Synthesis and antioxidant activities of 3,5-dialkoxy-4-hydroxycinnamamides

Article abstract of DOI:10.1016/j.bmcl.2008.01.061

A series of 3,5-dialkoxy-4-hydroxycinnamamides 6 and 7 was synthesized, and their antioxidant activity was assessed using the thiobarbituric acid reactive substance (TBARS) assay. Interestingly, cinnamamides with longer alkoxy groups on the C-3 and C-5 positions display enhanced inhibition, and most of the compounds in the series tested exhibit excellent lipid peroxidation inhibitory activities. Some cinamamides bearing hexyloxy or 2,6-di-tert-butyl-4-methyl phenol groups have submicromolar inhibitory activities.

Full text of DOI:10.1016/j.bmcl.2008.01.061

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1663–1667
Synthesis and antioxidant activities
of 3,5-dialkoxy-4-hydroxycinnamamides
Tae-Souk Kang,a Hyang-Ok Jo,a Woo-Kyu Park,a Jong-Pyung Kim,b
Yasuo Konishi,c Jae-Yang Kong,a No-Sang Parka and Young-Sik Junga,*
aDrug Discovery Division, Korea Research Institute of Chemical Technology, PO Box 107, Yuseong, Daejeon 305-600, South Korea
bKRIBB, 52 Oeun-dong, Yuseong, Daejeon 305-806, South Korea
cNational Research Council Canada, Biotechnology Research Institute, 6100 Royalmount Ave., Montreal, Que., Canada H4P 2R2
Received 12 November 2007; revised 20 December 2007; accepted 15 January 2008
Available online 19 January 2008
Abstract—A series of 3,5-dialkoxy-4-hydroxycinnamamides 6 and 7 was synthesized, and their antioxidant activity was assessed
using the thiobarbituric acid reactive substance (TBARS) assay. Interestingly, cinnamamides with longer alkoxy groups on the
C-3 and C-5 positions display enhanced inhibition, and most of the compounds in the series tested exhibit excellent lipid peroxida-
tion inhibitory activities. Some cinamamides bearing hexyloxy or 2,6-di-tert-butyl-4-methyl phenol groups have submicromolar
inhibitory activities.
ꢀ 2008 Elsevier Ltd. All rights reserved.
Reactive oxygen species (ROS), including the hydroxyl
radical, superoxide anion, hydrogen peroxide, and per-
oxynitric species are continuously generated in the
process of respiration as natural byproducts of oxygen
metabolism.1 ROS play an important roles in bio-
chemical processes of the immune system, cell differen-
tiation, and internal signal transduction.2 These
species are sufficiently reactive to cause injury to cells
through the destruction of components such as pro-
teins, lipids, sugars and nucleotides.3 Under normal
circumstances, cells are able to defend against ROS
damage through the use of enzymes such as superox-
ide dismutases and catalases.4 Small molecule antioxi-
dants such as ascorbic acid (vitamin C), uric acid, and
glutathione also play important roles as cellular anti-
oxidants.5 Thus, under normal conditions ROS are
maintained at constant levels in body by the antioxi-
dant defense mechanism. However, imbalances be-
tween the formation and detoxification of ROS can
result in significant damage to cells, a situation known
as oxidative stress. Oxidative stress leads to the initia-
tion and/or progression of various diseases, including
for example cancer,6 ischemia-reperfusion injury,7 ath-
erosclerosis,8 cardiovascular diseases,9 inflammation,10
and neurodegenerative disorders11 such as Alzheimer’s
and Parkinson’s diseases. Because of this, there is a
growing interest in the discovery of natural and
unnatural antioxidants that attenuate oxidative stress
and, as result, can serve as protective agents against
these diseases.12
In a previous report,13 we described the synthesis and
antioxidant activities of a series of N-{3-(3,4-dimethyl-
phenyl)propyl}-4-hydroxyphenylacetamides,
substi-
tuted at C-3 positions with various alkoxy groups
(1–3) and several N-{3-(3,4-dimethylphenyl)propyl}-4-
hydroxycinnamamides (4, 5). The results of this effort
showed that, among the substances investigated, the 4-
hydroxycinnamamides
properties.
had
potent
antioxidant
The structures of 1 and 2 are closely related to that of
capsaicin, a vanilloid receptor agonist.14 Previous stud-
ies with these amides showed that they are centrally
acting agent with potent analgesic activities.15 These
findings led to current study, aimed at the development
of new antioxidant as neuroprotective agents, which fo-
cus on novel 3,5-dialkoxy-4-hydroxycinnamamides 6
and 7.
