Synthesis of a novel radical trapping and carbonyl group trapping anti-AGE agent: A pyridoxamine analogue for inhibiting advanced glycation (AGE) and lipoxidation (ALE) end products

Article abstract of DOI:10.1021/ol0348147

(Matrix presented) Pyridoxamine is known to be an effective inhibitor of both advanced glycation (AGE) and advanced lipoxidation (ALE) end products. The synthesis of a novel multifunctional AGE and ALE inhibitor, 6-dimethylaminopyridoxamine (dmaPM, 11) is

Full text of DOI:10.1021/ol0348147

ORGANIC
LETTERS
2003
Vol. 5, No. 15
2659-2662
Synthesis of a Novel Radical Trapping
and Carbonyl Group Trapping Anti-AGE
Agent: A Pyridoxamine Analogue for
Inhibiting Advanced Glycation (AGE)
and Lipoxidation (ALE) End Products
Sean M. Culbertson,* Gary D. Enright, and K. U. Ingold
Steacie Institute for Molecular Sciences, National Research Council Canada,
Ottawa, Ontario K1A 0R6, Canada
sean.culbertson@nrc.ca
Received May 12, 2003
ABSTRACT
Pyridoxamine is known to be an effective inhibitor of both advanced glycation (AGE) and advanced lipoxidation (ALE) end products. The
synthesis of a novel multifunctional AGE and ALE inhibitor, 6-dimethylaminopyridoxamine (dmaPM, 11) is described. The 6-dimethylamino
substituent increases the radical trapping ability of pyridoxamine’s phenolic group. Results obtained during ribose glycations show that both
the new dmaPM and a known strong radical trapping agent, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), prevent intermolecular
protein cross-linking more effectively than pyridoxamine (PM).
Nonenzymatic protein glycation by reducing sugars, such
as glucose or ribose, (the Maillard reaction) is a complicated
cascade of condensations, rearrangements, fragmentations,
and oxidative modifications that lead to products collec-
tively called advanced glycation end products (AGE).1,2
During lipid peroxidation reactions, a number of reactive
carbonyl compounds are formed by the decomposition of
lipid hydroperoxides, including malondialdehyde (MDA) and
hydroxynonenal (HNE). Nucleophilic proteins such as lysine
can trap such lipid peroxidation products to form adducts
(e.g., MDA-Lys, HNE-Lys) collectively called advanced
lipoxidation end products (ALE).2,3 Once formed, both AGE
and ALE products may prevent normal protein function or
recognition or stimulate potentially detrimental interactions
in signaling pathways.4 The formation of AGE and ALE has
been increasingly implicated in the pathogenesis of diabetic
complications, Alzheimer’s disease, and atherosclerosis.2,5
Compounds that reduce AGE or ALE formation may prove
useful for limiting nonenzymatic protein modification as-
sociated with the pathology of these and other chronic age-
related diseases.
(1) Abbreviations: ABAP (or AAPH), 2, 2′-azobis(2-amidinopropane)
dihydrochloride; AGE, advanced glycation end products; ALE, advanced
lipoxidation end products; APC, allophycocyanin; BDE, bond dissociation
enthalpy; BSA, bovine serum albumin; CML, Nꢀ-carboxymethyllysine;
DTPA, diethylenetriaminepentaacetic acid; HNE, hydroxynonenal; IP,
ionization potential; MDA, malondialdehyde; PM, pyridoxamine; PN,
pyridoxine; SEC, size-exclusion chromatography; Tx, 6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid (Trolox).
(3) Requena, J. R.; Fu, M. X.; Ahmed, M. U.; Jenkins, A. J.; Lyons, T.
J.; Baynes, J. W.; Thorpe, S. R. Biochem. J. 1997, 322, 317-325.
(4) (a) Bierhaus, A.; Hofmann, M. A.; Siegler, R.; Nawroth, P. P.
CardioVasc. Res. 1998, 37, 586-600. (b) Schmidt, A. M.; Yan, S. D.;
Wautier, J.; Stern, D. Circ. Res. 1999, 84, 489-497.
(5) (a) Sing, R.; Barden, A.; Mori, T.; Beilin, L. Diabetologia 2001, 44,
129-146. (b) Picklo, M. J.; Montine, T. J.; Amarnath, V.; Neely, M. D.
