)
alongside DMSO, and etoposide alone was used as the control.
Cells were plated in 96-well plates. Th living cells were visualized
using AM-calcein; 10 µL of calcein (1/24) was added per well,
and 45 min after incubation, 15 µL of a solution of hemoglobin in
PBS (100 mg/mL) was used to quench the reaction. Images of the
whole wells were captured using a Flash Cytometer plate reader.
The number of surviving cells was automatically evaluated by
TYNA software. All experiments were done at least six times. No
toxicity of compounds was found at 10 µM. For TUNEL staining,
electroporated cortical neurons were fixed with 4% PFA 40 h after
electroporation. Following permeabilization with 0.1% Triton
X-100, TUNEL was performed according to manufacturer’s
instructions (Apoptag, In situ apoptosis detection kit, Serologicals
Corp.). Cover slips were mounted in Vectashield/Dapi (Vector).
All assays were performed in triplicate.
Immunohistochemistry. For protein expression studies, the
animals were sacrificed on day 1, 4, or 7 post axotomy by injecting
a chloral hydrate overdose, and the eyes were extracted. The cornea,
lens, and the vitreous body were removed, and the remaining eye
cup containing the retina was fixed in 4% para-formaldehyde for 1
h. The eye cups were then dehydrated in 30% sucrose at 4 °C for
24 h and immersed in cryostat mounting liquid at -20 °C. Retinal
sections of 16 µm were prepared using a Leica cryostat, collected
on gelatin-coated glass slides and stored at -20 °C. Tissue sections
were dehumidified at 37 °C for 1 h, and antigen retrieval was
performed for 4 h in Tris-buffered saline (TBS; pH 9,0) at 60 °C.
Unspecific binding was blocked by the application of 10% goat
serum, and primary antibodies were applied in a 1:100 dilution at
4 °C overnight. Secondary antibody was then applied for 45 min
at room temperature. The sections were then nuclear counter-stained
with DAPI (Sigma-Aldrich, Munich, Germany) and mounted in
Moviol (Hoechst, Frankfurt, Germany). All comparable images of
a series were taken using identical exposure times. Adjustments
for brightness and contrast were performed using Corel Draw 8 in
an identical manner for all comparable images of one series.
Statistical Analysis. All data is given as mean ( sem. The values
were compared using one-way ANOVA and considered as signifi-
cantly different at p < 0.05.
Western Blot Analysis. Total extracts were prepared using
boiling total protein extraction buffer (TPEB: 1 part of 10% SDS,
1 part of 1M Tris HCl at pH 6.8, 2 parts of water). Then, sonication
and protein determination were performed. Western blots were done
as previously described.31
Axotomy of the Optic Nerve, Retrograde Labeling, and
Animal Numbers. Optic nerve transection was performed as
previously described.32 In brief, adult male Wistar rats (300-400
g; Charles River, Sulzfeld, Germany) were anesthetized by intra-
peritoneal injection of chloral hydrate (420 mg/kg body weight).
The skin was incised close to the superior orbital rim, and the orbita
were opened leaving the supraorbital vein intact. The lacrimal gland
was moved aside, the superior extraocular muscles detached from
their tendinous insertion points, and the eye rotated to expose the
optic nerve. The optic nerve was exposed by longitudinal incision
of the perineurium and then transected ∼2 mm from the posterior
eye pole without damaging the retinal blood supply. A 1 × 1 mm
piece of gel foam was soaked in FluoroGold (FG; 5% dissolved in
0.9% NaCl; Fluorochrome Inc., Englewood, CO) and placed on
the optic nerve stump to retrogradely label retinal ganglion cells.
The retinal blood supply was verified by fundoscopy, and animals
with persistent retinal ischemia were excluded. For RGC counts, a
total of 26 animals were used, with a minimum of n ) 3 per
experimental group: control (DMSO 2%), n ) 6; PFT-r 6 µM
and 0.06 µM, n ) 3 each; 2b 6 µM and 0.06 µM, n ) 4 each;
0.006 µM, n ) 3; 4b 6 µM, n ) 3. For immuno-histological
visualization of protein regulation, two animals were operated on
for each condition, and the contra-lateral eyes served as nonaxo-
tomized controls. All animal experiments were carried out according
to the regulations of the local animal research council and state
legislation.
