Phenolic thiazoles as novel orally-active neuroprotective agents

Article abstract of DOI:10.1016/j.bmcl.2003.09.077

Novel phenolic thiazoles compounds were prepared which demonstrated potent antioxidant activity and potent in vivo neuroprotection in mitochondrial toxin models and also possess good oral bioavailability.

Full text of DOI:10.1016/j.bmcl.2003.09.077

Bioorganic & Medicinal Chemistry Letters 14 (2004) 157–160
Phenolic thiazoles as novel orally-active neuroprotective agents
Jeremiah J. Harnett,a,* Veronique Roubert,b Christine Dolo,a Christelle Charnet,b
Brigitte Spinnewyn,b Sylvie Cornet,b Alain Rolland,a Jean-Gregoire Marin,b
b
Dennis Bigga andPierre-E. Chabrier
aDepartment of Medicinal Chemistry, Ipsen Research Laboratories, Institute Henri Beaufour, 5, Avenue du Canada,
91966 Les Ulis Cedex, France
bDepartment of Biology, Ipsen Research Laboratories, Institute Henri Beaufour, 5, Avenue du Canada,
91966 Les Ulis Cedex, France
Received20 May 2003; revised2 September 2003; accepted29 September 2003
Abstract—Novel phenolic thiazoles compounds were prepared which demonstrated potent antioxidant activity and potent in vivo
neuroprotection in mitochondrial toxin models and also possess good oral bioavailability.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
andremarkable neuroprotection in in vivo models of
mitochondrial poisoning induced by different toxins.5
Neurodegenerative disorders, Alzheimer’s, Hunting-
ton’s andParkinson’s disease as well as amyotrophic
lateral sclerosis are characterisedby a selective or gen-
eralised neuronal dysfunction. The cascade of events
that leads to neuronal dysfunction and ultimately to cell
death is multifactorial involving mitochondrial dys-
function, neuro-inflammatory processes andoxidative
stress.1
This report describes the chemical synthesis of these
compounds, their in vitro antioxidant activities and
preliminary in vivo biological results.
2. Chemistry
Cells exposedto oxidative stress suffer DNA andlipid
damage which may overwhelm the cellular antioxidant
capacity resulting in cell death by apoptosis or necro-
sis.2 Free radicals are mainly produced during mito-
chondrial respiration and mitochondrial dysfunction
has been proposed as a cause of oxidative damage.3
The thiazole compounds 1 and 2 were preparedaccord-
ing to the synthetic pathway shown in Scheme 1.
Readily available N-Cbz or N-Boc protected a-amino
amides 1a and 2b were treatedwith phosphorous pen-
tasulphide in dimethoxyethane under basic conditions
to yield the corresponding thioamides 1b and 2c. The
use of the Lawesson’s reagent ledto lower yields of
thioamides. The N-Boc protectedintermediate 2b was
preparedfrom sarcosinamide 2a and 1a is commercially
available. The thioamides 1b and 2c in toluene were
then coupledto 3,5-i-d tert-butyl-4-hydroxyphenacyl
bromide 3, to affordthe thiazoles 1c and 2d. The car-
bamate protecting groups of 1c and 2d were removed
under acidic conditions to afford the primary and sec-
ondary free amines 1d and 2e.
Neuro-inflammatory processes have also been impli-
cated in the toxicity cycle leading to neuronal dysfunc-
tion.4 Inflammatory processes in the central nervous
system (CNS) leadto increasedformation of proin-
flammatory cytokines, formation of reactive oxygen
species (ROS) andreactive nitrogen species (RNS).
Recently in our laboratories we have discovered novel
orally-active thiazole containing compounds (1 and 2)
that demonstrate potent in vitro antioxidant activity
Removal of the N-Cbz protecting group from inter-
mediate 1c under catalytic palladium-hydrogenation
provedproblematic andbasic hyrdolysis (alcoholic
* Corresponding author. Fax: +33-1-6907-3802; e-mail: jeremiah.
harnett@ipsen.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.09.077