Toxicol. Appl. Phramacol. 2002, 184, 187-197. (c) Uchida, K. Free Rad.
Med. Biol. 2000, 28, 1685-1696.
(2) Baynes, J. W.; Thorpe, S. R. Free Rad. Med. Biol. 2000, 28, 1708-
1716.
10.1021/ol0348147 CCC: $25.00 Published 2003 by the American Chemical Society
Published on Web 07/02/2003

Specific mechanisms for the late Maillard reaction conver-
sion of the initial Amadori adducts to the irreversible
formation of AGE involve complex sequential and parallel
reactions that are believed to produce a number of reactive
carbonyl compounds, especially R-dicarbonyls.5c AGE for-
mation in vitro is typically accelerated by oxidative condi-
tions (glycoxidation) catalyzed by metal ions such as Fe3+
or Cu2+, and it is generally accepted that metals may play a
role during AGE formation in vivo.6
Although a variety of AGE inhibitors have been identified,
their mechanisms of action are not well understood.7 The
most common feature among the AGE inhibitors known to
date is the presence of a nucleophilic functionality, such as
an amine or hydrazone, which can intercept reactive carbonyl
compounds and prevent progression to AGE or ALE
products. Other classes of inhibitors, such as metal ion
chelators (diethylenetriaminepentaacetic acid (DTPA)) and
radical trapping antioxidants (R-tocopherol and ascorbate)
exhibit their activity by limiting oxidative acceleration of
glycation.7a Typically, AGE inhibitors have primarily been
identified using glycation models in the presence of transition
metal ions. Thus, it is very difficult to discern in the literature
whether some of the most promising AGE inhibitors are
providing protection as a result of their carbonyl trapping
or metal ion chelating activities.8 Surprisingly, little work
has been done on the effects of radical trapping antioxidants
on AGE chemistry.
the involvement of both reactive carbonyls and radical
intermediates in the complex pathways of the Maillard
reaction and lipid peroxidation. Considering that the known
carbonyl trapping AGE and ALE inhibitor, PM, may be
found bound to important glycation or lipoxidation inter-
mediates, we suspected that the phenol functionality might
serve as a local protective agent against radical AGE and
ALE intermediates. However, PM has been reported to have
only a weak hydrogen atom donating (radical trapping)
ability, making it only a marginally effective antioxidant.9b,12
Electron-donating para substituents lower phenolic O-H
bond dissociation enthalpies (BDE), which enhances radical
trapping rates. Unfortunately, such substituents also lower
the ionization potential (IP) of the phenol and can do so to
such an extent that, for example, 4-dimethylaminophenol
(which should be an extremely good radical trap) is of no
practical value as an antioxidant because it reacts directly
with dioxygen. Pratt et al.13 have shown that strong electron-
donating groups (such as dimethylamino) para to the phenolic
hydroxyl group of 3-pyridinols and 5-pyrimidinols decrease
the O-H BDE and greatly increase radical trapping rates
but do not lower the IP to the point that there is a direct
reaction with oxygen. Therefore, PM was modified by the
addition of a 6-dimethylamino substituent (11). A pyridoxine
(PN) derivative containing a 6-dimethylamino substituent (6)
was also prepared to study the importance of the nucleophilic
amine group during glycation reactions.
The synthesis of 6-dimethylaminopyridoxine hydrochloride
(6) is given in Scheme 1.14 The 6-aminopyridoxine hydro-
Pyridoxamine (PM) has been identified as an AGE
inhibitor for late stage (post-Amadori) glycation and as an
inhibitor of ALE.9 For example, PM has been reported to
decrease the formation of fluorescent AGE products signifi-
cantly and, specifically, to decrease the yield of Nꢀ-car-
boxymethyllysine (CML).7a,10 PM is a less active metal ion
chelator than many other known AGE inhibitors, but there
is evidence showing that it can trap the dicarbonyls gly-
oxal and methyl glyoxal.11 The mechanism of ALE inhibi-
tion by PM has been proposed to be by the formation of
PM adducts with 9- and 13-oxo-decadienoic acid intermedi-
ates formed during the peroxidation of linoleic acid, which
leads to the formation of hexanoic acid amide and nonane-
dioic acid monoamide derivatives of PM, respectively,
with substantially reduced levels of MDA-Lys and HNE-
Lys.9b
Scheme 1
The present work describes the synthesis of a novel
multifunctional AGE and ALE inhibitor designed to address
(6) (a) Monnier, V. M. J. Clin. InVest. 2001, 107, 799-801. (b) Wolff,
S. P.; Jiang, Z. Y.; Hunt, J. V. Free Rad. Med. Biol. 1991, 10, 339-352.
(7) (a) Khalifah, R. G.; Baynes, J. W.; Hudson, B. G. Biochem. Biophys.
