Novel p53 inactivators with neuroprotective action: Syntheses and pharmacological evaluation of 2-imino-2,3,4,5,6,7-hexahydrobenzothiazole and 2-imino-2,3,4,5,6,7-hexahydrobenzoxazole derivatives

Article abstract of DOI:10.1021/jm020044d

Tumor suppressor protein, p53, is an intracellular protein that is critical within the biochemical cascade that leads to cell death via apoptosis. Recent studies identified the tetrahydrobenzothiazole analogue, pifithrin-α (2), as a p53 inhibitor that was effective in protecting neuronal cells against a variety of lethal insults and reducing the side effects of anticancer drugs. As up-regulation of p53 has been described as a common feature of several neurodegenerative disorders, including Alzheimer's disease, 2 and novel analogues (3-16) were synthesized to (i) assess the value of tetrahydrobenzothiazole analogues as neuroprotective agents and (ii) define the structural requirements for p53 inactivation. Not only did 2 exhibit neuroprotective activity in both tissue culture and in vivo stroke models but also compounds 6, 7, 10, 13, 15, and 16 proved to be highly potent in protecting PC12 cells and compounds 3, 4, and 6 were highly potent in protecting primary hippocampal cells against death induced by the DNA-damaging agent, camptothecin.

Full text of DOI:10.1021/jm020044d

5090
J . Med. Chem. 2002, 45, 5090-5097
Novel p 53 In a ctiva tor s w ith Neu r op r otective Action : Syn th eses a n d
P h a r m a cologica l Eva lu a tion of 2-Im in o-2,3,4,5,6,7-h exa h yd r oben zoth ia zole a n d
2-Im in o-2,3,4,5,6,7-h exa h yd r oben zoxa zole Der iva tives
Xiaoxiang Zhu,† Qian-sheng Yu,† Roy G. Cutler,† Carsten W. Culmsee,†,‡ Harold W. Holloway,†
Debomoy K. Lahiri,§ Mark P. Mattson,† and Nigel H. Greig*,†
Laboratory of Neurosciences, Gerontology Research Center, National Institute on Aging, Intramural Research Program,
National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, Maryland 21224, and Department of Psychiatry,
Psychiatric Research Institute, Indiana University School of Medicine, Indianapolis Indiana 46202
Received J anuary 28, 2002
Tumor suppressor protein, p53, is an intracellular protein that is critical within the biochemical
cascade that leads to cell death via apoptosis. Recent studies identified the tetrahydroben-
zothiazole analogue, pifithrin-R (2), as a p53 inhibitor that was effective in protecting neuronal
cells against a variety of lethal insults and reducing the side effects of anticancer drugs. As
up-regulation of p53 has been described as a common feature of several neurodegenerative
disorders, including Alzheimer’s disease, 2 and novel analogues (3-16) were synthesized to (i)
assess the value of tetrahydrobenzothiazole analogues as neuroprotective agents and (ii) define
the structural requirements for p53 inactivation. Not only did 2 exhibit neuroprotective activity
in both tissue culture and in vivo stroke models but also compounds 6, 7, 10, 13, 15, and 16
proved to be highly potent in protecting PC12 cells and compounds 3, 4, and 6 were highly
potent in protecting primary hippocampal cells against death induced by the DNA-damaging
agent, camptothecin.
In tr od u ction
Tissue culture studies have established strong correla-
tions between p53 expression and neuronal death
induced by DNA-damaging agents and glutamate,7,8 and
studies of p53 deficient mice have confirmed an essential
role for p53 in neuronal apoptosis resulting from is-
chemic and excitotoxic insults.9,10 The involvement of
p53 in neurodegenerative disorders raises the possibility
that chemical inhibitors of p53 activation might prove
effective in suppressing the neurodegenerative process
in such disorders.
Because of improved preventative, diagnostic, and
therapeutic measures for cardiovascular disease and a
variety of cancers, the average age of the U. S. popula-
tion continues to gradually rise. Regrettably, accompa-
nying this increase in life span, there has been a
progressive escalation in the number of individuals
afflicted with age-related neurodegenerative disorders,
which includes Alzheimer’s disease (AD), Parkinson’s
disease (PD), and stroke. Whereas different brain areas
are primarily affected in each of these diseases, specif-
ically, hippocampal and cortical neurons in AD,1 sub-
stantia nigral and midbrain dopaminergic neurons in
PD,2 and cortical and striatal neurons in stroke,3 each
disorder likely develops from activation of a common
final cascade of biochemical and cellular events that
eventually lead to neuronal dysfunction and death.1 In
this regard, different triggers, including oxidative dam-
age to DNA, overactivation of glutamate receptors, and
disruption of cellular calcium homeostasis, can initiate
a cascade of intracellular events that proceed via p53
activation of a death program termed apoptosis.1,4
While considerable evidence implicates a central role
for the transcription factor p53 in the neuronal deaths
that occur in AD,5 PD,6 stroke,7 traumatic brain injury,8
and a variety of ischemic and excitotoxic insults,9-12 no
therapeutic agents have been specifically designed to
target and inactivate p53 as an interventive strategy.
Relating to this, we recently demonstrated that the
antihelminthic (antiparasitic) compound, pifithrin-R
(PFTR) 2 (Figure 1), proved effective in protecting
cultured hippocampal neurons against death induced
by the DNA-damaging topoisomerase I and II inhibitors,
camptothecin and etoposide,13,14 respectively.11 These
agents induce neuronal apoptosis via a mechanism that
involves p53 induction and caspase activation,12,13 which,
parenthetically, were suppressed in the presence of 2.11
Compound 2, additionally, protected neuronal cells
against fatality induced by glutamate and the poten-
tially toxic AD peptide, â-amyloid,11 two insults that
similarly induce p53-mediated neuronal death.1,15 How-
ever, it was ineffective in preventing cell death caused
by trophic factor withdrawal,11 which is known to kill
cells in a p53-independent manner.16,17 With regard to
â-amyloid, it would be interesting to examine the effect
of 2 on the levels of its precursor protein and its
C-terminal fragments, which form a transcriptionally
active complex with Fe65.18 These studies confirmed the
activity of compound 2 as a p53 inhibitor19 and provided
a lead compound to optimize in the development of
potentially useful neuroprotective agents for a variety
of common debilitating neurological disorders. The
current study describes a series of novel PFTR ana-
Dedicated to Dr. Arnold Brossi, Scientist Emeritus, National
Institutes of Health, Bethesda, MD, on the occasion of his 80th
birthday.
