Altering pyridinone N-substituents to optimise activity as potential prodrugs for Alzheimer's disease

Article abstract of DOI:10.1039/b815404j

Selective design modifications of specifically substituted 3-hydroxy-4(1H)-pyridinones show possibly advantageous ring freedom while maintaining metal-binding ability and antioxidant capacity, moving toward an efficient potential treatment for Alzheimer's disease.

Full text of DOI:10.1039/b815404j

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Altering pyridinone N-substituents to optimise activity as potential prodrugs
for Alzheimer’s disease†
Lauren E. Scott,‡ Brent D. G. Page,‡ Brian O. Patrick and Chris Orvig*
Received 3rd September 2008, Accepted 12th September 2008
First published as an Advance Article on the web 6th October 2008
DOI: 10.1039/b815404j
Selective design modifications of specifically substituted 3-
hydroxy-4(1H)-pyridinones show possibly advantageous ring
freedom while maintaining metal-binding ability and antioxi-
dant capacity, moving toward an efficient potential treatment
for Alzheimer’s disease.
in the brain, the glucose moiety can be enzymatically removed,
leaving an efficient metal binding agent that will sequester
metals through the a-hydroxy-keto functionality of the pyridinone
moiety.3 Among a range of other pyridinone compounds, 3-(b-D-
glucopyranosyloxy)-2-methyl-1-phenyl-4(1H)-pyridinone (Gppp)
has been previously synthesised in our group; enzymatic cleavage
of the glycosidic bond was demonstrated using Agrobacterium sp.
b-glucosidase.12
The medically interesting and testable forms of these com-
pounds are the active, un-glycosylated species that are capable of
binding metals. Members of our research group have proposed
a series of pyridinones showing moderate activity in in vitro
AD tests.3 Our work has focused on optimising the pro-ligand
structures to maximise their efficacy against the symptoms of
AD. Two pyridinones that have been tested against some of the
biomarkers of AD are shown in Fig. 2.
The recent success of metal-attenuating compounds (now in phase
2 clinical trials) for the treatment of Alzheimer’s disease (AD) has
brought further support to the amyloid cascade hypothesis and
emphasises the role that cerebral metals (Cu, Fe and Zn) play
in the pathology of AD.1,2 Metal-binding pyridinone prodrugs
(Fig. 1) that possess antioxidant characteristics and brain targeting
abilities have been suggested as treatments for AD that may have
better efficacy than treatments that only sequester metals.3
Fig. 1 Generic structure of a glycosylated pyridinone prodrug as a
potential multifunctional AD therapeutic.3
Pyridinones are chosen as the scaffold for elaboration because
they are generally non-toxic, and approved for therapeutic use
in many parts of the world (including Europe, where 3-hydroxy-
2-methyl-4(1H)-pyridinone is used to mitigate iron overload in
thalassemia patients).4 Hydroxypyridinones are effective metal
binders (logb2 = 20–22 for Cu2+,5 11–18 for Zn2+;6 and logb3 = 27–
37 for Fe3+)7 and their structure is easily modified by altering the
N-substituent; which may be varied without significantly affecting
the metal binding efficiency of the pro-ligand.8
Because the brain requires a large portion of the body’s
energy intake, it is often found that molecules incorporating a
glucose moiety will be transported into the brain;9 this targeting
strategy has been used to effectively elevate brain uptake of
compounds such as dopamine for Parkinson’s disease therapy
and opiate analgesics.10,11 In our design strategy, this is achieved
by attaching a glucose moiety at the 3-hydroxyl oxygen. Once
Fig. 2 Analogous hydroxypyridinones Hppp (left) and Hnbp (right).
3-Hydroxy-2-methyl-1-phenyl-4(1H)-pyridinone (Hppp) was
first studied as a potential AD treatment because it incorpo-
rates the desired antioxidant capacity and metal-binding ability.3
Adding a methylene spacer between the pyridinone ring and the
phenyl ring (such as in Hnbp) may alter the antioxidant capacity
and may also affect the ability to infiltrate and ameliorate toxic
beta-amyloid (Ab) deposits within the brains of AD patients.