Keywords: Antioxidant; 4-Hydroxycinnamamide; Lipid peroxidation;
Reactive oxygen species.
*
Corresponding author. Tel.: +82 42 860 7135; fax: +82 42 861
1291; e-mail: ysjung@krict.re.kr
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.061

1664
T.-S. Kang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1663–1667
O
H
H
N
MeO
HO
RO
HO
N
MeO
HO
N
H
O
O
OMe
2
1
3
O
O
O
MeO
HO
MeO
HO
RO
HO
R'
N
H
N
H
N
H
OMe
OR
4
5
6,7
In order to prepare the new 3,5-dialkoxy-4-hydroxyc-
innamamides 6 and 7 as antioxidants, we needed 3,5-
dialkoxy-4-hydroxycinnamic acids 11 with various
alkoxy groups at the C-3 and C-5 ring positions. To
the best of our knowledge, the synthesis of 3,5-dialk-
oxy-4-hydroxycinnamic acids 11 had not been reported
until our recent report,16 even though sinapic acid 11a
is commercially available.17 The 3,5-dialkoxy-4-
hydroxycinnamic acids 11 were generally prepared start-
ing with methyl 4-benzyloxy-3,5-dihydroxybenzoate 818
in 38–70% overall yields (Scheme 1).
Coupling of the acids 11 with 3-(3,4-dimethyl-
phenyl)propyl amine 12 or 13 using (benzotriazole-1-
yloxy)tripyrrolidinophosphonium hexafluorophosphate
(PyBOP)19 and triethylamine in CH2Cl2 and DMF at
0 ꢁC provided the cinnamamides 6 or 7 in 50–95% yields
after flash column chromatography (Scheme 2). Initial
attempts to perform these coupling reactions by using
1,3-dicyclohexycarbodiimide (DCC) and 1-hydroxyben-
zotriazole (HOBt) gave the amides 6 in less than 50%
yields. Amine 13, which is required for the synthesis of
7, was obtained from the benzyl bromide 15 that is
O
HO
CO2Me
a) RBr, K2CO3
RO
HO
CO2Me
a) LiAlH4
RO
HO
CHO
Malonic acid
piperidine
RO
HO
OH
BnO
b) PCC
b) H2, Pd/C
OH
OR
OR
OR
10
11
8
9
Scheme 1. Synthesis of 3,5-dialkoxy-4-hydroxycinnamic acids (PCC: pyridinium chlorochromate).
O
RO
N
H
NH
2
PyBOP, Et N
3
+
11
11
DMF/CH Cl
HO
2
2
12
OR
80-95%
6a R = CH(CH )(CH CH CH )
3
2
2
3
6b R = CH(CH CH )
2
3 2
6c R = (CH ) CH
2 5
3
O
n
n
NH
RO
HO
tBu
OH
tBu
HO
PyBOP, Et N
3
N
H
2
+
DMF/CH Cl
2
2
OR
tBu
tBu
50-75%
13
7
Compound
n
Compound
n
R
R
7a
7b
7c
7d
7e
7f
CH
CH
1
2
1
2
1
2
7g
7h
7i
CH(CH )(CH CH CH )
1
3
3
3
2
2
3
CH(CH )(CH CH CH )
2
3
2
2
3
(CH ) CH
CH(CH CH )
3 2
1
2 2
3
3
3
3
2
(CH ) CH
7j
CH(CH CH )
3 2
2
2 2
2
(CH ) CH
7k
7l
(CH ) CH
1
2 2
2 5
3
3
(CH ) CH
(CH ) CH
2
2 2
2 5
Scheme 2. Synthesis of 3,5-dialkoxy-4-hydroxycinnamamides 6.

