3,5-disubstituted-4-hydroxyphenyls linked to 3-hydroxy-2-methyl-4(1h)- pyridinone: Potent inhibitors of lipid peroxidation and cell toxicity

Full text of DOI:10.1021/jm990945v

J . Med. Chem. 2000, 43, 2779-2782
2779
Ch a r t 1. Structures of Typical Antioxidant (1, 8, 9) and
Fe Chelator (2, 7) Molecules
3,5-Disu bstitu ted -4-h yd r oxyp h en yls
Lin k ed to 3-Hyd r oxy-2-m eth yl-
4(1H)-p yr id in on e: P oten t In h ibitor s of
Lip id P er oxid a tion a n d Cell Toxicity
David Bebbington,*,† Nathaniel J . T. Monck,†
Suneel Gaur,† Alan M. Palmer,‡ Karen Benwell,‡
Victoria Harvey,‡ Craig S. Malcolm,‡ and
Richard H. P. Porter‡
Departments of Chemistry and Molecular Pharmacology,
Cerebrus, Oakdene Court, 613 Reading Road,
Winnersh, Wokingham RG41 5UA, U.K.
Received November 3, 1999
In tr od u ction . For many years radical scavenging
antioxidants have been successfully used to protect
synthetic materials and food products from the degrad-
ing process of oxidation.1 More recently a role as
neuroprotective agents for the treatment of disorders
known to involve oxidative stress (e.g. stroke, traumatic
brain injury, Parkinson’s disease, and Alzheimer’s
disease) has been proposed and supported by animal
models.2 The effectiveness of radical scavengers in
reducing oxidative stress within a living biological
environment is undermined, however, by the continual
production of radicals via mechanisms such as that
described by the Fenton reaction, a process that is
catalyzed by the presence of Fe2+ (eq 1).3 We postulated
lipophilicity to the Fe chelator component (typically
hydrophilic in nature, e.g. desferrioxamine (2)), giving
the molecule a greater potential to penetrate and
sequester Fe from areas susceptible to oxidative damage
(i.e. lipids, proteins, and DNA).11,12
Ch em istr y. Favorable structural components for the
hybrid molecules were selected by the evaluation of
several classes of Fe chelator and radical scavenger in
in vitro lipid peroxidation and cell toxicity assays.13,14
This investigation, together with an assessment of
amenability to synthetic manipulation, led to the iden-
tification of 3-hydroxy-4(1H)-pyridinone15,16 and 2,6-
disubstituted phenol17 as preferred structural units for
incorporation into hybrid molecules and to the design
of target structures 3a -f, where the two structural
features are linked via simple alkyl or alkylthio chains.
The key intermediate amines 4a -f in the synthesis of
3a -f were prepared in 15-63% overall yield from the
commercially available phenols 5a -d , using standard
synthetic methods (Scheme 1). The amines 4a -f were
then reacted with benzyl maltol (6),18 followed by
deprotection, to give the target compounds 3a -f in 17-
56% yield from 4a -f (Scheme 2).
Resu lts a n d Discu ssion . Lipid peroxidation in rat
brain homogenates was used to measure the antioxidant
capacity of the molecules in a biological environment
(Table 1).13 All the compounds (3a -f) were more potent
inhibitors of lipid peroxidation than the Fe chelator
deferiprone (7)19 or the antioxidants BHT (1), Trolox (8),
and LY231617 (9).20 The two 2,6-di-tert-butyl-substi-
tuted compounds 3a ,f were the most potent inhibitors
of lipid peroxidation within the series (3a -f). Replace-
ment of the tert-butyl groups in compounds 3a ,b with
methoxy groups led to compounds 3d ,e and a conse-
quent reduction in potency. An increase in the linker
chain length as seen in compounds 3a -c also resulted
in a decrease in potency. Substitution of the benzylic
CH2 group in 3b with a sulfur atom gave the more
potent compound 3f.
(1)
that treatment with both a radical scavenger and an
Fe chelator might protect living tissue from oxidative
stress to a greater degree than is achievable with either
a radical scavenger or an Fe chelator alone (for ex-
amples of each, see Chart 1).4
A further rationale, that radical scavengers and Fe
chelators may be able to interact synergistically,5 is
supported by reports of tert-butylphenolic antioxidants
interacting with methoxyphenol,6 phosphites,7 ascor-
bate,8 thiols,8 and sulfites9 to produce synergistic anti-
oxidant effects. In addition it was hypothesized that a
significant advantage might be gained over the com-
bined administration of separate radical scavengers and
Fe chelators, by combining the structural features for
radical scavenging and Fe chelation within one “hybrid”
molecule. First, a hybrid molecule could have the
potential to behave synergistically via intramolecular
interaction, a more favorable process than one which
relies upon intermolecular interaction.10 Second, by
creating a hybrid molecule, it was likely that the
antioxidant component of the molecule (typically lipo-
philic in nature, e.g. BHT (1)) would bestow more
* Current address for correspondence: David Bebbington, DPhil.,
Vertex Pharmaceuticals (Europe) Ltd., 88 Milton Park, Abingdon,
Oxfordshire OX14 4RY, U.K. Tel: +44 (0) 1235 438 838. Fax: +44 (0)
1235 820 440. E-mail: david_bebbington@vpharm.com.
† Department of Chemistry.
‡ Department of Molecular Pharmacology.
10.1021/jm990945v CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/08/2000