* To whom correspondence should be addressed. Tel: 410-558-8278.
Fax: 410-558-8323. E-Mail: GreigN@vax.grc.nia.nih.gov.
† National Institutes of Health.
‡ Current address: Philipps University, Marburg, Germany.
§ Indiana University School of Medicine.
10.1021/jm020044d CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/03/2002

Novel p53Inactivators with Neuroprotective Action
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5091
of iodine was dissolved in boiling water, extracted with
ether, and made basic with solid NaHCO3 to give pale
yellow crystals as compound 1. Compound 2 was syn-
thesized from compound 1 according to a published
procedure.21 Both agents then were tested for their
activity to protect rat hippocampal neurons and PC12
cells against the toxic agents, camptothecin and etopo-
side (Figures 2 and 3).
F igu r e 1. Chemical structures of 1 and of 2.
At concentrations of 500 nM and lower, compounds 1
and 2 were not toxic when administered alone to
neurons but were toxic to neurons at higher concentra-
tions. We therefore employed concentrations of 400 nM
and lower in subsequent cell culture experiments. As
shown in Figure 2, both camptothecin (5 µM) and
etoposide (2.5 µM), known to induce neuronal apoptosis
by a mechanism involving DNA damage and p53 induc-
tion, killed approximately 75 and 85% of neurons,
respectively. Pretreatment of cells with 2, but not 1 (200
µM), resulted in improved cell survival (60-65%) as
compared to vehicle controls, demonstrating the potent
neuroprotective activity of compound 2 in vitro. Like-
wise, and as illustrated in Figure 3 (initial two blocks)
in a concentration-dependent manner, 2 but not 1
protected PC12 cells from camptothecin (40 µM)-induced
cell death.
Design a n d Syn th esis of Novel p 53 In a ctiva tor s.
The inactivity of precursor 1 indicates that the N-
substituent group of compound 2 is important for its
neuroprotective action. Modifications were therefore
primarily made to this moiety to elucidate structure/
activity relations for neuroprotective activity. In this
regard, analogues were synthesized to assess the im-
portance of the carbonyl and aryl groups, together with
substitution of electron-donating and -withdrawing
moieties on the latter (Scheme 1). In addition, the
heterocycle was also modified to assess the influence of
S to O atom replacement on neuroprotective activity
(Scheme 2).
F igu r e 2. PFTR protects cultured hippocampal neurons
against death induced by DNA-damaging agent. Hippocampal
cell cultures were pretreated for 1 h with 0.5% DMSO (vehicle),
200 nM 2, or 200 nM 2 precursor. Cultures were then exposed
for 24 h to the DNA-damaging agents camptothecin (5 µM)
and etoposide (2.5 µM). Neuron survival in each culture was
quantified (n ) 4-6 cultures + the standard deviation).
Survival following administration of 2 was significantly dif-
ferent from that resulting from vehicle and precursor 1 (p <
0.01 ANOVA with Scheffe posthoc tests).
logues synthesized to both maximize and elucidate the
p53 inactivation associated with 2.
Resu lts
Syn th esis a n d Neu r op r otective Activity for 2
a n d Its P r ecu r sor (1) in Tissu e Cu ltu r e. Synthesis
of the precursor, 2-amino-4,5,6,7-tetrahydrobenzothia-
zole hydrogen iodide (1) was carried out following the
method of King et al.20 The crude product from the
reaction of cyclohexanone with thiourea in the presence
As shown in Scheme 1, compounds 3-5 and 7-14
were synthesized using cyclohexanone and thiourea as
starting materials. 2-Amino-4,5,6,7-tetrahydrobenzothi-
F igu r e 3. PFTR analogues protect cultured PC12 cells against death induced by the DNA-damaging agent camptothecin. PC12
cells were pretreated for 6 h with compounds 1-16 (prepared in 0.5% DMSO at concentrations between 100 and 400 nM) and
were then exposed for 24 h to the DNA-damaging agents camptothecin (40 µM). Neuron survival in each culture was quantified
(n ) 3 cultures), and the mean is presented. In each case, the standard deviation was 5% or less and is not shown in the figure.
For some compounds, studies were repeated at higher or lower concentrations to obtain an EC50 value. No toxicity was associated
with the use of the PFTR analogues alone, as assessed by MTT and LDH assays (data not shown).

5092 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Zhu et al.
Ta ble 1. Concentration of Compounds Required to Induce 25%
Protection (EC25) and 50% Protection (EC50) of PC12 Cells from
Camptothecin-Induced Cell Death and Clog D Valuea
Sch em e 1
EC25 EC50
EC25 EC50
compds (nM) (nM) ClogD
compds (nM) (nM) ClogD
1
2
3
4
5
6
7
8
855 8750
9
340
80
420
170
770
380
190
410
1.33
1.11
3.01
1.47
1.79
0.99
120
415
410
230
100
150
250
250
740
570
595
305
180
350
1.75
0.86
0.26
0.16
2.05
1.28
1.26
10
11
12
13
14
15
16
340
300
120
230
35
70 -0.30
145 -1.15
a
EC50 and EC25 values were determined from logit plots of
cellular survival vs compound concentration and were calculated
from mean values (n ) 3, standard error of the mean e5%) at
three concentrations (in each, correlation coefficients r2 g 0.95).