Hppp was prepared by previously reported methods.13 1-Benzyl-
3-hydroxy-2-methyl-4(1H)-pyridinone (Hnbp) was synthesised
based on methods reported by Kitagawa et al.14 and Barta et al.15
All products were isolated as colourless solids; intermediate species
1
were characterised using H NMR spectroscopy, final products
were additionally characterised by 13C{H} NMR spectroscopy
and elemental analysis, and Hnbp was further characterised by
X-ray crystallography.
Medicinal Inorganic Chemistry Group, Department of Chemistry, University
of British Columbia, Vancouver, Canada V6T 1Z1. E-mail: orvig@chem.
ubc.ca
† Electronic supplementary information (ESI) available: experimental
details of syntheses, turbidity, TEAC and MTT assays; selected crys-
tallographic data for Hnbp, Cu(ppp)2 and Cu(nbp)2. CCDC reference
numbers 697416–697418. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/b815404j
Demonstration of metal-binding ability was achieved through
synthesis of metal complexes of Hppp and Hnbp. Copper com-
plexes are of particular interest because copper is one of the redox-
active metals involved in AD, and has higher affinity for Ab than
does iron.16 In this light, copper complexes were synthesised by
adding two equivalents of deprotonated pro-ligand to a solution
of Cu(ClO4)·6H2O in a mixture of dichloromethane and methanol.
Stirring the mixture for 6 h afforded the copper complex as a fine
‡ Co-authorship statement: L.E.S. and B.D.G.P. contributed equally to
this work and shall be recognised as co-first authors.
6364 | Dalton Trans., 2008, 6364–6367
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Table 1 Selected crystallographic data for Hnbp·CHCl3, Cu(ppp)2·2CHCl3 and Cu(nbp)2·2CHCl3
Hnbp·CHCl3
C13H13NO2·CHCl3
334.61
Monoclinic, P21/c (#14)
11.7981(13)
10.8093(13)
12.5786(15)
107.760(6)
1527.7(3)
4
173(2)
16 003/3665 (Rint = 0.025)
wR2 = 0.110
R1 = 0.045
Cu(ppp)2·2CHCl3
C24H20N2O4Cu·2CHCl3
702.70
Orthorhombic, Pbca (#61)
11.5466(11)
11.6975(11)
20.9936(18)
90
2835.5(5)
4
173(2)
35 879/2789 (Rint = 0.046)
wR2 = 0.071
R1 = 0.028
Cu(nbp)2·2CHCl3
Formula
M
C26H24N2O4Cu·2CHCl3
730.75
Monoclinic, P21/n (#14)
6.3840(3)
21.0223(12)
11.7249(7)
96.787(2)
1562.53(15)
2
Crystal system, space group
˚
a/A
˚
b/A
˚
c/A
b/◦
V/A
Z
T/K
3
˚
173(2)
Refln collcd/unique
13 415/3545 (Rint = 0.033)
wR2 = 0.163
R1 = 0.053
Residuals (F2, all data)
Residuals (F, I > 2s(I))
green precipitate; X-ray quality crystals were obtained via liquid–
liquid diffusion of chloroform and diethyl ether.
investigation is required to determine the magnitude of this effect
in vivo.
Green prism crystals of Cu(ppp)2 and Cu(nbp)2 were analyzed
using X-ray diffraction techniques (see Table 1) and assigned
the structures shown in Fig. 3. These complexes co-crystallised
with two molecules of chloroform per copper centre, which is not
unknown for bis-pyridinonato–copper complexes crystallised in
this manner.17 The trans and nearly square planar coordination
geometry that is seen in these complexes is also exhibited
in the few other structurally characterised pyridinone–copper
complexes.5b,17,18 Comparing the solid-state structures of Cu(ppp)2
and Cu(nbp)2 we see that they are very similar in their coordination
geometries; however, Cu(nbp)2 possesses additional ring freedom
compared to that found in Cu(ppp)2.