Acknowledgment. We greatfully acknowledge Innate Phar-
ma (Marseille, France), H. Hassani, and T. Drenth for helpful
discussions and K. Dudley for the critical reading of the
manuscript. We also thank G. Que´le´ver, C. Garino, Y. Laras,
and V. Moret for helpful discussions regarding the synthesis of
the new compounds and S. Candeias for p53 mutant mice, R.
Demoulin and D. Michel for administrative assistance, and S.
Corby and L. Bardouillet for their technical contribution with
animal care and genotyping. This work was funded by the
“Association Franc¸aise contre les Myopathies” (AFM), the
“Fondation pour la Recherche Me´dicale” (FRM), the “Associa-
tion pour la Recherche sur le Cancer” (ARC), the “Fondation
de France” (FdF), and the ACI grant. N.P. was supported by
INSERM and a “Conseil Re´gional Provence Alpes Coˆte d’Azur”
fellowship, A.M. by “Ligue contre le cancer” fellowships, and
R.D. by a grant from “FRM” and from the Marie Curie Host
Fellowship for the Transfer of Knowledge (MTKD-CT-2004-
509804).
Supporting Information Available: Elemental analysis of
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
Intraocular Drug Administration. Intravitreal injections of p53-
inhibitors and vehicles were performed on day 0, 3, and 7 following
axotomy. Prior to injection, the animals were briefly anesthetized
with diethyl ether. Using a glass micropipet, 3 µL of the inhibitor
or vehicle (DMSO 2%) were injected into the vitreous space. The
sclera was punctured ∼1 mm away from the cornea-sclera junction
with the tip of the pipet directed toward the posterior eye pole,
taking care not to injure the lens. Animals with cataractogenic lens
injury were excluded from the study because lens injury has been
shown to have protective effects on RGC survival.
References
(1) Jenner, P.; Olanow, C. W. Understanding cell death in Parkinson’s
disease. Ann. Neurol. 1998, 44, S72-84.
(2) Dirnagl, U.; Iadecola, C.; Moskowitz, M. A. Pathobiology of
ischaemic stroke: an integrated view. Trends Neurosci. 1999, 22,
391-397.
(3) Mattson, M. P. Apoptosis in neurodegenerative disorders. Nat. ReV.
Mol. Cell. Biol. 2000, 1, 120-129.
(4) Green, V. J.; Kokkotou, E.; Ladias, J. A. Critical structural elements
and multitarget protein interactions of the transcriptional activator
AF-1 of hepatocyte nuclear factor 4. J. Biol. Chem. 1998, 273,
29950-29957.
Retinal Flat Mounts and Cell Counting. For RGC survival
assays, the animals received an overdose of chloral hydrate, and
the eyes were extracted on day 14 post axotomy. The cornea, lens,
and the vitreous body were removed, and the remaining eye cup
(5) Blum, D.; Wu, Y.; Nissou, M. F.; Arnaud, S.; Alim Louis, B.; Verna,
J. M. p53 and Bax activation in 6-hydroxydopamine-induced apo-
ptosis in PC12 cells. Brain Res. 1997, 751, 139-142.

3652 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Pietrancosta et al.
(6) Sakhi, S.; Bruce, A.; Sun, N.; Tocco, G.; Baudry, M.; Schreiber, S.
S. Induction of tumor suppressor p53 and DNA fragmentation in
organotypic hippocampal cultures following excitotoxin treatment.
Exp. Neurol. 1997, 145, 81-88.
(7) Xiang, H.; Kinoshita, Y.; Knudson, C. M.; Korsmeyer, S. J.;
Schwartzkroin, P. A.; Morrison, R. S. Bax involvement in p53-
mediated neuronal cell death. J. Neurosci. 1998, 18, 1363-1373.
