2,3-Dimethoxy-5-methyl-1,4-benzoquinones and 2-methyl-1,4-naphthoquinones: Glycation inhibitors with lipid peroxidation activity

Article abstract of DOI:10.1016/j.bmcl.2004.12.029

Anti-glycation activity of our anti-oxidant quinone library was measured and several 2,3-dimethoxy-5-methyl-1,4-benzoquinones and 2-methyl-1,4- naphthoquinones were identified as novel inhibitors of glycation, of which 2,3-dimethoxy-5-methyl-1,4-benzoquinones 13b is the most potent glycation inhibitor with around 50 μM of the IC₅₀ value.

Full text of DOI:10.1016/j.bmcl.2004.12.029

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1125–1129
2,3-Dimethoxy-5-methyl-1,4-benzoquinones and
2-methyl-1,4-naphthoquinones: glycation inhibitors with
lipid peroxidation activity
Young-Sik Jung,a,* Bo-Young Joe,a Sung Ju Chob and Yasuo Konishib
aKorea Research Institute of Chemical Technology, PO Box 107, Yusong, Taejon 305-606, Republic of Korea
bNational Research Council Canada, Biotechnology Research Institute, 6100 Royalmount Ave., Montreal, QC, Canada H4P 2R2
Received 7 October 2004;accepted 6 December 2004
Abstract—Anti-glycation activity of our anti-oxidant quinone library was measured and several 2,3-dimethoxy-5-methyl-1,4-benzo-
quinones and 2-methyl-1,4-naphthoquinones were identified as novel inhibitors of glycation, of which 2,3-dimethoxy-5-methyl-1,4-
benzoquinones 13b is the most potent glycation inhibitor with around 50 lM of the IC50 value.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
related complications.6 Several reports indicate the pro-
duction of free radicals and highly reactive oxidants
from glycated proteins under physiological conditions.7
Free radicals are known to stimulate AGE production
by autoxidation of sugars.8 Oxidative stress has been
linked to diabetic complications and diabetic atherogen-
esis.9 a-Tocoperol is a potent anti-oxidant and is an
anti-glycating substance in vitro.10 More recently the
combination of vitamins C and E more efficiently inhib-
ited glycation and AGE than the single vitamins, and
this is an evidence for the inhibition of glycation by
anti-oxidants.11
Nonenzymatic protein glycation initiated by the interac-
tion between amino groups of protein and reducing sug-
ars, such as glucose leads to chemical modification of
proteins. The resulting reaction products;advanced gly-
cation end products (AGEs) have been implicated in
health complications associated with the normal aging
process and the development of diabetes related compli-
cations.1 It was already proposed that the design and
discovery of inhibitors of the glycation cascade should
offer a promising therapeutic approach for the preven-
tion of diabetic or other pathogenic complications.2
For examples, administration of amino compounds,
such as alanine, can react with glucose and counteract
the potentially deleterious consequences of hyperglyce-
mia in diabetes.3 Hydrazine reagent is introduced to trap
the reactive carbonyls formed during the Maillard reac-
tion, especially Amadori intermediates, thus impeding
their conversion to AGEs.4 As a similar approach,
aminoguanidine is well known as an inhibitor of
AGEs.4,5
In the continuing our efforts to search the possibility
of anti-oxidants as therapeutic agents, we are interested
in the development of anti-oxidants that have potent
anti-glycation activity. In the screening of our anti-oxi-
dant compound library to anti-glycation assay system,
several 2,3-dimethoxy-5-methyl-1,4-benzoquinones and
2-methyl-1,4-naphthoquinones provided inhibitory
activity of glycation even though some other phenol
anti-oxidant compounds showed low anti-glycation
activity.
It is also known that glycation generates reactive oxygen
species, free radicals, reactive a-dicarbonyl species, and
protein cross-links, causing age-, diabetes-, and smoking-
The present paper deals with the synthesis and the bio-
logical evaluation of a series of 2,3-dimethoxy-5-
methyl-1,4-benzoquinones substituted at the C-6 posi-
tion with various groups and 2-methyl-1,4-naphthoqui-
nones substituted at the C-3 position with various
halogen groups.
*
Corresponding author. Tel.: +82 42 860 7135;fax: +82 42 861
1291;e-mail: ysjung@krict.re.kr
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.12.029

1126
Y.-S. Jung et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1125–1129
2. Chemistry
ever acylations with halogen substituted benzoyl chlo-
rides were not succeeded. We have overcome this
problem by three consequent steps;formylation, addi-
tion of Grignard reagents, and oxidations to give 6. Biaryl
compound 9 was prepared by bromination of 2
followed by Suzuki coupling with phenyl boronic acid.