Res. Comm. 1999, 257, 251-258. (b) Miyata, T.; Van Ypersele De Strihou,
C.; Ueda, Y.; Ichimori, K.; Inagi, R.; Onogi, H.; Ishikawa, N.; Nangaku,
M.; Kurokawa, K. J. Am. Soc. Nephrol. 2002, 13, 2478-2487. (c)
Costantino, L.; Rastelli, G.; Vianello, P.; Cignarella, G.; Barlocco, D. Med.
Res. ReV. 1999, 1, 3-23.
chloride (3) was synthesized by the methods of Korytnyk et
al.15 Pyridoxine hydrochloride (1) was reacted with diazotized
(8) Price, D. L.; Rhett, P. M.; Thorpe, S. R.; Baynes, J. W. J. Biol. Chem.
2001, 276, 48967-48972.
(9) (a) Booth, A. A.; Khalifah, R. G.; Todd, P.; Hudson B. G. J. Biol.
Chem. 1997, 272, 5430-5437. (b) Onorato, J. M.; Jenkins, A. J.; Thorpe,
S. R.; Baynes, J. W. J. Biol. Chem. 2000, 275, 21177-21184.
(10) Fu, M.-X.; Requena, J. R.; Jenkins, A. J.; Lyons, T. J.; Baynes, J.
W.; Thorpe, S. R. J. Biol. Chem. 1996, 271, 9982-9986.
(11) Voziyan, P. A.; Metz, T. O.; Baynes, J. W.; Hudson, B. G. J. Biol.
Chem. 2002, 277, 3397-3403.
(12) DeLange, R. J.; Glazer, A. N. Anal. Biochem. 1989, 177, 300-
306.
(13) (a) Pratt, D. A.; DiLabio, G. A.; Brigati, G.; Pedulli, G. F.;
Valgimigli, L. J. Am. Chem. Soc. 2001, 123, 4625-4626. (b) Wijtmans,
M.; Pratt, D. A.; DiLabio, G. A.; Pedulli, G. F.; Porter, N. A. Angew. Chem.,
Int. Ed. 2003, in press.
(14) See Supporting Information for details.
2660
Org. Lett., Vol. 5, No. 15, 2003

aniline to produce 6-phenylazopyridoxine hydrochloride (2),
which was then hydrogenated to yield 3. Treatement of 3
with imidazole (2 equiv) and tert-butyldimethylsilyl chloride
(1 equiv) provided the TBDMS ether at the unexpected R4
position, 4 in a 52% yield. The amine 4 was then alkylated
by reductive amination with formaldehyde, sodium triace-
toxyborohydride, and sodium cyanoborohydride. After ex-
traction the 6-dimethylamino derivative 5 was purified by
flash chromatography (5 can also be crystallized from ethyl
acetate). The position of the TBDMS ether at the R4 position
was confirmed by the X-ray crystal structure of 5.16 The
purified compound 5 was carried forward to complete the
synthesis of 6-dimethylaminopyridoxamine (11, Scheme 2).
conditions generated the product cleanly and starting material
was easily recovered and resubjected to the same reaction
conditions. The aldehyde 9 was then converted to the oxime
10. There was some loss of the methoxymethyl ether
protecting groups during the formation of the oxime, but
removal of the remaining MOM protecting groups at this
stage was avoided because of the undesirable hydrolysis of
the oxime under acidic conditions. Instead, the oxime 10 and
other protected versions were hydrogenated to the amine in
the presence of a small amount of hydrochloric acid, which
catalyzed the hydrogenation, and then the methoxymethyl
ether protecting groups were removed. The 6-dimethylami-
nopyridoxamine (11, or dmaPM) was purified to yield an
off-white solid as a dihydrochloride salt.
