Antioxidant properties of Mannich bases

Article abstract of DOI:10.1016/j.bmcl.2012.08.080

The biological importance of antioxidants influenced to synthesize some curcumin-related compounds as potential antioxidants. Accordingly, a series of 2,4-diaryl-3-azabicyco[3.3.1]nonan-9-ones were synthesized with polyphenolic and/or polymethoxyphenyl groups by modified Mannich condensations. The yield was significantly improved using BF₃·SiO₂ as heterogeneous catalyst under mild conditions. Stereochemistry of all the synthesized compounds was established as twin-chair with an equatorial disposition of the aryl groups, through their NMR and XRD interpretations. The ABNs 8 (curcumin analog) and 10 (bis-demethoxycurcumin analog) showed an effective profile over curcumin, α-tocopherol, and vitamin C by chemical methods. Further, the efficiency of one of the active molecules, ABN 10, was demonstrated by its intracellular ROS inhibition activity on RAW 264.7 macrophage cells by FACS analysis in dose-dependent manner.

Full text of DOI:10.1016/j.bmcl.2012.08.080

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6362–6367
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antioxidant properties of Mannich bases
Dong Ho Park a, Jayachandran Venkatesan b, Se-Kwon Kim b, Venkatachalm Ramkumar c,
Paramasivam Parthiban a,
⇑
a Department of Biomedicinal Chemistry, Inje University, Gimhae 621-749, Gyeongnam, South Korea
b Department of Chemistry and Marine Bioprocess Research Center, Pukyong National University, Nam-Gu, Busan 608-737, South Korea
c Department of Chemistry, IIT-Madras, Chennai 600036, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 January 2012
Revised 25 July 2012
Accepted 21 August 2012
Available online 29 August 2012
The biological importance of antioxidants influenced to synthesize some curcumin-related compounds as
potential antioxidants. Accordingly, a series of 2,4-diaryl-3-azabicyco[3.3.1]nonan-9-ones were synthe-
sized with polyphenolic and/or polymethoxyphenyl groups by modified Mannich condensations. The
yield was significantly improved using BF3ꢀSiO2 as heterogeneous catalyst under mild conditions. Stereo-
chemistry of all the synthesized compounds was established as twin-chair with an equatorial disposition
of the aryl groups, through their NMR and XRD interpretations. The ABNs 8 (curcumin analog) and 10
Keywords:
(bis-demethoxycurcumin analog) showed an effective profile over curcumin, a-tocopherol, and vitamin
Mannich base
BF3ꢀSiO2
C by chemical methods. Further, the efficiency of one of the active molecules, ABN 10, was demonstrated
by its intracellular ROS inhibition activity on RAW 264.7 macrophage cells by FACS analysis in dose-
dependent manner.
Crystal structure
DPPH assay
Ó 2012 Elsevier Ltd. All rights reserved.
Total antioxidant assay
Reducing power assay
FACS assay
An essential part of search for the new leads in drug design pro-
gram is the synthesis of novel molecules that still resemble known
biologically active molecules, by the presence of some influential
structural features.1 Accordingly, a series of 2,4-diaryl-3-azabi-
cyco[3.3.1]nonan-9-ones (ABNs) were synthesized as heterocyclic
analogs of curcumin2 with polymethoxy/methoxy-phenolic/poly-
phenolic groups, to obtain efficient antioxidant molecules. ABN nu-
cleus is present in the molecular structure of various alkaloids, and
it has been frequently reported for interesting chemical reactions
and important biological actions.3
As reported by Prostakov and Lijinsky, nature and position of
the substituents are important factors for exciting biological ac-
tions of the molecules.4 Correspondingly, the reports on flavo-
noids with three hydroxyl substitutions indicated the increased
antioxidant activity, which in turn depending on the position
of the substitutions.5 The phenolic/polyphenolic as well as meth-
oxy/alkoxy phenyl and amino groups are considered important
key units to expose the antioxidant property.6 In this respect,
we reported a series of ABN derivatives with a variety of alkoxy
substituents such as OCH3, SCH3, OCH2CH3, O(CH2)2CH3,
O(CH2)3CH3, OCH(CH3)2, OCH2CH@CH2, OCOCH3 and OCH2Ph at
various positions of the phenyl group.3g Hence, we now focused
on the synthesis of novel ABNs with polymethoxyphenyl, meth-
oxy/ethoxy/bromo substituted phenols and polyhydric phenolic
groups. Polyphenols are chain-breaking antioxidants that func-
tion by scavenging radicals7 and thus, they prevent the damage
of lipid membranes, proteins and DNA. The chain-breaking path-
way is one of the two important mechanisms of antioxidant
process.8
The ABNs were conveniently synthesized in better yield by the
addition of Lewis acid catalyst (Scheme 1) through the Mannich
condensation.9 Moreover, previously optimized conditions for the
synthesis of ABNs without catalyst3e–g is adopted and found to
be the best over elevated temperatures.
The 1H and 13C chemical shifts of ABNs 1 and 2 are shown in Fig-
ure 1 with stereochemistry. Figure 1c provides better understand-
ing of ortho substitution effect. The ABNs 1 and 2 are positional
isomers; in ABN 1, the OCH3 groups on ortho position interacted
with H-2a/4a and H-1/5 by their vicinity. The vicinity is a conse-
quence of circumventing the dipole–dipole interaction between
the C–O and C–N bonds. Thus, the C–O bond of OCH3 prefers to
be syn to H-4a as shown in Figure 1c. Hence, the non-bonded
interactions established between the C–O bond of ortho-OCH3
and H-2a/4a as well as C–O and H-1/5. Of them, H-2a (2.462 Å)
and H-4a (2.550 Å) are closer than bridge-head protons H-1/5
(2.635/2.552 Å) that resulted more deshielding on the former.
Because of these non-bonded interactions in ABN 1, the nitrogen
lone pair also deshielded the ortho-protons. This is almost equal
(0.37 ppm) to the 0.37 ppm deshielding on H-2a/4a by the non-
bonded interactions. The NMR data10 of ABNs 3–10 were assigned
similar to that of ABN 2.
⇑
Corresponding author.
E-mail address: parthisivam@yahoo.co.in (P. Parthiban).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.080