A turbidity assay was performed to investigate the ability of
Hnbp to dissolve aggregated Ab species in vitro. Synthetic human
Ab1–40 was dissolved in a buffered aqueous solution to which was
added metal ions (Cu2+ or Zn2+, in different trials). Copper and zinc
were used in this assay as they are found in elevated concentrations
in Alzheimer’s amyloid plaques.19,20 Metal ion addition causes Ab
aggregation, which, through formation of a turbid solution, is
measureable by absorption spectroscopy; suspended solids (such
as aggregated Ab) scatter light and give an increase in apparent
absorbance. Addition of metal binding pro-ligands reduces this
apparent absorption and thus a comparison of Ab disaggregating
ability can be made between pro-ligands; lower absorbance indi-
cates greater efficacy. Diethylenetriaminepentaacetic acid (DTPA)
was used as a positive control; it is a hexadentate ligand known to
bind metals and dissolve metal-containing Ab aggregates.3,21
Hppp and Hnbp significantly reduce the amount of aggregated
Ab compared to the “Ab and metal” negative control for both the
copper and zinc trials (Fig. 4). In both metal trials, the abilities of
Hppp and Hnbp to reduce Ab aggregates in solution were found
to be statistically equivalent.
Fig. 3 Ellipsoid plots (50% probability, H-atoms omitted for clarity) of
Cu(ppp)2 (above) and Cu(nbp)2 (below).
In the solved structure of Cu(nbp)2, the benzyl ring is found in
a variety of orientations within the crystal lattice; the structure
shown in Fig. 3 is representative of one of these orientations. In
the Cu(ppp)2 complex, the phenyl ring is oriented at an angle
of 63◦ relative to the pyridinone ring. The additional freedom
of ring orientations seen in solid state Cu(nbp)2 and presumably
in Hnbp itself, may allow the compound to better infiltrate Ab
aggregates and permit improved access to the problematic metal
species involved in AD.
It is unlikely that either of these pyridinones remain in a fixed-
ring orientation while in physiological conditions, however the
solid state structures hint that there is a smaller energy barrier to
rotation with Hnbp which may prove to be advantageous. Further
Fig. 4 Dissolving Ab aggregates; absorbance taken at 45 min after
addition of test pro-ligands to metal-aggregated Ab. Bars indicate mean
absorbance of solutions at 405 nm (n ≥ 3) and error bars indicate standard
deviation.
The antioxidant capacities of Hppp and Hnbp were determined
using a trolox equivalent antioxidant capacity (TEAC) assay.22
2,2¢-Azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) is
easily made into a radical cation (ABTS∑+) that absorbs strongly
at 745 nm, but does not absorb at this wavelength in its neutral
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and non-radical form.22 Measuring the reduction of absorbance
upon addition of a compound gives a value for the antioxidant
capacity of that compound. The antioxidant capacities of Hppp
and Hnbp are compared with that of a-tocopherol (vitamin E, a
known antioxidant) in Fig. 5. For all test compounds, the TEAC
was calculated by normalising the effective radical quenching of
each test compound to that of trolox, at time points of 1, 3
and 6 min. At all time points, the TEAC values for Hppp and
Hnbp are statistically equivalent, and are comparable to that of
a-tocopherol.
There is evidence from the solid-state structures of Cu(ppp)2 and
Cu(nbp)2 that the addition of the methylene unit may allow more
rotational freedom which may improve the compound’s ability to
infiltrate large Ab aggregates in vivo; further investigation is called
for.
The synthetic route utilised to produce Hnbp presents some
synthetic advantages over other methods developed to synthesise
pyridinones. It is thought that this synthetic method could be
used to make a library of pyridinone pro-ligands similar to Hnbp,
allowing this project to more fully examine the effect of N-
substituent variation on pro-ligand activity.
Acknowledgements
This work was supported by the Natural Sciences and Engineering
Research Council (NSERC). L.E.S. acknowledges the Alzheimer’s
Society of Canada for a Doctoral Training Award and B.D.G.P.
acknowledges NSERC for an Undergraduate Student Research
Award. The authors wish to thank Prof. C. Overall and lab.
for allowing us the use of their plate reader for turbidometric
assays performed in the Centre for Blood Research, University of
British Columbia. The authors would also like to acknowledge Dr
Elena Polishchuk and Jessie Chen at UBC Biological Services for
assistance with MTT assays.
Fig. 5 Antioxidant activity of hydroxypyridinones Hppp and Hnbp
compared to a-tocopherol (vitamin E) at the 6 min time point. Shown
are means of three trials and error bars represent standard deviation.
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