Chloromethlyation of 2 followed by treatment with aryl
lithium reagent provided 12a, and the debenzylation un-
der reduction condition and alkylations with alkyl ha-
lides gave 12b and 12c. Amide functional groups were
formed from substitution of ester with amines to give
15. Finally the conversion of the fully substituted tolu-
For the preparation of tetramethoxytoluene 2 as our ini-
tial starting material, the reduction of benzoquinone 1
was carried out under the condition of either the cata-
lytic hydrogenation under high pressure (30–45 psi) or
the reduction with Na2S2O4, and then methylation was
conducted with dimethylsulfide (Scheme 1). Various
substituents were introduced to give fully substituted
toluenes such as the substitution with acyl-, phenyl-,
aralkyl-, or carbamide-group. For examples, acylation
with benzoyl chloride provided benzophenone 3, how-
OCH3
O
O
O
O
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
a, b
c
d
CH3
OCH3
CH3
OCH3
CH3
CH3
O
O
1
2
3
4
OCH3
CHO
O
O
O
O
H3CO
H3CO
H3CO
H3CO
f, g
e
d
H3CO
H3CO
2
R
R
CH3
OCH3
CH3
OCH3
CH3
7a
R = 4-F
5
6
7b R = 2-Cl
OCH3
OCH3
O
H3CO
H3CO
Br
H3CO
H3CO
H3CO
H3CO
d
h
i
2
2
CH3
OCH3
CH3
OCH3
CH3
O
8
9
10
OCH3
OCH3
O
H3CO
H3CO
H3CO
H3CO
H3CO
j
k
d
Cl
CH3
H3CO
CH3
OCH3
OR
CH3
OR
OCH3
O
11
13a R = CH2Ph
13b R = CH2CH3
12a
12b
12c
R = CH2Ph
R = CH2CH3
R = CH2COC2H5
l
m
13c
R = CH2COC2H5
OCH3
OCH3
O
O
H3CO
H3CO
CO2CH3
H3CO
H3CO
CONHR
H3CO
H3CO
o
n
d
NHR
CH3
2
CH3
CH3
OCH3
OCH3
15
O
16a R = CH2Ph
16b R = (CH2)2Ph
14
16c
R = (CH2)3Ph
Scheme 1. Reagents: (a) H2, Pd/C, MeOH;(b) (CH O)2SO2, NaOH/MeOH;(c) ArCOCl, AlCl , CH2Cl2;(d) CAN/H O–MeCN;(e) Cl CHOCH3,
3
3
2
2
TiCl4, CH2Cl2;(f) ArMgBr, THF;(g) PCC, CH 2Cl2;(h) NBS/CH 2Cl2;(i) PhB(OH) 2, Pd(PPh3)4, Na2CO3, C6H6;(j) HCHO, HCl;(k)
LiC6H4OCH2Ph, THF;(l) H 2, Pd/C then BrCH2CH3, K2CO3;(m) H 2, Pd/C then BrCH2COCH3, K2CO3;(n) n-BuLi/THF then ClCO2CH3;(o)
RNH2, DCC, HOBT, CH3CN.

Y.-S. Jung et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1125–1129
OCH3
1127
O
O
O
O
OCH3
X
X
a, b
c or d
e
CH3
CH3
CH3
OCH3
CH3
OCH3
17
18
19a
19b
20a X = Cl
20b X = Br
X = Cl
X = Br
O
I
f
17
CH3
O
21
Scheme 2. Reagents: (a) H2, Pd/C, MeOH;(b) (CH 3O)2SO2, NaOH/MeOH;(c) C 6H5I(OAc)2, TBSCl, CH2Cl2 for 19a;(d) NBS/CH 2Cl2 for 19b;(e)
CAN/H2O–MeCN;(f) TMSN 3, I2/pyridine.
enes (3, 6, 9, 12, 15) was conducted with ceric ammo-
nium nitrate (CAN) oxidation to give the derivatives
of 2,3-dimethoxy-5-methyl-1,4-benzoquinone (4, 7, 10,
13, 16).