D. H. Park et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6362–6367
6363
1.93, dd
J=13.75,
4.75 Hz
2.86
nine-line
1.32
quint
O
m
1.74-1.66
Ha He
m
He
He
1.74-1.66
m
H
Ha
Ha
2.65, br s
2.462 Å
21.02
7
O
8
6
CH3
30.04
55.69
30.04
218.83
O
H
O
9
55.69
3.837, s
3.832, s
OCH3
OCH3
1
2
5
4
OCH3
OCH3
50.02
58.07
50.02
58.07
3
150.47
110.85
150.47
110.85
N
H
6.78, d
=4.00
Hz
6.78, d
J =4.00
Hz
N
J
130.92
130.92
N
H
H
1.46, br s
115.07
153.47
OCH3
55.61
115.07
153.47
111.60
111.60
H
4.74, d
J= 2.50 Hz
3.76, s
N lone pair
deshield
3.76, s
OCH3
OCH3
OCH3
55.61
7.54, d
H3CO
the ortho-H
J=2.50 Hz
(c)
(a)
(b)
1.97, dd
J=14.00,
5.00 Hz
2.86
nine-line
m
1.41
quint
Ha He
He
He
1.76-1.68
1.76-1.68
21.13
m
m
Ha
Ha
7
2.45, br s
29.12
29.12
217.90
O
8
6
O
7.02,d
7.02, d
9
J=2.0
J =2.0
1
2
5
4
3.92, s
54.26
64.57
3.92, s
3.90, s
54.26
64.57
Hz
Hz
55.98
OCH3
55.98
111.13
111.13
H3CO
H3CO
OCH3
3
H3CO
148.99
148.38
H3CO
N
H
N
133.91
133.91
148.99
148.38
OCH3
H
1.65, brs
118.84 118.84
OCH3
4.35, d
6.92, d
J=8.5
Hz
6.92,d
J=8.5
Hz
110.10
110.10
3.90, s
J= 2.50 Hz
55.91
55.91
7.17, dd
J=8.5, 2.0 Hz
(e)
(d)
Figure 1. (a) and (b): 1H and 13C NMR data of ABN 1 (2,4-bis(2,5-dimethoxyphenyl)-3-azabicyclo[3.3.1]nonan-9-one), which also adopts twin-chair conformation; (c) Spatial
interactions in ABN 1; (d) and (e): 1H and 13C NMR data of ABN 2 (2,4-bis(3,4-dimethoxyphenyl)-3-azabicyclo[3.3.1]nonan-9-one), which also adopts twin-chair conformation.
The stereochemistry of ABN 1 is further confirmed through the
single-crystal XRD (Fig. 2). The detailed XRD analysis shows that
the piperidone ring N1–C1–C2–C8–C6–C7 of the 3-azabicycle
adopts a near ideal chair conformation with the deviation of ring
atoms N1 and C8 from the best plane C1–C2–C6–C7. The Nardel-
li’s11 smallest displacement asymmetry parameters q2 and q3 are
0.0930 (15) and ꢁ0.6011 (15) Å, respectively. The Cremer and Po-
ple12 ring puckering parameters such as total puckering amplitude
‘QT’ and phase angle ‘h’ are 0.6083 (15) Å and 171.19 (14)°. Thus all
parameters strongly support the near ideal chair conformation for
piperidone. Similarly, the analysis of cyclohexanone C2–C3–C4–
C5–C6–C8 indicates its chair conformation; however, deviates
from the ideal chair as follows. The displacement asymmetry
parameters q2 and q3 are 0.1022 (16) and ꢁ0.5354 (16) Å, respec-
tively, and puckering parameters ‘QT’ and ‘h’ are 0.5451 (16) Å and
169.20 (17)°. The torsion angles of 2,5-dimethoxyphenyl rings C8–
C6–C7–C15 and C8–C2–C1–C9 are 177.30(10) and 178.13(12)°,
respectively. Thus, all parameters clearly indicate the twin-chair