The experimentally determined log D values of compounds 2 and
15 were 1.49 and 0.48.
drobenzoxazole according to the known procedure.22
2-Amino-4,5,6,7-tetrahydrobenzoxazole then was stirred
with R-bromomethyl ketone at room temperature for 2
days, and the precipitate was collected by filtration and
recrystallized from MeOH/EtOAC to give compounds 15
and 16 in yields of 51 and 57%, respectively.
Assa ys for Neu r op r ot ect ive Act ivit y in P C12
Cells. Compounds 3-16 were then assayed for neuro-
protective activity in PC12 cells to which a lethal dose
of camptothecin (40 µM) had been added. Neuronal
survival, expressed as a percent of untreated cells (100%
survival) and cells administered camptothecin alone (0%
survival), is shown in Figure 3. The concentration of
each analogue required to protect 25 and 50% of cells
(determined as a EC25 and EC50 value, respectively) is
shown in Table 1. Both values are included as in our
prior studies involving constitutively expressed proteins;
between 25 and 50% inactivation was required for
clinical activity. With regard to Table 1, compounds
6-8, 10, and 13-15 proved to be highly potent.
Sch em e 2
Parenthetically, we initially investigated analogues
with substituted phenyl groups to assess how such
substituents affected activity. For example, analogue 10
has an unsubstituted phenyl moiety as its aryl group
and is more potent (EC50 170 nM) than the parent
compound 2, which additionally possesses a para-
substituted methyl group (EC50 250 nM). In contrast,
11, which has a particularly bulky (biphenyl) aryl group,
with a phenyl group in the para position of the phenyl
ring (EC50 770 nM), is less potent than 2. Alternatively,
for compounds with an aryl ring having an electron-
donating substituent in the para position, such as
compound 7 with a methoxy group (EC50 180 nM),
neuroprotective potency was elevated vs parent com-
pound 2. However, when the methoxy group was moved
to the ortho position (8) or meta position (9), the
resulting compounds possessed a reduced potency (EC50
350 and 420 nM, respectively) vs 2. Then again, for the
compounds with an electron-withdrawing group on their
aryl ring, analogue 13 proved to be more potent (EC50
185 nM), whereas 12 and 14 were less potent (EC50 380
and 410 nM, respectively) than 2, indicating that a
chloro group was favored and nitro and fluoro ones were
detrimental to activity.
azole, prepared from the neutralization of compound 1
with Na2CO3, was stirred with R-bromomethyl ketone
in benzene at room temperature for 24 h to give the
crude compounds 4, 5, and 7-14, which were recrystal-
lized from MeOH/EtOAc or EtOH/EtOAc to afford
compounds 4, 5, and 7-14 as crystals. Furthermore,
2-amino-4,5,6,7-tetrahydrobenzothiazole was refluxed
with benzyl bromide for 2 days, and the precipitate was
collected by filtration and recrystallized from EtOH/
Et2O to obtain compound 3 as a white crystal. Com-
pound 6 was synthesized from 4-methylcyclohaxanone
and thiourea according to a published procedure.21
Compounds 15 and 16 were prepared from 2-hydroxy-
cyclohexanone dimer and cyanamide. Specifically, 2-hy-
droxycyclohexanone dimer was reacted with cyanamide
in a 95 °C oil bath to give 2-amino-4,5,6,7-tetrahy-
In the event that the aryl group was replaced with
an aliphatic one, such as by an ethyl moiety (4) or an
ethoxy (5), potency was decreased by some 2-3-fold

Novel p53Inactivators with Neuroprotective Action
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5093
F igu r e 5. Primary hippocampal neurons were pretreated
with compounds 3 and 4 (0.04 µM) 1 h before exposure to
glutamate (20 µM) in Locke’s medium, respectively. The
percentage of neuronal survival 24 h after the onset of insult
is given as a mean value (( the standard deviation). The
survival associated with 3 was significantly greater than that
of the vehicle (p < 0.05 ANOVA with Scheffe posthoc tests).
Cellular survival associated with exposure to 3 and 4 alone
was no different from untreated controls, indicating a lack of
toxicity (data not shown).
F igu r e 4. Protective effect of PFT-R derivatives against
neuronal apoptosis induced by DNA damage. Compounds 2-4
and 6 (100 nM) were added 1 h before exposure of primary
hippocampal neurons to the DNA-damaging compounds (a)
camptothecin (5 µM) or (b) etoposide (2.5 µM). The percentage
of neuronal survival 24 h after the treatment is given for each
group as a mean value (( the standard deviation). The
survivals associated with all agents were significantly greater
than that associated with vehicle (p < 0.05 ANOVA with
Scheffe posthoc tests). Cellular survival associated with ex-
posure to compounds alone was no different from vehicle alone,
indicating a lack of toxicity (shown in control column).
(EC50 570 and 595 nM, respectively). Absence of the
carbonyl moiety, as in 3, resulted in a 4-fold loss of
activity as compared to related analogue 10 (EC50 740
vs 170 nM, respectively). Finally, modifications in the
heterocyclic structure (Scheme 2) were undertaken to
assess effects on potency. In this regard, 6, a 6-methyl-
substituted derivative of compound 2, proved to be
equipotent to the parent, 2. In contrast, for compounds
15 and 16, replacement of S by O resulted in a 3-fold
greater potency than the related thiazole derivatives, 2
and 4, respectively.
Assa ys for Neu r op r otective Activity in Hip p oc-
a m p a l Cells. Selected compounds then were assessed
for their ability to protect hippocampal cells against
camptothecin (5 µM) or etoposide (2.5 µM) alongside
compounds 1 and 2, which were used as external
controls. As illustrated in Figure 4, none of the com-
pounds were toxic at concentrations of 100 nM following
administration to cells alone. In response to camptoth-
ecin challenge (inducing 84% cell death), neuronal
survival for 2-4 and 6 was 62, 61, 65, and 29%,
respectively, with 3 and 4 equipotent to parent, 2. In
response to etoposide challenge (inducing 74% cell
death), all compounds possessed compelling neuropro-
tective action at the concentration assessed, with 3 and
4 proving to be more potent and 6 equipotent to 2 (78,
71, 60, and 60%, respectively).