(8) Cregan, S. P.; MacLaurin, J. G.; Craig, C. G.; Robertson, G. S.;
Nicholson, D. W.; Park, D. S.; Slack, R. S. Bax-dependent caspase-3
activation is a key determinant in p53-induced apoptosis in neurons.
J. Neurosci. 1999, 19, 7860-7869.
(9) Uberti, D.; Belloni, M.; Grilli, M.; Spano, P.; Memo, M. Induction
of tumour-suppressor phosphoprotein p53 in the apoptosis of cultured
rat cerebellar neurones triggered by excitatory amino acids. Eur. J.
Neurosci. 1998, 10, 246-254.
(10) Crumrine, R. C.; Thomas, A. L.; Morgan, P. F. Attenuation of p53
expression protects against focal ischemic damage in transgenic mice.
J. Cereb. Blood Flow Metab. 1994, 14, 887-891.
(11) Li, R.; Waga, S.; Hannon, G. J.; Beach, D.; Stillman, B. Differential
effects by the p21 CDK inhibitor on PCNA-dependent DNA
replication and repair. Nature 1994, 371, 534-537.
(12) Napieralski, J. A.; Raghupathi, R.; McIntosh, T. K. The tumor-
suppressor gene, p53, is induced in injured brain regions following
experimental traumatic brain injury. Brain Res. Mol. Brain Res. 1999,
71, 78-86.
(13) de la Monte, S. M.; Sohn, Y. K.; Wands, J. R. Correlates of p53-
and Fas (CD95)-mediated apoptosis in Alzheimer’s disease. J. Neurol.
Sci. 1997, 152, 73-83.
(14) Jeffers, J. R.; Parganas, E.; Lee, Y.; Yang, C.; Wang, J.; Brennan,
J.; MacLean, K. H.; Han, J.; Chittenden, T.; Ihle, J. N.; McKinnon,
P. J.; Cleveland, J. L.; Zambetti, G. P. Puma is an essential mediator
of p53-dependent and -independent apoptotic pathways. Cancer Cell
2003, 4, 321-328.
(15) Moll, U. M.; Wolff, S.; Speidel, D.; Deppert, W. Transcription-
independent pro-apoptotic functions of p53. Curr. Opin. Cell Biol.
2005, 17, 631-636.
preserve dopamine neurons and motor function in experimental
parkinsonism. Ann. Neurol. 2002, 52, 597-606.
(20) Pietrancosta, N.; Maina, F.; Dono, R.; Moumen, A.; Garino, C.; Laras,
Y.; Burlet, S.; Quelever, G.; Kraus, J. L. Novel cyclized Pifithrin-
alpha p53 inactivators: synthesis and biological studies. Bioorg. Med.
Chem. Lett. 2005, 15, 1561-1564.
(21) Barchechath, S. D.; Tawatao, R. I.; Corr, M.; Carson, D. A.; Cottam,
H. B. Inhibitors of apoptosis in lymphocytes: Synthesis and biological
evaluation of compounds related to pifithrin-alpha. J. Med. Chem.
2005, 48, 6409-6422.
(22) Johnson, M.; Lajiness, M.; Maggiora, G. Molecular similarity: a basis
for designing drug screening programs. Prog. Clin. Biol. Res. 1989,
291, 167-171.
(23) Evans, B. E.; Rittle, K. E.; Bock, M. G.; DiPardo, R. M.; Freidinger,
R. M.; Whitter, W. L.; Lundell, G. F.; Veber, D. F.; Anderson, P.
S.; Chang, R. S.; et al. Methods for drug discovery: development of
potent, selective, orally effective cholecystokinin antagonists. J. Med.
Chem. 1988, 31, 2235-2246.
(24) King, L. C.; Hlavacek, R. The reaction of ketones with iodine and
thiourea. J. Am. Chem. Soc. 1950, 72, 3722-3725.