The radical trapping activities of the dimethylamino
derivatives dmaPN (6) and dmaPM (11) were evaluated by
following the peroxyl radical induced quenching of allophy-
cocyanin (APC) fluorescence. APC is a naturally fluorescent
protein that rapidly loses its fluorescence when exposed to
a source of free radicals.18 Thermal decomposition of 2,2′-
azobis(2-amidinopropane) dihydrochloride (ABAP) in aer-
ated aqueous systems generates positively charged peroxyl
radicals at known and reproducible rates. Radical trapping
antioxidants competitively trap ABAP-derived peroxyl radi-
cals before their reaction with the protein. Results in Figure
1 demonstrate that the novel dimethylamino derivatives
Scheme 2
Compound 5 was deprotected with tetrabutylammonium
fluoride (TBAF) in THF to yield 6 as a recrystallized product.
The X-ray crystal structure of 6-dimethylaminopyridoxine
hydrochloride 6 was also obtained.16
Scheme 2 shows the synthetic route to 11 from the
TBDMS ether 5. Methoxymethyl ether protecting groups
were introduced at the 3-phenol and the R5-hydroxyl groups
to give 7. The TBDMS ether was then selectively deprotected
with TBAF in THF solution, and the resulting R4-hydroxyl
derivative 8 was oxidized by manganese(IV) oxide in
acetone. This standard procedure for oxidation of pyridoxine
to pyridoxal15,17 was unusually slow for the conversion of 8
to the pyridoxal derivative 9. However, these oxidation
(15) Korytnyk, W.; Srivastava, S. C. J. Med. Chem. 1973, 16, 638-
642.
(16) Crystal Data. The X-ray diffraction data were collected on a Bruker
SMART CCD diffractometer using graphite-monochromated Mo KR
radiation at 172 K. Compound 5: C16H30N2O3Si, monoclinic, space group
P21/c, a ) 15.6916(12), b ) 8.0862(6), c ) 16.9360(18) Å, â ) 118.437-
(10)°, Dc ) 1.148 g cm-3, µ ) 0.137 mm-1, Z ) 4, final residual R1(Rw)
) 0.0430 (0.1149) for 3755 reflections with I > 2σ(I), and 319 parameters.
Compound 6: C10H17N2O3Cl, monoclinic, space group P21/c, a ) 10.4039-
(10), b ) 12.9429(8), c ) 8.9220(6) Å, â ) 91.346(10)°, Dc ) 1.375 g
cm-3, µ ) 0.313 mm-1, Z ) 4, final residual R1(Rw) ) 0.0411 (0.1069)
for 2429 reflections with I > 2σ(I), and 213 parameters. See also Supporting
Information.
Figure 1. Radical trapping antioxidants (Tx, dmaPM, and dmaPN)
retard peroxyl radical induced quenching of allophycocyanin
fluorescence. Conditions were 64.4 nM APC, ABAP 12 mM, and
inhibitor 50 µM, as indicated, in 50 mM phosphate buffer pH 7.4
at 37 °C under air (n ) 2 with high reproducibility ( 1-2%).22
dmaPM (11) and dmaPN (6) are comparable in their ability
to retard APC fluorescence quenching to the well-known
water-soluble R-tocopherol analogue 6-hydroxy-2,5,7,8-
(17) Ueda, T.; Nakaya, K.; Nagai, S.; Sakakibara, J. J. Heterocycl. Chem.
1989, 26, 33-37.