F igu r e 6. Protective effect of 2 in a model of transient and
permanent focal cerebral ischemia in mice. Compound 2 (2 mg/
kg) was administered intraperitoneally 1 h before transient
and between 1 h before and 3 h after permanent middle
cerebral artery occlusion in C57BL/6 mice. Twenty-four hours
later, mice were euthanized and the infarct size was quantified
after TTC staining of 2 mm brain sections. Values are the
mean (( the standard deviation) of 12 animals per group. *p
< 0.05 as compared to controls (Student’s t-test).
activity in a rodent model of stroke (Figure 6). In models
of transient and permanent focal ischemia, the infarct
volume for animals pretreated with 2 systemically (2
mg/kg, ip) an hour prior to middle cerebral artery
occlusion was significantly smaller (p < 0.05) than
vehicle-treated controls by approximately 50 and 25%,
respectively. When mice were treated with 2 either at
the time of artery occlusion or 3 h after, infarct volume
was similarly reduced (p < 0.05).
Con clu sion
As compounds 3 and 4 possessed neuroprotective
action against camptothecin and etoposide, they were
selected for further study against glutamate-induced cell
death. In response to an 18% cell survival after glutamate
(20µM) administration, 3 significantly elevated survival
rate to 43% (Figure 5).
In Vivo Stu d ies. To assess whether potency in the
described tissue culture models translated into in vivo
action, compound 2 was assessed for neuroprotective
Several recent studies suggest that the DNA binding
phosphoprotein, p53, plays multiple roles in cells,
particularly with regard to cell cycle regulation.1,23
Expression of high levels of endogenous wild-type (but
not mutant) p53 has two primary outcomes, specifically,
G1 cell cycle arrest or apoptosis.1,23 The observation that
DNA-damaging agents, such as etoposide and radiation,
induce levels of p53 in cells led to the characterization

5094 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Zhu et al.
of p53 as a checkpoint factor. While dispensable for
viability, in that p53 disrupted transgenic mice develop
normally,24 in response to genotoxic stress, p53 acts as
an “emergency brake” causing either arrest of prolifera-
tion or apoptosis, ultimately to protect the genome from
accumulating excess mutations. Consistent with this
notion, cells that lack p53 are typically genetically
variable and thus more prone to tumors.24,25 Conversely,
overexpression of p53 results in premature aging in
mice.26 Clearly, the value of p53-regulated apoptosis in
protecting progenitor cells with proliferative potential
(e.g., precursor B cells) from genetic instability must be
counterbalanced by the disadvantage of the same ma-
chinery in end-differentiated cells (e.g., neurons) that
lack proliferative ability and cannot be replaced.
Our current molecular studies are focused on eluci-
dating the interaction of 2 and analogues with p53 and
on the individual components within the biochemical
cascade downstream of its activation. In this regard,
X-ray crystallographic and NMR analyses have been
undertaken on p53 to define its tertiary structure and
elucidate potential ligand binding sites.27-32 For biologi-
cal function, p53 is active as a tetramer and must,
therefore, self-associate to elicit interactions with other
cellular proteins. Each p53 protein is comprised of a
transactivating region, a DNA binding region, and an
oligomerization region. Ligand binding or interaction to
any of these would potentially result in a loss of action.
For tetramerization to occur, individual p53 monomers
form relatively stable dimers via specific amino acid
interactions within their oligomerization domains;27-29
thereafter, dimers associate entirely via hydrophobic
interactions, which are particularly susceptible to dis-
ruption by small molecule compounds.32 Parenthetically,
our studies thus far have demonstrated that incubation
of 2 with camptothecin-treated neuronal cells reduces
their intracellular levels of p53, as assessed by Western
blot utilizing an antigoat p53 polyclonal antibody (Santa
Cruz Biotechnology, Santa Cruz, CA). In addition,
nuclear extracts of cells subjected to gel-shift analysis
demonstrated that 2 suppresses camptothecin-induced
increases in the level of p53 DNA binding activity.11 The
precise mechanism through which these effects are
achieved, either by direct interaction with p53 or via a
binding protein,33 remains to be elucidated. In this
regard, structure/activity analysis, as described in the
current study, may allow appropriate labeling of the
pharmacophore to define whether, as well as how, 2 and
analogues bind to p53 to modulate its function. Ir-
respective of this, downstream elements of p53 inactiva-
tion by 2 and analogues are clearly affected, with
suppression in the levels of the death protein, Bax, as
well as of caspase-3.11,34
The development of reversible inhibitors of p53 activ-
ity can fine-tune this balance and potentially restore it
in clinical conditions, such as stroke, where cell death
would be detrimental to survival. In this regard, our
synthetic and pharmacological studies have thus far
demonstrated that specific tetrahydrobenzothiazole ana-
logues possess the ability to protect neuronal cells
against toxic insults that induce cell death via p53-
dependent apoptosis. Our understanding of this series
of compounds leads to the identification of several highly
potent analogues, some of which are more potent than
the parent compound 2.