T.-S. Kang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1663–1667
1665
tBu
HO
tBu
HO
tBu
HO
a) NaN3, DMAP
DMF
OH
Br
NH2
PBr3
CH2Cl2
86%
b) H2/Pd-C
MeOH, 75%
tBu
14
tBu
tBu
15
13a
KCN, DMAP
DMF, 90%
tBu
HO
tBu
HO
NH2
CN
(CH3)2S-BH3
THF,56%
tBu
tBu
13b
16
Scheme 3. Synthesis of 3,5-di-tert-butyl-4-hydroxyphenyalkyl amines 13.
Table 1. Inhibition test of lipid peroxidation by TBARS assay
To evaluate the antioxidant properties of the newly syn-
thesized amides, we examined the lipid peroxidation in a
rat brain homogenate by using the thiobarbituric acid
reactive substances (TBARS) assay20 according to the
method of Stocks.21 We first examined antioxidant
property of the cinamicacids 11 with alkoxy chains of
various lengths (Table 1). Generally, longer alkoxy
groups enhanced inhibition. Also, the order of inhibi-
tion potency was 11e > 11b > 11a. The inhibitory poten-
cies of all the cinnamic acids 11 were comparable with
that of 2,6-di-tert-butyl-4-methylphenol (BHT) or
a-tocopherol, a likely result of the stabilization of the
phenyloxy radicals by the ortho substituted alkoxy
groups.
O
RO
OH
HO
11
OR
Compound
R
Inhibition of lipid
peroxidation (IC50, lM)
1
57.17
25.90
9.28
2aa
CH3
(CH2)3CH3
2ba
3a
11.77
9.94
3.41
4a
5a
11a
CH3
(CH2)3CH3
4.77
3.68
The preliminary results encouraged an examination of
the cinnamamides 6. We observed that all three amides
6 were more potent inhibitors than the corresponding
acids 11 (Table 2). It is clear that the 3,4-dimethylphe-
nylpropyl amine moiety is responsible for this gain in
inhibitory activity. Among the amides, 6c with n-hexyl-
oxy groups was the most potent compound toward inhi-
bition of lipid peroxidation, and 6c is more potent than
5, a substance containing with methoxy substituents.
Important information was gained from these results.
Specifically, we learned that inhibitory activities are
strongly influenced by longer, straight chain alkyoxy
groups rather than shorter, branched alkoxy groups.
11b
11c
CH(CH3)(CH2CH2CH3) 4.84
11d
11e
CH(CH2CH3)2
(CH2)5CH3
2.21
2.24
4.68
4.03
a-Tocopherol
BHT
a See Ref. 13a.
obtained from the corresponding alcohol 14. Reaction
of 15 with NaN3 followed by Pd/C reduction provided
benzyl amine 13a, and reaction of 15 with KCN fol-
lowed by reduction with (CH3)2S–BH3 gave phenylethyl
amine 13b, in moderate yields (Scheme 3).
Since the amides 6 were found more potent than the
acids 11, we next focused on new cinnamamides 7 that
are linked to with the well-known synthetic radical scav-
enger, BHT. As shown at Table 2, most of these amides
7 were very potent inhibitors. The higher activities of
7a–7j over 6 are probably a consequence of the presence
of BHT group in the amide. Generally the inhibition po-
tency of 7 was not strongly affected by the alkoxy
groups. The amide 7k and 7l which contain n-hexyoxy
substituents were also investigated, but lower potencies
were observed in for these substances as compared to
other amides. Among these amides, 7c and 7d with pro-
pyloxy groups showed the most potent inhibition
properties.