Table 1. Inhibition of lipid peroxidation by 2,3-dimethoxy-5-methyl-
1,4-benzoquinones
O
H3CO
H3CO
X
Y
The naphthoquinones (20, 21) are synthesized via
known methods from 2-methyl-1,4-naphthoquinone 17
as shown in Scheme 2. Our initial material 18 which pre-
pared from 2-methyl-1,4-naphthoquinone 1 7 was halo-
genated to give chlorine- and bromine-containing
compounds 19a and 19b. Compound 19a was obtained
in high yield from 18 with phenyliododiacetate and trim-
ethylsilyl chloride in CH2Cl2. Bromination of 18 with
NBS in CH2Cl2 at rt for 0.5 h afforded 19b in 95% yield.
Following ceric ammonium nitrate (CAN) oxidation of
19 should proceed to naphthoquinone 20. Iodine substi-
tuted 2-methyl-1,4-naphthoquinone 21 was prepared
from treatment of 17 with trimethylsilyl azide and a mix-
ture of iodine and pyridine in CH2Cl2 followed by CAN
oxidation.12
CH3
O
Compound
X
Y
Inhibition of
lipid peroxidation
(IC50, lM)
4
7a
C@O
C@O
C@O
—
H
4-F
11.84
8.36
7b
10
2-F
H
15.30
39.43
64.17
57.70
13a
13b
CH2
CH2
4-OBn
4-OEt
13c
16a
CH2
CONHCH2
4-OCH2COEt 102.00
H
H
H
31.91
29.21
16.25
4.68
16b
16c
CONH(CH2)2
CONH(CH2)3
a-Tocopherol
NA: no activity was observed in our test concentrations.
3. Results and discussion
The evaluation of anti-oxidant property of 2,3-dime-
thoxy-5-methyl-1,4-benzoquinones and 2-methyl-1,4-
naphthoquinones was examined by the effects on lipid
peroxidation in rat brain homogenate by thiobarbituric
acid reactive substances (TBARS) assay13 according to
the method of Stocks et al.,14 as modified by Barrier
et al.15 The IC50 values for 2,3-dimethoxy-5-methyl-
1,4-benzoquinones are shown in Table 1. The substituents
of benzoquinones at the C-6 position are benzoyl (4,
7), phenyl (10), benzyl (13), and N-aralkylamide (16),
and the inhibitory activity was strongly influenced by
the substituents. The inhibitory activity of lipid peroxi-
dation was highest when the substituent is benzoyl (4,
7) in this series of compounds, and the order is ben-
zoyl > N-benzyl amido > phenyl > benzyl.16 Therefore
more electron withdrawing substituents increase the
inhibition activity of lipid peroxidation. The mechanism
of anti-oxidant activity of the quinone has been pro-
posed to be by formation of reduced phenols, which
can scavenge free radicals.17 For 2-methyl-1,4-naphtho-
quinone compounds, the substituents at the C-3 position
enhanced the inhibition of lipid peroxidation compared
with the unsubstituted quinone 17 (Table 2). The po-
tency is 10 times higher than that of the unsubstituted
quinone 17 regardless of the kind of halogen atom.
The naphthoquinones (20, 21) are also more potent than
a-tocopherol.
We then examined their anti-glycation activity. As the
glycation reactions progressively develop Maillard fluo-
rescence (kex = 370 nm and kem = 440 nm) that is attrib-
uted to the AGE formation of heterocyclic aromatic
structures, the anti-glycation activity was measured as
the inhibition of the Maillard fluorescence formation.
The anti-glycation activity for selected compounds are
shown in Table 3.18

1128
Y.-S. Jung et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1125–1129
Table 2. Inhibition of lipid peroxidation by 2-methyl-1,4-naphthoqui-
nones
and 2-methyl-1,4-naphthoquinones as novel inhibitors
of glycation.
O
X
Acknowledgements
CH
3
This work was supported by grants from Korea Science
& Engineering Foundation (R01-2001-00160), and from
the Ministry of Science and Technology in Korea. Taira
Kiyota and Dr. Woo-Kyu Park are acknowledged for
their technical assistance on glycation assay and anti-
oxidant assay.
O
Compound
X
Inhibition of lipid
peroxidation (IC50, lM)
17
20a
H
Cl
Br
I
12.24
1.41
1.10
1.23
4.68
20b
21
a-Tocopherol
References and notes
1. (a) Monnier, V. M.;Cerami, A. Science 1981, 211, 491;(b)
Bunn, H. F. Am. J. Med. 1981, 70, 325;(c) Brownlee, M.;
Vlassara, H.;Cerami, A. Ann. Int. Med. 1984, 101, 527.