Org. Lett., Vol. 5, No. 15, 2003
2661

tetramethylchroman-2-carboxylic acid (Trolox, or Tx). PN
provided only a slight prevention of fluorescence loss, while
PM was found to accelerate the loss of APC fluorescence.19
These radical trapping comparisons were made in buffer at
pH 7.4 where the dimethylamino substituent or the pyridine
(for PN and PM) may be protonated. Such protonation would
induce a strong electron-withdrawing effect and decrease the
hydrogen atom donating ability of the phenol. Protonation
of the aliphatic amine of dmaPM may also have some
influence on the hydrogen atom donating ability of the
phenol.20 In working with dmaPN (6) and dmaPM (11) as
hydrochloride salts, no significant decomposition was ob-
served until the pH was adjusted with buffer. However, after
a 7-day incubation at 37 °C under air in pH 7.4 phosphate
buffer, ∼60% dmaPM (11) and ∼20% dmaPN (6) had
decomposed.21
Table 1. Radical Trapping Antioxidants Prevent Intermolecular
Protein Cross-Links during Ribose Glycations14,23
experiment
(at 7 days)
[inhibitor]
(mM)
% protein
cross-linked
control (0 days)
control
Tx
Tx
Tx
dmaPM, 11
dmaPM, 11
dmaPM, 11
dmaPN, 6
PM
0
0
13.7 ( 0.5
40.3 ( 3.7
14.4 ( 1.4
15.9 ( 1.7
27.0 ( 0.1
14.8 ( 0.4
19.1 ( 1.2
30.5 ( 0.1
25.8 ( 0.6
18.4 ( 0.7
27.2 ( 0.1
32.8 ( 0.1
5.0
1.0
0.2
5.0
1.0
0.2
5.0
5.0
1.0
5.0
PM
PN
The novel carbonyl group trapping and radical trapping
AGE inhibitor dmaPM (11) was also tested for its ability to
act as a radical trapping antioxidant in a model glycation
system. Intermolecular protein cross-links formed during the
glycation of bovine serum albumin (BSA) with ribose in a
metal ion free buffer23 were determined by size-exclusion
chromatography (SEC) with refractive index detection.14 The
radical trapping antioxidants dmaPM (11) and Tx provided
the greatest inhibition of intermolecular protein cross-linking
(Table 1). The carbonyl trapping inhibitor PM was less
effective at preventing cross-links and PN was a poor cross-
link inhibitor. The new AGE and ALE inhibitor dmaPM (11)
was nearly as effective at preventing protein cross-linking
as Tx. The radical trapping dmaPN derivative (6) showed
less inhibition of cross-links than the carbonyl and radical
group trapping dmaPM. The results from Tx experiments
show that radical trapping provides better cross-link preven-
tion than carbonyl group trapping (PM). We suggest that
dmaPM (11) is comparable to Tx in cross-link prevention
largely because of its radical trapping ability.24 However,
its carbonyl group trapping ability may also play some role
in cross-link prevention.
(18) (a) Nakagawa, T.; Yokozawa, T.; Terasawa, K.; Shu, S.; Juneja, L.
R. J. Agric. Food Chem. 2002, 50, 2418-2422. (b) Courderot-Masuyer,
C.; Dalloz, F.; Maupoil, V.; Rochette, L. Fundam. Clin. Pharmacol. 1999,
13, 535-540.
(19) PM incubated with APC and the absence of ABAP showed no
quenching of APC fluorescence (i.e., result identical to No ABAP).
(20) We speculate that protonation of the R4-amino group of dmaPM,
11, may free the phenol from an intramolecular hydrogen bond and result
in improved radical trapping when compared to the hydrogen bond accepting
R4-hydroxyl group of dmaPN, 6.
(21) HPLC conditions: Discovery C18 column, solvents A (water) and
B (acetonitrile) both containing 0.1% heptafluorobutyric acid delivered at
1 mL/min as gradient; time (%A) 0 min (100); 30 (40). Quantification of
inhibitors was at 293 nm UV against a calibration curve.
(22) Fluorescence of allophycocyanin (Ex ) 633 nm, Em ) 660 nm) at
37 °C was measured in a 1 cm cell just before addition of ABAP, and at
5 min intervals, on a Photon Technology International C-700 fluorescence
lifetime system.
(23) Conditions were 10 mg/mL BSA and 50 mM ribose in 0.2 M
phosphate buffer pH 7.4 containing 1 mM DTPA and 1 mM phytic acid
(chelators added after buffer was stored over Chelex-100 resin for 24 h).
Supporting Information Available: Experimental pro-
cedures for the synthesis and characterization of the com-
pounds in Schemes 1 and 2, including data in CIF format .
This material is available free of charge via the Internet at
http://pubs.acs.org.
OL0348147
(24) (a) For more complete examination of dmaPM (11) and Tx in
glycation models, see: Culbertson, S. M.; Vassilenko, E.; Morrison, L.;
Ingold, K. U. manuscript submission to J. Biol. Chem. (b) For example,
results from ribose derived pentosidine yields also support radical trapping
activity for dmaPM, 11, and dmaPN, 6.
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Org. Lett., Vol. 5, No. 15, 2003