Specifically, analogues 6, 7, 10, 13, 15, and 16 proved
to be highly effective in protecting PC12 cells. In this
regard, the N-substituent group of the parent com-
pound, 2, is essential for biological action. Substitution
on the distal aryl ring exerts a remarkable influence
on activity, preferring p-chloro, p-methoxy, or a lack of
substitution. Methyl substitution in position 6 (com-
pound 6) was well-tolerated and provided an agent that
proved active in all assays. In addition, an oxazole vs a
thiazole ring was preferred in the modification of the
heterocyclic portion of the primary pharmacophore, as
the tetrahydrobenzoxazole derivatives, 15 and 16, proved
to be remarkably more potent than their corresponding
tetrahydrobenzothiazole derivatives (2 and 4). Interest-
ingly, compounds 3 and 4, which were not particularly
potent neuroprotective agents in the PC12 cell assay,
proved to be highly potent in protecting primary hip-
pocampal cells from apoptosis induced by the same
insult, camptothecin, as well as by etoposide. Of further
interest and shown in Figure 4, compound 6, although
equipotent to parent 2 and more potent than 3 and 4 in
protecting PC12 cells against camptothecin, proved less
active than equimolar amounts of each in protecting
primary hippocampal cells from the same insult. This
suggests that different cell lines likely possess disparate
vulnerabilities to apoptosis-inducing agents and express
dissimilar levels of p53. Equally intriguing, in primary
hippocampal cells, 6 proved to be more protective
against apoptosis induced by etoposide, a topoisomerase
II inhibitor, vs camptothecin, a topoisomerase I inhibi-
tor. This suggests that within the same cells, the
common biochemical pathway leading to cell death via
p53 is differentially stimulated by different apoptotic
insults with divergent pharmacological targets. Such
caveats clearly would have to be considered and further
examined when choosing specific compounds to take
forward for potential clinical assessment.
From a pharmacokinetic perspective, compound 2 and
analogues are sufficiently lipophilic to allow high brain
penetration. In this regard, their computed log D values
(octanol/water partition coefficient at physiological pH),
which correlate with permeability at the blood-brain
barrier in the absence of an inward or outward trans-
porter,35 are shown in Table 1 and cover the same
approximate range as caffeine and haloperidol that
readily enter brain.36 These computed values favorably
compare with experimentally determined values (Table
1 legend). Although the tetrahydrobenzoxazole deriva-
tives, 15 and 16, are more water soluble than their
thiazole counterparts, they clearly are sufficiently per-
meable at the cell membrane to provide potent neuro-
protective activity, and their brain uptake is the focus
of current investigation. As substitution in the 6-posi-
tion, as in 6, was well-tolerated, it could provide a site
to increase the lipophilicity of oxazole analogues to
optimize their brain penetration. The high lipophilicity
of 2 (ClogD 1.75, measured 1.49) is in accord with its in
vivo activity, after systemic administration, to reduce
stroke volume by 50 and 25% in a classical mouse model
of transient and permanent focal cerebral ischemia,
respectively. The magnitude of this reduction is consis-
tent with that, ≈40%, found in p53 deficient mice, vs
their normal littermates, in a focal ischemia stroke

Novel p53Inactivators with Neuroprotective Action
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5095
model12 and is in accord with p53 inactivation being the
mechanism of action of compound 2 and analogues in
vivo. The lower, but nevertheless significant, activity
of 2 in the permanent vs transient focal ischemia model
is in accord with the greater amount of necrosis- vs
apoptosis-induced cell death associated with the former.
Even so, the reduction in infarct volume determined at
3 h post permanent artery occlusion is suggestive of
therapeutic value, and longer time intervals prior to
treatment are the focus of current studies to define the
window of treatment opportunity.
In summary, we describe herein a novel series of
potent neuroprotective agents that may aid in providing
a better understanding of the involvement in p53 in the
molecular mechanisms underlying neuronal cell death
associated with nervous system development, aging,
injury, and disease. In addition, depending on the
results of further work to characterize any adverse
effects associated with either acute or chromic admin-
istration, the targeted inactivation of p53 function
through the use of specific analogues of 2 may provide
an innovative strategy to reduce or prevent neurode-
generation and neurological disabilities as well as to
protect against the side effects associated with cancer
therapy.37
was refluxed for 2 days. The precipitate was collected by filter,
washed with a small amount of THF, and recrystallized from
ethanol/ethyl ether to afford compound 3 (75 mg, 75%) as pale
yellow crystals; mp 271 °C. H NMR (DMSO-d6): δ 9.63 (s, 1
H), 7.45-7.35 (m, 3 H), 7.15 (d, J ) 7.1 Hz, 2 H), 5.29 (s, 2 H),
2.50-2.30 (m, 4 H), 1.76 (br, 4 H). HRMS m/z calcd for
1
C
14H17N2S, 245.1112; found, 245.1104. Anal. (C14H17BrN2S) C,
H, N.
1-(4,5,6,7-Tetr a h yd r o-2-im in o-3(2H)-ben zoth ia zolyl)-2-
bu ta n on e Hyd r obr om id e (4). Yield (50%); mp 124-125 °C
1
(EtOH/EtOAc). H NMR (DMSO-d6): δ 9.45 (s, 1 H), 5.06 (s,
2 H), 2.64 (q, J ) 7.2 Hz, 2 H), 2.41-2.52 (m, 2 H), 2.29 (br,
2H), 1.73 (br, 4H), 0.99 (t, J ) 7.2 Hz, 3 H). HRMS m/z calcd
for C11H17N2OS, 225.1062; found, 225.1071. Anal. (C11H17BrN2-
OS) C, H, N.
E t h yl 2-(4,5,6,7-Tet r a h yd r o-2-im in o-3(2H )-b en zot h i-
a zolyl)a ceta te Hyd r obr om id e (5). Yield (52%); mp 224 °C
1
(MeOH/EtOAc). H NMR (DMSO-d6): δ 9.63 (s, 1H), 4.93 (s,
2H), 4.21 (q, J ) 7.1 Hz, 2H), 2.38 (m, 2H), 1.73 (m, 4H), 1.24
(t, J ) 7.1 Hz, 3H). Anal. (C11H17BrN2O2S) C, H, N.