The design of 3,5-dialkoxy-4-hydroxycinnamamides 6
and 7 was based on the lipid peroxidation inhibitory
activity data shown in Table 1. The results showed that
(1) homovanillic amide 2a (25.90 lM) was a better inhi-
bition than 1 (57.17 lM), (2) 3,5-dialkoxy-4-hydroxy-
phenylacetamides 3 (11.77 lM) were more active than
3-alkoxy-4-hydroxyphenylacetamides 2 (25.90 lM for
2a), and (3) cinnamamides
4 (9.94 lM) and 5
(3.41 lM) have higher potencies than phenylacetamides
2a (25.90 lM) and 3 (11.77 lM). In addition, longer alk-
oxy substituents at the C-3 position of amides 2 were
shown to lead to enhanced inhibition, although the po-
tency was still lower than that of 2,6-di-tert-butyl-4-
methylphenol (BHT) or a-tocopherol.13 Based on these
observed structure-activity relationships, we designed
and prepared 3,5-dialkoxy-4-hydroxycinnamamides 6.
The results obtained for assessment of the antioxidant
activities of amides 6 and 7 by using ABTS•+ and DPPH

1666
T.-S. Kang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1663–1667
Table 2. Inhibition of lipid peroxidation by the newly synthesized amides
O
O
n
RO
HO
tBu
OH
RO
N
H
N
H
HO
OR
tBu
OR
6
7
Compound
R
n
Yield(%) from 11
Inhibition of lipid peroxidation (IC50, lM)
6a
6b
CH(CH3)(CH2CH2CH3)
CH(CH2CH3)2
(CH2)5CH3
80
84
95
68
74
56
58
50
52
58
75
50
52
72
64
1.33
1.73
0.29
1.46
0.69
0.27
0.26
0.42
0.41
0.57
0.36
0.82
0.44
3.52
2.74
4.68
4.03
6c
7a
CH3
CH3
1
2
1
2
1
2
1
2
1
2
1
2
7b
7c
(CH2)2CH3
(CH2)2CH3
7d
7e
(CH2)3CH3
(CH2)3CH3
7f
7g
CH(CH3)(CH2CH2CH3)
CH(CH3)(CH2CH2CH3)
CH(CH2CH3)2
CH(CH2CH3)2
(CH2)5CH3
7h
7i
7j
7k
7l
(CH2)5CH3
a-Tocopherol
BHT
Table 3. ABTS and DPPH radical scavenging activity of the amides
6, 7
yellow and is monitored spectrophotometrically. In
these tests, the amides 7a and 7b were found to be the
most active compounds.
Compound
ABTS•+ (IC50, lM)
DPPH (IC50, lM)
6a
6b
6c
7a
7b
7c
7d
7e
7f
26.3
49%a
28.5
11.3
15.6
24%a
37%a
57%a
—
47%a
64%a
44%a
—
68.4
84.4
77.7
80.3
69.0
73.0
114.1
176.8
—
In summary, we have synthesized a series of 3,5-dialk-
oxy-4-hydroxycinnamamides 6 and 7 bearing 3,4-dime-
thyphenylpropyl amine and BHT moieties. Studies
have shown that the amides exhibit excellent antioxidant
activities, especially in their inhibition of lipid
peroxidation.
7g
7h
7i
66.6
85.1
67.7
55%b
75%b
75.6
Acknowledgments
This work was supported by the Korea Foundation for
International Cooperation of Science & Technology
(KICOS) through a grant provided by the Korean Min-
istry of Science & Technology (No. K20711000016-
07A0100-01610).
7j
7k
7l
23.2
27.4
a % inhibition at 150 lM.
b % inhibition at 300 lM.