2. (a) Brownlee, M. Diabetes 1994, 43, 836–841;(b) Vlassara,
H.;Bucala, R.;Striker, L. Lab. Invest. 1994, 70, 138–151.
3. Monnier, V. M. Prog. Clin. Biol. Res. 1989, 304, 1–22.
4. Brownlee, M.;Vlassara, H.;Kooney, A.;Ulrich, P.;
Cerami, A. Science 1986, 232, 1629–1632.
5. (a) Ruderman, N. B.;Williamson, J. R.;Brownlee, M.
FASEB J. 1992, 6, 2905–2914;(b) Soulis-Liparota, T.;
Cooper, M. E.;Dunlop, M.;Jerums, G. Dibetologia 1995,
38, 387–394;(c) Li, Y. M.;Steffes, M.;Donnelly, T.;Liu,
Table 3. Anti-glycation activities of 2,3-dimethoxy-5-methyl-1,4-ben-
zoquinones and 2-methyl-1,4-naphthoquinones
Compound
Inhibition of
Inhibition of BSA
glycation (IC50, lM) glycation (IC50, lM)
4
10
120
85
269
218
120
74
13a
13b
83
36
20a
20b
115
98
199
144
125
NA
NA
C.;Fuh, H.;Basgen, J.;Bucala, R.;Vlassara, H.
Natl. Acad. Sci. U.S.A. 1996, 93, 3902–3907.
Proc.
21
a-Tocopherol
Trolox methyl ester 468
93
NA
6. Elgawish, A.;Glomb, M.;Friedlander, M.;Monnier,
V. M. J. Bio. Chem. 1996, 271, 12964–12971.
7. (a) Gillery, P.;Monboisse, J. C.;Maquart, F. X.;Borel, J.
P. Diabetes Metab. (Paris) 1988, 14, 25–30;(b) Sakurai,
NA: no activity was observed in our test concentrations (3.80 nM to
1.00 mM).
T.;Sugioka, K.;Nakano, M.
1990, 1043, 27–33.
Biochem. Biophys. Acta
8. Wolff, S. P.;Jiang, X. Y.;Hunt, J. V. Free Rad. Biol. Med.
1991, 10, 339–352.
9. (a) Baynes, J. W. Diabetes 1991, 40, 405–412;(b) Mullar-
key, C. J.;Edelstein, D.;Brownlee, M. Biochem. Biophys.
Res. Commun. 1990, 173, 932–939.
10. Ceriello, D.;Giugliano, D.;Quatraro, A.;Dello Russo, P.;
Torella, R. Diabetes Metab. (Paris) 1988, 14, 40–
42.
11. Vinson, J.;Howard, T. B., III, Nut. Biochem. 1996, 7, 659–
663.
2,3-Dimethoxy-5-methyl-1,4-benzoquinones and 2-
methyl-1,4-naphthoquinones decrease the formation of
fluorescent AGEs significantly at micromolar concentra-
tion ranges. Phenyl group substituted benzoquinone 10
and benzyl group substituted benzoquinones (13a, 13b)
show strong activity. Therefore electron donating sub-
stituents may contribute to the anti-glycation activity.
For the naphthoquinone compounds, all halogen atom
substituted compounds at C-3 position (20a, 20b, 21)
show anti-glycation activity. It is interesting that 13b,
which is rather weak anti-oxidant in this series, is the
most potent anti-glycation agent in this series, whereas
the reference anti-oxidants a-tocopherol and trolox
methyl ester showed no and weak anti-glycation activ-
ity, respectively. Glycation consists of complex network
reactions of which many are not well understood. Also,
fluorescent AGEs are formed through multiple reaction
pathways such that blocking multiple reaction steps may
be more efficient rather than blocking a particular reac-
tion. The mechanism of anti-glycation activity of the
quinone is likely related to that of anti-oxidant activity;
however, other mechanisms should also be considered as
the correlation between the anti-oxidant potency and
the anti-glycation potency is rather poor.
12. Jung, Y. S.;Joe, B. Y.;Seong, C. M.;Park, N. S.
Korean Chem. Soc. 2000, 21, 462–463.
Bull.