1-(4-Me t h ylp h e n yl)-2-(4,5,6,7-t e t r a h yd r o-2-im in o-6-
m eth yl-3(2H)-ben zoth ia zolyl)eth a n on e Hyd r obr om id e
(6). Following a procedure similar to the one described in
general procedure I, the product was obtained as white crystals
in the yield of 54%; mp 256-257 °C (MeOH/EtOAc) (lit.21 278
1
°C). H NMR (DMSO-d6): δ 9.60 (s, 1 H), 7.95 (d, J ) 8.2 Hz,
2 H), 7.45 (d, J ) 8.2 Hz, 2 H), 5.70 (s, 2 H), 2.71-2.64 (m, 1
H), 2.51-2.31 (m, 5 H), 2.21-2.12 (m, 1 H), 1.83-1.79 (m, 2
H), 1.36 (br, 1 H), 1.02 (d, J ) 6.5 Hz, 3 H). Anal. (C17H21
BrN2OS) C, H, N.
-
Exp er im en ta l Section
1-(4-Met h oxylp h en yl)-2-(4,5,6,7-t et r a h yd r o-2-im in o-
3(2H)-ben zoth ia zolyl)eth a n on e Hyd r obr om id e (7). Yield
(61%); mp 205 °C (MeOH/EtOAc). 1H NMR (DMSO-d6): δ 9.47
(s, 1 H), 8.03 (d, J ) 8.9 Hz, 2 H), 7.16 (d, J ) 8.9 Hz, 2 H),
5.67 (s, 2 H), 3.89 (s, 3 H), 2.55 (br, 2 H), 2.32 (br, 2 H), 1.73
(br, 4 H). HRMS m/z calcd for C16H19N2O2S, 303.1167; found,
303.1158. Anal. (C16H19BrN2O2S) C, H, N.
Ch em istr y. Melting points were determined with a Fisher-
1
J ohns apparatus and are uncorrected. H NMR and 13C NMR
were recorded on a Bruker AC-300 spectrometer. Mass spectra
and high-resolution mass spectra (HRMS) were recorded on
VG 7070 mass spectrometer and Finnigan-1015D mass spec-
trometer. All exact mass measurements show an error of less
than 5 ppm. Elemental analyses were performed by Atlantic
Microlab, Inc., Norcross, GA.
1-(2-Met h oxylp h en yl)-2-(4,5,6,7-t et r a h yd r o-2-im in o-
3(2H)-ben zoth ia zolyl)eth a n on e Hyd r obr om id e (8). Yield
2-Am in o-4,5,6,7-tetr ah ydr oben zoth iazole Hydr ogen Io-
d id e (1). A mixture of cyclohexanone (1.96 g, 0.02 mmol),
thiourea (3.04 g, 0.04 mmol), and iodine (5.08 g, 0.02 mmol)
was stirred in a 110 °C oil bath for 12 h. The reaction mixture
then was cooled, dissolved in boiling water, and extracted with
ether to remove ketone and iodine. The solution was neutral-
ized with solid NaHCO3, and pale yellow crystals thereafter
precipitated and were collected by filter to give 1 (3.2 g, 57%);
1
(17%); mp 160-161 °C (MeOH/EtOAc). H NMR (DMSO-d6):
δ 7.70-7.12 (m, 4 H), 5.42 (s, 2 H), 3.99 (s, 3 H), 2.50-2.39
(m, 4 H), 1.81 (br, 4 H). HRMS m/z calcd for C16H19N2O2S,
303.1167; found, 303.1173. Anal. (C16H19BrN2O2S) C, H, N.
1-(3-Met h oxylp h en yl)-2-(4,5,6,7-t et r a h yd r o-2-im in o-
3(2H)-ben zoth ia zolyl)eth a n on e Hyd r obr om id e (9). Yield
1
(52%); mp 143-145 °C. H NMR (DMSO-d6): δ 9.50 (s, 1 H),
1
mp 185-187 °C. H NMR (DMSO-d6): δ 2.50-2.48 (m, 4 H),
7.67-7.37 (m, 4 H), 5.74 (s, 2 H), 3.86 (s, 3 H), 2.55-2.33 (m,
4H), 1.73 (br, 4 H). HRMS m/z calcd for C16H19N2O2S, 303.1167;
found, 303.1171. Anal. (C16H19BrN2O2S) C, H, N.
1.78-1.75 (m, 4 H). 13C NMR (DMSO-d6): δ 168.6, 140.7,
116.3, 26.1, 24.1, 24.0, 23.6.
Gen er al P r ocedu r e I. P r epar ation of 2-Im in o-2,3,4,5,6,7-
h exa h yd r oben zoth ia zole Der iva tives by Alk yla tion of
2-Am in o-4,5,6,7-t et r a h yd r ob en zot h ia zole w it h r-Br o-
m om eth yl Keton e. Compound 1 was dissolved in hot satu-
rated aqueous Na2CO3, and after it was cooled, 2-amino-
4,5,6,7-tetrahydrobenzothiazole was gained as white needle
crystals (93%); mp 87-88 °C (lit.20 87-88 °C). A mixture of
2-amino-4,5,6,7-tetrahydrobenzothiazole (1 mmol) and R-bro-
momethyl ketone (1 mmol) in dry benzene (20 mL) was stirred
at room temperature for 2 days. Thereafter, the precipitate
was filtered off from the reaction mixture, washed with a small
amount of benzene, and recrystallized from MeOH/EtOAc or
EtOH/EtOAc.
1-P h en yl-2-(4,5,6,7-tetr a h yd r o-2-im in o-3(2H)-ben zoth i-
a zolyl)eth a n on e Hyd r obr om id e (10).38 Yield (58%); mp
1
151-152 °C (MeOH/EtOAc). H NMR (DMSO-d6): δ 9.53 (s,
1 H), 8.07-8.04 (m, 2 H), 7.79-7.74 (m, 1 H), 7.66-7.62 (m, 2
H), 5.75 (s, 2 H), 2.55-2.33 (m, 4H), 1.72 (br, 4 H). HRMS m/z
calcd for C15H17N2OS, 273.1162; found, 273.1058. Anal. (C15H17
BrN2OS) C, H, N.