References and notes
assays22 are given in Table 3. The 2,20-azinobis (3-ethyl-
benzothiazoline-6-sulfonate) (ABTS) assay involves the
long-lived ABTS cation radical, which is chemically pro-
duced by oxidation of the corresponding colorless sul-
fonic acid with potassium persulfate. The green-blue
ABTS•+ has excellent spectral characteristics, is stable
over a wide range of pH and is applicable to the study
of both water-soluble and lipid-soluble antioxidants that
convert ABTS•+ back to the initial sulfonic acid.23 The
2,2-diphenyl-1-picrylhydrazyl (DPPH) assay measures
the ability of antioxidants to donate a hydrogen atom
in the conversion of the stable DPPH free radical to
1,1-diphenyl-2-picrylhydrazine.24 The reaction is accom-
panied by a change in color from deep-violet to light-
1. Boveries, A.; Cadenas, E. Cellular sources and steady-
state levels of reactive oxygen species. In Oxygen, Gene
Expression, and Cellular Function; Clerch, L. B., Massaro,
D. J., Eds.; Marcel Dekker: New York, 1977; pp 1–25.
2. Droge, W. Physiol. Rev. 2002, 82, 47.
3. (a) Halliwell, B.; Gutteridge, J. M. C. In Free Radical
in Biology and Medicine; Halliwell, B., Gutteridge, J.
M. C., Eds., 2nd ed.; Oxford University Press: Claren-
don: Oxford, 1989; pp 1–20; (b) Braughler, J. M.;
Duncan, L. A.; Chase, R. L. J. Biol. Chem. 1986, 261,
10282.
4. (a) Abid, M. R.; Schoots, I. G.; Spokes, K. C.; Wu, S. Q.;
Mawhinney, C.; Aird, W. C. J. Biol. Chem. 2004, 279,
44030; (b) Schiavone, J. R.; Hassan, H. M. J. Biol. Chem.

T.-S. Kang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1663–1667
1667
1988, 263, 4269; (c) Li, S.; Yan, T.; Yang, J. Q.; Oberley,
T. D.; Oberley, L. W. Cancer Res. 2000, 60, 3927.
5. (a) Marcinik, G.; Petty, M. A. Drugs Future 1996, 21,
1037; (b) Harrison, S. C.; Lynch, J. J.; Rose, K.; Choi, D.
W. Brain Res. 1994, 639, 102; (c) Saunders, R. D.; Dugan,
L. L.; Demediuk, P.; Means, E. D.; Horrocks, L. A.;
Anderson, D. K. J. Neurochem. 1987, 49, 24.
6. Lamson, D. W.; Brignall, M. S. Altern. Med. Rev. 1999, 4,
304.
7. (a) Flamm, E. S.; Demopoulos, H. B.; Seligman, M. L.;
Poser, R. G.; Ransohoff, J. Stroke 1978, 9, 445; (b) Green,
A. R.; Shuaib, A. Drug Discov. Today 2006, 11, 681.
8. Gey, K. F. Bibl. Nutr. Dieta 1986, 37, 53.
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 absorbance at 532 nm was
measured. Typical TBARS formation in brain homoge-
nate was calculated from the absorbance at 532 nm using
1,1,3,3-tetraethoxypropane as an external standard.
21. Stocks, J.; Gutteridge, J. M. C.; Sharp, R. J.; Dormandy,
T. L. Clin. Sci. Mol. Med. 1974, 47, 215.
22. Lertvorachon, J.; Kim, J.-P.; Soldatov, D. V.; Boyd, J.;
Roman, G.; Cho, S. J.; Popek, T.; Jung, Y.-S.; Lau, P. C.
K.; Konishi, Y. Bioorg. Med. Chem. 2005, 13, 4627.
23. DPPH radical scavenging activity: DPPH radical scaveng-
ing activity was determined by using the method of Brand-
Williams et al. [25] with minor modifications. The solution
of the sample (10 lL) in ethanol was added to 90 lL of a
0.15 mM DPPH radical in ethanol in a 96-well plate. The
sample solution refers to the tested compounds and the
reference antioxidants at various concentrations, as well as
ethanol as a control. The solutions of the tested com-
pounds had concentrations ranging from 3 to 1000 lg/ml,
whereas the concentrations of the solutions of the refer-
ence compounds varied from 0.1 to 1000 lg/ml. The
reaction leading to the scavenging of DPPH radical was
complete within 10 min at 25 ꢁC. The absorbance of the
mixture was then measured at 517 nm using a microplate
reader. The reduction of DPPH radical was expressed as
percentage:
9. Vivekananthan, D. P.; Penn, M. S.; Sapp, S. K.; Hsu, A.;
Topol, E. J. Lancet 2003, 361, 2017.