2780 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Sch em e 1. Synthesis of Amines 4a -fa
Communications to the Editor
Ta ble 1. Biological Activities of Compounds
inhibition of lipid
peroxidationa
IC50, µM
protection of CGC from
IAA-induced oxidative stressb
EC50, µM (rel efficacy)c
compd
1
5.9
0.3
1.0
2.9
3.3
2.0
0.4
3.9
28.7
14.8
6.0 (0.9)
0.3 (0.9)
0.3 (0.8)
0.6 (0.7)
33.3 (0.9)
26.8 (0.8)
0.4 (0.6)
46.7 (1.0)
77.8 (0.9)
5.0 (0.9)
3a d
3be
3c
3d
3e
3ff
7
8
9
a
Compounds were tested in duplicate, and results are the
b
average of at least two independent experiments. Compounds
were tested in duplicate, and results are the average of at least
three independent experiments. c Rel efficacy: an indication of the
percent of viable cells at the maximal efficacious concentration
d
(i.e. 1.0 ) 100% viability). Tested as the mesylate salt. e Tested
as the mesylate salt. Anal. (C23H33NO3‚CH3SO3H) C, H; N: calcd,
f
2.99; found, 2.46. Tested as the hydrochloride salt. Anal.
(C22H31NO3S‚HCl) C, N; H: calcd, 7.57; found, 7.07.
F igu r e 1. Inhibition of IAA-induced oxidative stress by
compound 3b. The intracellular oxidative stress induced
during the IAA cell toxicity assay was measured using the
oxidant-sensitive fluorescent dye 2′,7′-dichlorodihydrofluores-
cein diacetate (DCFH-DA). The nonfluorescent DCFH-DA
readily crosses cell membranes whereupon it is trapped within
the cytoplasm by deacetylation as the non-membrane-perme-
able form 2′,7′-dichlorodihydrofluorescein (DCFH). Upon oxi-
dation, DCFH yields the highly fluorescent product 2′,7′-
dichlorofluorescein (DCF). Compound 3b was tested as its
mesylate salt and inhibited IAA-induced oxidative stress with
an EC50 ) 0.28 µM.
a
Reagents and conditions: i. BH3‚SMe2, THF, reflux, 63%; ii.
H2, Pd/C, EtOH, 50 psi, rt, 98%; iii. (a) SOCl2, DMF (cat.), PhMe,
CH2Cl2, rt, (b) NH4OH, THF, 0 °C, 98%; iv. LiAlH4, Et2O, reflux,
65%; v. LiAlH4, THF, reflux, 100%; vi. MsCl, Et3N, CH2Cl2, rt,
74%; vii. NaCN, DMF, 100 °C, 63%; viii. BH3‚SMe2, THF, reflux,
80%; ix. BnCl, K2CO3, DMF, 80 °C, 81%; x. MeNO2, NH4OAc,
reflux, 76%; xi. LiAlH4, THF, reflux, 86%; xii. MeCN, KOH, reflux,
46%; xiii. H2, Pd/C, MeOH, rt, 94%; xiv. LiAlH4, THF, reflux, 44%;
xv. n-BuLi, TMSCl, THF, -78 °C to rt, 97%; xvi. (a) t-BuLi, S8,
THF, -78 to -30 °C, then 2-chloroacetamide, (b) Bu4NF, MeOH,
reflux, 57%; xvii. BH3‚SMe2, THF, reflux, 100%.
Sch em e 2. Synthesis of Compounds 3a -fa
effective than BHT (1) and LY231617 (9) but more
effective than deferiprone (7) and Trolox (8). The relative
efficacy of compounds 3a -e decreased with increasing
chain length. Within the 2,6-di-tert-butyl-substituted
series, 3a ,b provided the best protection against IAA-
induced cellular toxicity, in terms of potency and rela-
tive efficacy. Using the oxidant-sensitive fluorescent dye
2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA),
the neuronal toxicity induced by IAA was shown to be
a result of oxidative stress, and 3b inhibited the
oxidation of DCFH to DCF in a similar concentration-
dependent manner to its inhibition of IAA-induced cell
death, confirming that 3b protects neuronal cells from
oxidative stress (Figure 1).21
a
Reagents and conditions: i. 5 N NaOH, H2O, EtOH, 6,18 reflux,
24-94%; ii. H2, Pd/C, EtOH, rt, 18-100% for 3a -e; iii. BCl3‚SMe2,
CH2Cl2, rt, 95% for 3f.
In addition to the significant neuroprotection offered
by the 2,6-di-tert-butyl compounds 3a -f, compound 3b
showed marked enhancement in neuroprotection over
the combination of the 3-hydroxy-2-methyl-4(1H)-pyri-
dinone 7 and di-tert-butylphenol 9. (Note: 9 was chosen
as an appropriate comparative agent for 3b because of
their similar lipophilicity.) Thus, compound 3b (mlog P
) 3.9) showed significantly enhanced neuroprotection
The ability of the compounds to protect cerebellar
granule cells (CGC) from iodoacetate (IAA)-induced
toxicity was measured (Table 1).14 The 2,6-di-tert-butyl-
substituted compounds 3a -c,f protected cells from IAA-
induced toxicity at lower concentrations than defer-
iprone (7), BHT (1), Trolox (8), and LY231617 (9). The
2,6-dimethoxy-substituted compounds 3d ,e were less

Communications to the Editor
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2781
Su p p or tin g In for m a tion Ava ila ble: Chemistry and biol-
ogy experimental details are available free of charge via the
Internet at http://pubs.acs.org.
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#
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3-hydroxy-4(1H)-pyridinone 7.19 Compounds of this
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Ack n ow led gm en t. We thank Ken Heatherington
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A. Cliffe for his help in preparing this manuscript.

2782 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Communications to the Editor
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J M990945V