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J. J. Harnett et al. / Bioorg. Med. Chem. Lett. 14 (2004) 157–160
pounds are potent inhibitors of LPO. As antioxidants
they are approximately 30–60 times more potent than
BHT and the antioxidant drug edaravone,7 a recently
launchedneuroprotective agent.
The mitochondrial protecting activities of the com-
pounds were evaluated in specific in-vivo mitochondrial
toxin models. 1-Methyl-4-phenyl-1,2,3,6-tetrahydro-
pyridine (MPTP) and potassium cyanide (KCN) are
neurotoxins that block ATP production at different sites
of the mitochondrial respiratory chain.8 The toxins
inhibit respectively complex I (NADH dehydrogenase)
andIV (cytochrome c oxidase) of the respiratory chain
causing free radical generation and ultimately leading to
neuronal cell death.
The compounds were evaluated for their ability to pro-
tect against KCN induced toxicity in mice as a measure
of their anti-anoxic effect.9 Compounds were admini-
stratedorally 1 h before the KCN challenge andthe
time between KCN injection andthe last gasp (the sur-
vival time) was determined.
The antioxidants BHT and edaravone orally admini-
stratedwere inactive while the thiazole derivatives were
found to dramatically delay death (see Fig. 1A). The
compounds 1 and 2 were foundto have ED 50’s of 9.3
and9.7 mg/kg.
Scheme 1. (i) BocOBoc, diisopropylethylamine, dichloromethane, 0–
20 ꢀC, 72%; (ii) (a) P2S5, dimethoxyethane, solid NaHCO3, 0–20 ꢀC,
48 h, 75%; (ii) (b) 65%; (iii) (b) toluene, 90 ꢀC, 3 h, 72%; (iii) (c) 28%;
(iv) (c) concentratedHCl, glacial acetic aci,d 100 ꢀC, 1 h, basic
workup, 88%; (iv) (d) trifluoroacetic acid, dichloromethane, triethyl-
silane, 0 ꢀC–rt, 1 h, basic workup, 73%; (v) 1 N HCl in anhydrous
diethyl-ether, diethyl-ether as solvent, quantitative yields.
The remarkable protection observedfor these com-
pounds in this acute model of intoxication was shown
where the mice treatedwith the highest dose of 30 mg/
kg were still alive 24 h after cyanide intoxication.
potassium or sodium hydroxide solutions) afforded low
yields. The free amine were then transformed into their
respective dihydrochloride salts using hydrochloric acid
in anhydrous diethyl ether to provide 1 and 2 as readily
water-soluble compounds.
MPTP administration to rodents induces a selective and
extensive destruction of dopaminergic neurons in the
substantia nigra pars compacta andprovokes the
depletion of brain dopamine thus providing a striking
pathological analogy to Parkinson’s disease.10 Mono-
amine oxidase-B (MAO-B) mediates the conversion of
MPTP to 1-methyl-4-phenylpyridinium (MPP+), its
3. Results and discussion
The butylated hydroxy toluene (BHT) (2,6-di-tert-butyl-
4-methylphenol) moiety of compounds 1 and 2 is a fea-
ture common to many antioxidants. The antioxidant
activity of BHT is believedto be partly due to its capa-
city to scavenge free radicals and by doing so ultimately
terminate the free-radical chain oxidation cycle. The
capacity of 1 and 2 to act as antioxidants was measured
by their ability to inhibit iron-induced lipid peroxi-
dation (LPO) in rat brain microsomes.6 LPO induced by
Fe2+ is due to the generation of free radicals where the
antilipoperoxidant activity of 1 and 2 is measuredby
their ability to reduce the concentration of mal-
ondialdehyde, a product of unsaturated fatty acid per-
oxidation.
Table 1. The effect of compounds 1, 2, BHT andEdaravone on the
inhibition of lipidperoxidation andprotection against potassium cya-
nide induced lethality and the neuroprotective effect of 1 and 2 on
MPTP-mediated loss of dopamine.
1
CompdR
LPO
inhibition
IC50, mMa
KCN induced
lethality
MPTP mediated
dopamine loss
ED50, mg/kgb
ED50, mg/kgb
1
H
CH3
0.54
0.93
30.00
32.00
9.3
9.7
6.5
4.3
—
The compounds 1 and 2 were expectedto have
improvedantioxidant activity comparedto BHT. This
may be partly attributedto the extra stabilisation of the
phenoxy radical (product of free radical scavenging)
provided by the p-conjugatedthiazole system in 1 and 2
comparedto the simple aromatic nucleus of the BHT
compound. As can be seen from Table 1, the com-
2
BHT
Edaravone
NSc
NSd
—
a Values are from one experiment in quadruplicate.
b Dose producing 50% activity, n=9–10, see Figure 1.
c NS, non significant effect when testedat 30 mg/kg (po).
d NS, non significant effect when testedup to 100 mg/kg (po).