10. Ziakas, G. N.; Rekka, E. A.; Gavalas, A. M.; Eleftheriou, P.
T.; Kourounakis, P. N. Bioorg. Med. Chem. 2006, 14, 5616.
11. (a) Mattson, M. P. Neuromol. Med. 2003, 3, 65–94; (b)
Stavrovskaya, I. G.; Kristal, B. S. Free Radic. Biol. Med.
2005, 38, 687.
12. Fong, J.; Rhoney, D. Ann. Pharmacother. 2006, 40, 461.
13. (a) Jung, Y.-S.; Kang, T.-S.; Yoon, J.-H.; Joe, B.-Y.; Lim,
H.-J.; Seong, C.-M.; Park, W.-K.; Kong, J.-Y.; Cho, J.;
Park, N.-S. Bioorg. Med. Chem. Lett. 2002, 12, 2599; (b)
Jung, Y.-S.; Kang, T.-S.; Lee, J.-C.; Seong, C.-M.; Ham,
W.-H.; Park, N.-S. Synth. Commun. 2001, 31, 455.
14. LaHann, T. R.; Farmer, R. W. Proc. West. Pharmacol.
Soc. 1983, 26, 145.
15. Lim, H.-J.; Jung, Y.-S.; Ha, D.-C.; Seong, C.-M.; Lee,
J.-C.; Choi, J.; Choi, S. W.; Han, M.-S.; Lee, K.-S.;
Park, N.-S. Arch. Pharm. Res. 1996, 19, 246.
16. Jo, H.-O.; Jung, Y.-S. J. Appl. Biomed. 2007, 5, 13.
17. (a) Freudenberg, K.; Dillenburg, R. Chem. Ber. 1951, 84,
67; (b) Yu, M. J.; McCowan, J. R.; Phebus, L. A.; Towner,
R. D.; Ho, P. P. K.; Keith, P. T.; Luttman, C. A.;
Saunders, R. D.; Ruterbories, K. J.; Lindstrom, T. D.;
Wikel, J. H.; Morgan, E.; Hahn, R. J. Med. Chem. 1993,
36, 1262.
Scavenged DPPHð%Þ ¼ ð1 ꢀ Atest=AcontrolÞ ꢁ 100;
where Atest is the absorbance of a sample at a given con-
centration after 10 min. reaction time, and Acontrol is the
absorbance recorded for 10 lL ethanol. The EC50 value
is defined as the concentration of sample that causes 50%
loss of the DPPH radical.
18. Freudenberg, K.; Dillenburg, R. Chem. Ber. 1951, 84, 67.
19. Seebach, D. Helv. Chim. Acta 1994, 77, 1313.
20. TBARS: Brain tissue from male Sprague–Dawley rats
(about 10-week old) was obtained after decapitation and
homogenated in ice-cold 10 mM Tris–HCl buffer (pH. 7.4)
using a Teflon-glass homogenizer. The homogenate was
centrifuged for 10 min at 1000g, and the supernatant was
used in the test. Lipid peroxidation was stimulated in
assays containing 250 lL rat brain homogenate by addi-
tions of 0.02 mM FeCl2 and 0.25 mM ascorbic acid, and
the mixture was incubated for 30 min at 37 ꢁC. The
24. ABTS cation radical scavenging activity: The ABTS cation
radical was produced by the reaction between 7.0 mM
ABTS/water and 2.45 mM potassium persulfate for 12 h in
the dark at room temperature. The ABTS solution was
diluted with PBS until A734 = 0.7. The reaction was
initiated by adding 190 lL of ABTS to 10 lL sample
solution at 25 ꢁC. The percentage of reduction of A734 was
recorded and was plotted as a function of the sample’s
concentration.
25. Brand-Williams, W.; Cuvelier, M. E.; Berset, C.
Lebensm.-Wiss. u.-Technol. 1995, 28, 25.