-
1-(4-P h en ylph en yl)-2-(4,5,6,7-tetr ah ydr o-2-im in o-3(2H)-
ben zoth ia zolyl)eth a n on e Hyd r obr om id e (11). Yield (58%);
mp 188-189 °C (MeOH/EtOAc) (lit.21 195 °C). 1H NMR
(DMSO-d6): δ 9.55 (s, 1 H), 8.15(d, J ) 8.5 Hz, 2 H), 7.81 (d,
J ) 7.0 Hz, 2 H), 7.57-7.44 (m, 4 H), 5.80 (s, 2 H), 2.56-2.36
(m, 4 H), 1.74 (br, 4 H).
Compounds 2, 4, 5, and 7-14 were prepared according to
general procedure I.
1-(4-Flu or oph en yl)-2-(4,5,6,7-tetr ah ydr o-2-im in o-3(2H)-
ben zoth ia zolyl)eth a n on e Hyd r obr om id e (12). Yield (44%);
1-(4-Meth ylph en yl)-2-(4,5,6,7-tetr ah ydr o-2-im in o-3(2H)-
ben zoth ia zolyl)eth a n on e Hyd r obr om id e (2). Yield (67%);
1
mp 255 °C (MeOH/EtOAc). H NMR (DMSO-d6): δ 9.51 (s, 1
1
mp 180 °C (EtOH/EtOAc) (lit.21 182 °C). H NMR (DMSO-d6):
H), 8.14 (d, J ) 5.5 Hz and J ) 8.7 Hz, 2 H), 7.49 (t, J ) 8.7
Hz, 2 H), 5.74 (s, 2 H), 2.55-2.34 (m, 4 H), 1.73 (m, 4 H).
HRMS m/z calcd for C15H16FN2OS, 291.0967; found, 291.0964.
Anal. (C15H16BrFN2OS) C, H, N.
δ 8.85 (s, 1 H), 7.96 (d, J ) 8.1 Hz, 2 H), 7.45 (d, J ) 8.1 Hz,
2 H), 5.70 (s, 2 H), 2.55-2.30 (m, 7 H), 1.73 (m, 4 H).
3-(P h en ylm et h yl)-4,5,6,7-t et r a h yd r o-2(3H )-b en zot h i-
a zolim in e Hyd r obr om id e (3). A mixture of 2-amino-4,5,6,7-
tetrahydrobenzothiazole (101 mg, 0.66 mmol) and benzyl
bromide (115 mg, 0.67 mmol) in tetrahydrofuran (THF, 5 mL)
1-(4-Ch lor oph en yl)-2-(4,5,6,7-tetr ah ydr o-2-im in o-3(2H)-
ben zoth ia zolyl)eth a n on e Hyd r obr om id e (13). Yield (70%);
mp 248-249 °C (MeOH/EtOAc) (lit.21 140 °C). 1H NMR

5096 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Zhu et al.
(DMSO-d6): δ 9.52 (s, 1 H), 8.06 (d, J ) 8.6 Hz, 2 H), 7.73 (d,
J ) 8.6 Hz, 2 H), 5.74 (s, 2 H), 2.55-2.33 (m, 4 H), 1.72 (br,
4 H).
the value from
a
correlation between
a
plot of the log
concentration of compound vs logit activity. A lack of cellular
toxicity was confirmed in separate studies in which PC12 cells
(2.0 × 106 per 60 mm dish) were incubated with a selected
group of analogues for 24 h and subjected to a sensitive lactate
dehydrogenase (LDH) assay. The LDH results were consistent
with the MTT assay that was used to determine cell viability.40
Hip p oca m p a l Cu ltu r es, Exp er im en ta l Tr ea tm en t, a n d
Qu a n tifica tion of Cell Su r viva l. Dissociated hippocampal
cell cultures were established from embryonic day 18 Spra-
gue-Dawley rats (Harlan, Inc.), as described previously.11
Cells were grown in polyethyleneimine-coated plastic dishes
or 22 mm2 glass coverslips and incubated in Neurobasal
medium containing B-27 supplements, 2 mM L-glutamine, 25
mg/mL gentamycin, 1 mM Hepes (Gibco BRL), and 0.001%
gentamicin sulfate. All experiments were performed using
9-10 day old cultures. Camptothecin and etoposide (Sigma)
were prepared as 500× stocks in DMSO. Glutamate was
freshly prepared as a 200× stock in saline (pH 7.2). Neuron
survival was quantified by established methods.41 Briefly,
viable neurons in premarked fields (10× objective) were
counted before experimental treatment and at specified time
points thereafter. Neurons with intact neurites of uniform
diameter and soma with a smooth round appearance were
considered viable. In contrast, neurons with fragmented neu-
rites and vacuolated soma were considered nonviable.
F oca l Cer ebr a l Isch em ia . Surgery was performed in
anesthetized (xylazine 5 mg/kg, chloral hydrate 350 mg/kg)
mice (3 month old C57Bl/6) in accord with a NIH Animal Care
and Use Committee-approved protocol. The focal ischemia/
reperfusion model has been described previously.42 Briefly, the
method involves occluding the middle cerebral artery for 1 h
with a nylon thread and then removing it to allow reperfusion.
In the permanent focal ischemia model, the same procedure
was followed without allowing reperfusion. Compound 2 (2 mg/
kg ip) and vehicle (saline controls) were administered either
prior to (1 h), during, or after (3 h) the induction of ischemia.
During the procedure, mice were maintained at 37 °C and
blood gases, flow, and pressure were monitored. At 24 h after
ischemia, mice were anesthetized and decapitated, and their
brains were cut into 2 mm coronal sections, which then were
stained with triphenyltetrazolium for 30 min and 37 °C.