J. J. Harnett et al. / Bioorg. Med. Chem. Lett. 14 (2004) 157–160
159
Figure 1. In vivo mitochondria protecting models: (A) the neuroprotective effect of 1 and 2 (po) on the cyanide intoxication in mice. *Significance,
comparedwith control group (CT), ** p<0.01 and***= p<0.001 (Student and Dunnett tests ), n=9–10; (B) the neuroprotective effect of 1 and 2
(po) on MPTP-mediated loss of dopamine in the mouse striatum. *Significance, compared with MPTP group, *p<0.05 and*** p<0.001 (Student
andDunnett tests), n=5–6.
toxic metabolite accumulating in the mitochondria
where it disrupts cellular respiration.
4. (a) Floyd, R. Free. Radical. Biol. Med. 1999, 26, 1346. (b)
Gonzalez-Scarano, F.; Baltuch, G. Annu. Rev. Neurosci.
1999, 22, 219. (c) Hirsch, E. C.; Hunot, S.; Damier, P.;
Faucheux, B. Ann. Neurol. 1998, 44, S115. (d) McGeer,
P. L.; McGeer, E. G. Alzheimer. Dis. Assoc. Disord. 1998,
12 (Suppl. 2), S1.
5. (a) Harnett, J. J.; Auguet, M.; Viossat, I.; Dolo, C.; Bigg,
D.; Chabrier, P.-E. Biorg. Med. Chem. Lett. 2002, 12,
1439. (b) Chabrier, P. E.; Roubert. V.; Harnett, J. J.;
Cornet, C.; Delaflotte, S.; Charnet, C.; Spinnewyn. B.;
Auguet, M. New Neuroprotective Agents Are Potent Inhi-
bitors of Mitochondrial Toxins: In Vivo and In Vitro Stud-
ies; 31st Annual Meeting of the Society for Neuroscience,
San Diego, CA, Nov. 10–15, 2001. (c) Spinnewyn, B.;
Cornet, S.; Auguet, M.; Chabrier, P.-E. J. Cereb. Blood
Flow Metab. 1999, 19, 139.
Oral administration of 1 at 1, 3, 10 mg/kg and 2 at 1, 3,
10, 30 mg/kg antagonised, in a dose-dependent manner,
dopamine depletion in the mouse striatum11 induced by
MPTP (see Fig. 1B). The ED50 for 1 was foundto be
6.5 mg/kg andfor 2 was 4.3 mg/kg. The remarkable
protection afforded by these compounds in this model
can be seen where 1 provided a 68% protection against
brain dopamine depletion at 10 mg/kg and 2 at the same
dose provided 100% protection.
In conclusion, we have synthesisednew phenolic thi-
azoles 1 and 2 which are potent antioxidants. They are
orally bioavailable andaffordpotent in vivo neuropro-
tection in models of mitochondrial toxin administra-
tion. Since the neurotoxins administrated act at different
levels of the mitochondrial respiratory chain it may be
considered that these compounds are acting as mito-
chondrial protecting agents.5b Moreover, it has recently
been shown that 1 provided increased survival and
neuroprotective effects in a transgenic mouse model of
Huntington’s disease.12
6. Esterbauer, H.; Cheeseman, K. H. Methods Enzymol.
1990, 186, 407.
7. (a) Edaravone (MCI-186, RadicutTM): Graul, A.; Cas-
tane, J. Drugs Future 1996, 21, 1014. (b) Tabrizchi, R.
Curr. Opin. Investig. Drugs 2000, 1, 347.
8. (a) Nicklas, W. J.; Vyas, I.; Heikkila, R. E. Life. Sci. 1985,
36, 2503. (b) Webb. J. L. In Enzymatic and Metabolic
Inhibitors; Academic: New York, 1966. (c) Araki, H.;
Karasawa, Y.; Nojiri, M.; Aihara, H. Methods Find. Exp.
Clin. Pharmacol. 1988, 10, 349.
In the light of these promising results, compounds 1 and
2 merit further study.
9. CD1 male mice were used, weighing 23–27 g (Charles
River, France). The test drugs were administrated orally
(20 mL/kg) 1 h before a bolus injection of KCN (4 mg/kg,
iv, the LD100). The mice were then observedfor 120 s,
noting the survival time, which representedthe time
between KCN injection andthe last gasp.
Acknowledgements
The authors wouldlike to thank Michel Auguet, Jose
Camara andtheir teams for their helpful advice and
support.
10. (a) Burns, R. S.; Chiueh, C. C.; Markey, S. P.; Ebert,
M. H.; Jacobowitz, D. M.; Kopin, I. J. Proc. Natl. Acad.
Sci. U.S.A. 1983, 80, 4546. (b) Langston, J. W.; Forno,
L. S.; Rebert, C. S.; Irwin, I. Brain. Res. 1984, 292, 390.
(c) Tetrud, J. W.; Langston, J. W. Science 1989, 245, 519.
11. The study was performed on C57bl6 mice. Striatal dopa-
mine levels were measuredby HPLC one day after treat-
ment with MPTP (three intraperitoneal injections of
20 mg/kg at 2-h intervals). Compounds 1 and 2 were dis-
solvedin water (10 mL/kg) andadministeredorally 90 min
before each MPTP injection, 30 min after the last MPTP
injection and24 h after the beginning of the experiment.
References and notes
1. (a) Alexi, T.; Borlongan, C. V.; Faull, R. L.; Williams,
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Neurobiol. 2000, 60, 409. (b) Albers, D. S.; Beal, M. F. J.
Neural. Transm. 2000, 59, 133. (c) Beal, M. F. Biochim.
Biophys. Acta 1998, 1366, 211.
2. Powis, G.; Briehl, M. J. Pharmacol. Ther. 1995, 68, 149.
3. Hosokawa, M.; Ueno, M. Neurobiol. Aging 1999, 2, 117.