(25) Tulasne, D.; Deheuninck, J.; Lourenco, F. C.; Lamballe, F.; Ji, Z.;
Leroy, C.; Puchois, E.; Moumen, A.; Maina, F.; Mehlen, P.; Fafeur,
V. Proapoptotic function of the MET tyrosine kinase receptor through
caspase cleavage. Mol. Cell. Biol. 2004, 24, 10328-10339.
(26) Thompson, T.; Tovar, C.; Yang, H.; Carvajal, D.; Vu, B. T.; Xu, Q.;
Wahl, G. M.; Heimbrook, D. C.; Vassilev, L. T. Phosphorylation of
p53 on key serines is dispensable for transcriptional activation and
apoptosis. J. Biol. Chem. 2004, 279, 53015-53022.
(27) Arriola, E. L.; Lopez, A. R.; Chresta, C. M. Differential regulation
of p21waf-1/cip-1 and Mdm2 by etoposide: etoposide inhibits the
p53-Mdm2 autoregulatory feedback loop. Oncogene 1999, 18, 1081-
1091.
(28) Bae, B. I.; Xu, H.; Igarashi, S.; Fujimuro, M.; Agrawal, N.; Taya,
Y.; Hayward, S. D.; Moran, T. H.; Montell, C.; Ross, C. A.; Snyder,
S. H.; Sawa, A. p53 mediates cellular dysfunction and behavioral
abnormalities in Huntington’s disease. Neuron 2005, 47, 29-41.
(29) Greene, W.; Wunts, P. G. ProtectiVe Groups in Organic Synthesis,
3rd ed.; John Wiley and Sons: New York, 1999; Vol. 1.
(30) Gary, R. K.; Jensen, D. A. The p53 inhibitor pifithrin-alpha forms a
sparingly soluble derivative via intramolecular cyclization under
physiological conditions. Mol. Pharm. 2005, 2, 462-474.
(31) Maina, F.; Pante, G.; Helmbacher, F.; Andres, R.; Porthin, A.; Davies,
A. M.; Ponzetto, C.; Klein, R. Coupling Met to specific pathways
results in distinct developmental outcomes. Mol. Cell 2001, 7, 1293-
1306.
(16) Komarov, P. G.; Komarova, E. A.; Kondratov, R. V.; Christov-
Tselkov, K.; Coon, J. S.; Chernov, M. V.; Gudkov, A. V. A chemical
inhibitor of p53 that protects mice from the side effects of cancer
therapy. Science 1999, 285, 1733-1737.
(17) Culmsee, C.; Zhu, X.; Yu, Q. S.; Chan, S. L.; Camandola, S.; Guo,
Z.; Greig, N. H.; Mattson, M. P. A synthetic inhibitor of p53 protects
neurons against death induced by ischemic and excitotoxic insults,
and amyloid beta-peptide. J. Neurochem. 2001, 77, 220-228.
(18) Zhu, X.; Yu, Q. S.; Cutler, R. G.; Culmsee, C. W.; Holloway, H.
W.; Lahiri, D. K.; Mattson, M. P.; Greig, N. H. Novel p53 inactivators
with neuroprotective action: syntheses and pharmacological evalu-
ation of 2-imino-2,3,4,5,6,7-hexahydrobenzothiazole and 2-imino-
2,3,4,5,6,7-hexahydrobenzoxazole derivatives. J. Med. Chem. 2002,
45, 5090-5097.
(32) Kermer, P.; Klocker, N.; Weishaupt, J. H.; Bahr, M. Transection of
the optic nerve in rats: studying neuronal death and survival in vivo.
Brain Res. Brain Res. Protoc. 2001, 7, 255-260.
(19) Duan, W.; Zhu, X.; Ladenheim, B.; Yu, Q. S.; Guo, Z.; Oyler, J.;
Cutler, R. G.; Cadet, J. L.; Greig, N. H.; Mattson, M. P. p53 inhibitors
JM060318N