Images of the stained brains were captured by digital camera
for quantitative analysis of infarct area and volume.
1-(4-Nitr op h en yl)-2-(4,5,6,7-tetr a h yd r o-2-im in o-3(2H)-
ben zoth ia zolyl)eth a n on e Hyd r obr om id e (14). Yield (61%);
1
mp 239-240 °C (MeOH/EtOAc). H NMR (DMSO-d6): δ 9.54
(s, 1 H), 8.46 (d, J ) 8.9 Hz, 2 H), 8.28 (d, J ) 8.9 Hz, 2 H),
5.80 (s, 2 H), 2.56-2.37 (m, 4 H), 1.74 (br, 4 H). HRMS m/z
calcd for
C15H16N3O3S, 318.0912; found, 318.0903. Anal.
(C15H16BrN3O3S‚0.5H2O) H, N; C: calcd, 44.23; found, 44.67.
Gen er al P r ocedu r e II.P r epar ation of 2-Im in o-2,3,4,5,6,7-
h exa h yd r oben zoxa zole Der iva tives.39 2-Amino-4,5,6,7-tet-
rahydrobenzoxazole (1 mmol) prepared from 2-hydroxycyclo-
hexanone and cyanamide was stirred with R-bromomethyl
ketone (1 mmol) in dry benzene (20 mL) at room temperature
for 2 days. Thereafter, the precipitate was filtered off from the
reaction mixture and recrystallized from MeOH/EtOAc.
Compounds 15 and 16 were prepared according to general
procedure II.
1-(4-Meth ylph en yl)-2-(4,5,6,7-tetr ah ydr o-2-im in o-3(2H)-
ben zoxa zolyl)eth a n on e Hyd r obr om id e (15). Yield (51%);
mp 237 °C (MeOH/EtOAc). 1H NMR (DMSO-d6): δ 9.64 (br,
1H), 7.95 (d, J ) 8.0 Hz, 2H), 7.46 (d, J ) 8.9 Hz, 2H), 5.78 (s,
2H), 2.52 (br, 2H), 2.44 (s, 3H), 2.33 (m, 2H), 1.77 (m, 4H).
MS (DEI) m/z 270 [M]+. HRMS (DEI) m/z calcd for C16H18N2O2,
270.1368; found, 270.1366. Anal. (C16H19BrN2O2) C, H, N.
1-(4,5,6,7-Tet r a h yd r o-2-im in o-3(2H )-b en zoxa zolyl)-2-
bu ta n on e Hyd r obr om id e (16). Yield (57%); mp 206 °C
1
(MeOH/EtOAc). H NMR (DMSO-d6): δ 9.56 (s, 1H), 4.95 (s,
2H), 2.59 (q, J ) 7.2 Hz, 2H), 2.27 (m, 2H), 1.78-1.69 (m, 4H),
0.99 (t, J ) 7.2 Hz, 3H). HRMS (DEI) m/z calcd for C11H16N2O2,
208.1212; found, 208.1205. Anal. (C11H17BrN2O2) C, H, N.
Biologica l Activity. P C12 Cell Ap op tosis P r otection .
PC12 cells were cultured in RPMI1640 media containing 10%
horse serum and 5% fetal calf serum (37 °C, pH 7.4) until 70-
80% confluent. Thereafter, 2.2 × 104 cells per mL was added
to each trough of a 96 well plate (approximately 6270 cells
per well 285 µL) and were left to grow for 12 h. The media
then was removed and was replaced by media with vehicle or
with one of compounds 1-16 (concentration 100-400 nM,
prepared in dimethyl sulfoxide (DMSO) to a final dilution of
0.4%, eight wells per concentration). Following 6 h of incuba-
tion, camptothecin (40 µM) was added to four of each eight
wells, and the cells were incubated for a further 24 h. Cells
then were gently washed twice with phosphate-buffered saline
(PBS: 0.1 M, 37 °C, pH 7.4) and were incubated in PBS
containing the live-cell indicator dye, 2′,7′-bis(2-carboxyethyl)-
5-(and-6)-carboxyfluorescein AM ester (BCECF AM: 5 µM)
(Molecular Probes, CA) for 45 min in minimal light. This
fluorescent dye was taken up and retained by live cells.
Finally, the cells were washed twice in PBS and their
fluorescence then was quantified (excitation, 490 nm; emission,
535 nm; Perkin-Elmer HTS 7000).
Octa n ol-Wa ter P a r tition Coefficien t. Five milliliters of
0.2 mM octanol solutions of compound 2 was prepared, and
their UV absorbencies, A1, were determined by spectropho-
tometer at 254 nm wavelength. The octanol solutions then
were vigorously mixed with an equal volume of water for 15
min. Following separation, the absorbency of the octanol was
again measured, A2. An octanol-water partition coefficient,
P, was calculated from the formula: P ) A2/A1 - A2.
Refer en ces
One hundred percent cell survival was determined from cells
incubated in the absence of both any compound and camp-
tothecin. One hundred percent cellular death was determined
from cells incubated in the absence of any compound and
presence of camptothecin, which in prior studies was found to
occur at a minimum concentration of 40 µM. Compound-
induced toxicity was assessed by comparing cells treated with
compound at various concentrations in the absence of camp-
tothecin with cells incubated in the absence of both compound
and camptothecin. No compound was found to induce PC12
cell toxicity at the maximal concentration assessed (400 nM).
Compound-induced cellular protection was assessed by com-
paring cells incubated with camptothecin with cells incubated
with camptothecin in the presence of compound, in a concen-
tration-dependent manner. Finally, the natural fluorescence
of each compound was quantified in the absence of cells and
BCECF AM and was found to be negligible. A 50% effective
concentration for neuroprotection (EC50), commensurate with
an IC50 (concentration required to inhibit 50% of p53 activity),
was calculated by transforming the data into a logit format
(logit ) ln(% protection/100% - % protection)) and calculating
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