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24 h after the last MPTP injection the whole brain was
ratedusing a refrigeratedWaters 717 plus Ultrawisp
autoinjector at 6 ꢀC; a Beckman Ultrasphere ODS C18
reverse phase, 5-m column (4.5 Â 150 mm) at 24 ꢀC, using
a ANTEC Leyden electrochemical detector set at a
potential of 770 mV. Samples were elutedat 1 mL/min.
The system was recalibratedafter every six samples. The
mobile phase consistedof 90% of 70 mM KH 2PO4,
0.1 mM EDTA, 2.1 mM TEA and1.25 mM soidum
octane sulfate pH=3.7 and10% methanol. Methanol was
added after pH adjustment.
removed, the striatum dissected, frozen in solid CO2 and
storedat À80 ꢀC. Dopamine was measured by adding
homogenisation solution containing l-cysteine, Na2S2O5
andHClO 4 to the striatum (40 mg/mL). The striatum was
then homogenisedwith Ultraturax 30 s in ice andthe
homogenate centrifugedfor 10 min at 2000 g at 4 ꢀC. The
supernatant was re-centrifugedat 4000 g for 1 min to
ensure removal of solidmatter. Aliquots (20 mL) of the
striatum extracts were then analysedby HPLC. A HPLC
apparatus with electrochemical detection was used to
measure striatal levels of dopamine. Dopamine was sepa-
12. Klivenyi, P.; Ferrante, R. J.; Gardian, G.; Browne, S.;
Chabrier, P.-E.; Beal, M. F. J. Neurochem. 2003, 86, 267.