13. Lipid peroxidation assays: To assess lipid peroxidation,
thiobarbituric acid reactive substances (TBARS) assay
was adopted in the present study. TBARS were assayed
according to the method of Stocks et al. (1974), as
modified by Barrier et al. (1998). In brief, a mixture
containing 250 lL brain homogenate (5 mg protein/mL),
10 lL test compound and 20 lL assay buffer was pre-
incubated at 37 °C for 20 min with shaking. Lipid perox-
idation was stimulated by additions of 0.02 mM FeCl2 and
0.25 mM ascorbic acid, and the mixture was incubated for
30 min at 37 °C. The reaction was stopped by the addition
of 0.05 mL of 35% perchloric acid. After centrifugation for
10 min at 1000g, 200 lL of the resulting supernatant was
added to 100 lL of an aqueous solution containing 0.5%
TBA and reacted at 80 °C for 1 h. Then, the reaction
mixture was cooled to room temperature, and its absor-
bance at 532 nm was measured. Typical TBARS forma-
tion in brain homogenate was calculated from the
absorbance at 532 nm using 1,1,3,3-tetraethoxypropane
In summary, from the anti-glycation screening of our
anti-oxidant compound library, we found several anti-
oxidants
2,3-dimethoxy-5-methyl-1,4-benzoquinones

Y.-S. Jung et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1125–1129
1129
as an external standard. The IC50 value is determined as
the concentration of malondialdehyde (MDA) to give 50%
inhibition.
shaking, in the dark. The final concentrations of BSA
and D-ribose were 0.075 mM and 50 mM, respectively.
The range of hydroxyquinone concentrations were
3.80 nM to 1.00 mM. After incubation, the Maillard
fluorescence (kex = 370 nm and kem = 440 nm) of each well
was measured to determine the IC50 values of inhibition of
glycation in Table 3 as described below. Then, aliquots of
the sample solutions were applied to a size exclusion
chromatographic column (Superrose 12PC 3.2/30,
100 mM phosphate buffer pH 7.4) to separate the pro-
teins and the small molecules and to measure the
Maillard fluorescence (kex = 370 nm and kem = 440 nm)
of the glycated BSA. The inhibitory activity of the
hydroxyquinone analogs was calculated using the
equation:
Inhibition percentage ð%Þ ¼ ½ðA À BÞ=AŠ  100
A: nmol of MDA/mg protein (control);B: nmol of MDA/
mg protein (test compound).
14. Stocks, J.;Gutteridge, J. M. C.;Sharp, R. J.;Dormandy,
T. L. Clin. Sci. Mol. Med. 1974, 47, 215–222.
15. Barrier, L.;Page, G.;Fauconneau, B.;Juin, F.;Tallineau,
C. Free Rad. Res. 1998, 28, 411–422.
16. This order can be explained that the quinones substituted
with more electron negative group (C@O) have higher
reduction potential (E1/2(À)) and they can be transferred
to the hydroquinones more easily. The hydroquinones
might be actual molecules, which inhibit the lipid perox-
idation. Thermodynamic feasibility of electron transfer
can be predicted by consideration of the redox potentials
(E1/2(+), E1/2(À)) of the donor and acceptor components
as shown in Eq. 1.
Inhibition ð%Þ ¼ f1 À ðF À F 0Þ=ðF 100 À F 0Þg  100
where F0 is the fluorescence of incubated BSA alone, F100
is the fluorescence of incubated BSA and D-ribose, F is the
DGSET ¼ E1=2ðþÞ À E1=2ðÀÞ
ð1Þ
17. Beyer, R. E.;Segura-Aguilar, J.;di Bernardo, S.;Cava-
zzoni, M.;Fato, R.;Fiorentini, D.;Galli, M. C.;Setti, M.;
Landi, L.;Lenaz, G. Mol. Aspects Med. 1997, 18, s15–s23.
18. Glycation assay: The stock solutions of bovine serum
albumin (BSA, 67 kDa) and D-ribose were prepared
separately into PBS (Phosphate-buffered saline). All
hydroxyquinone compounds were initially dissolved in
DMSO and diluted by PBS to prepare the appropriate
concentrations. Then, stock solutions of BSA, D-ribose
and hydroxyquinone compounds were mixed in a 96-well
plate and incubated for 5 days at 37 °C under mild
fluorescence of incubated BSA, D-ribose and hydroxy-
quinone analog. The inhibition of glycation and inhibition
of BSA glycation were determined by using the fluores-
cence of the reaction solution and of the glycated
BSA after the size exclusion chromatography, respec-
tively. The inhibition (%) is plotted against logarithm of
the concentration of hydroxyquinone analog. Sigmoidal
curve fitting is applied to the data by using software
ÔOrigin 6.0Õ, and the IC50 value is determined as the con-
centration of hydroxyquinone analog to give 50%
inhibition.