conformation of the bicycle with an equatorial orientation of the
phenyl groups (Figs. 2 and 3).
droxyl radical scavenging, nitric oxide radical scavenging, xanthine
oxidase, cytochrome C, etc., the in vitro antioxidant activity of
ABNs 1–10 were evaluated by four important methods. They are,
DPPHÅ scavenging activity, total antioxidant assay, reducing power
method and Fluorescence activated cell sorting (FACS) assay.
Among them, DPPH method is the best and very common over
the chemical methods in practice.14 The data of all methods used
in this study were performed by three independent experiments
and expressed as mean SD; All the statistically significant vari-
ances (P <0.005) were calculated using one-way ANOVA (Graph-
Pad, Prism 5.0).
DPPH assay was performed by the method of Je et al.,15 which is
based on the measurement of 2,2-diphenyl-1-picrylhydrazyl radi-
cal (DPPHÅ) scavenging ability of antioxidants. ABNs 8 and 10 are
the analogs of curcumin and bis-demethoxycurcumin, respec-
tively. They exhibited higher DPPH scavenging activity (Fig. 4)
compared to the curcuminoids and a-tocopherol, which can prob-
ably be favored by the contribution of amine group along with the
polyphenolic groups. Of the two active compounds ABNs 8 and 10,
the DPPHÅ scavenging activity of ABN 10 is very effective as indi-
Among the large number of existing methods such as ORAC,
ABTS, DMPD, FRAP, TRAP, TBA, superoxide radical scavenging, hy-
cated by its IC50 of 26.1
l
l
M, which is almost half of the required
IC50 of -tocopherol (59.0
a
M) and better than 35.1
l
M16 of curcu-

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D. H. Park et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6362–6367
O
O
N
H
O
O
H
a
R
R
R
R
H
O
ABN 1-10
O
NH4
O
CH3
O
O
O
O
O
O
O
ABN 1
3
ABN
2
ABN
O
O
O
O
O
OH
ABN 4
ABN 6
Figure 3. The intermolecular NH. . .O interactions stabilize the crystal packing and
the short contact between N(1)-H(1 N). . .O(5) is 2.441 Å.
ABN 5
min. The superior activity of ABN 10 is due to the presence of poly-
phenolic (four OH groups) and secondary amine groups in the mol-
ecule. However, to know the importance of ABN skeleton, the
DPPHÅ scavenging activity of the direct phenolic compound, that
is, catechol (ortho-dihydricphenol) was estimated under identical
O
O
O
Br
OH
OH
OH
OH
OH
7
10
ABN
ABN
8
ABN
ABN 9
conditions and it was found to be 36
ABN 10 did not show cytotoxicity against RAW 264.7 macrophage
cells up to 100 M concentration, whereas the catechol is classified
lM. On the other hand,
Scheme 1. Reagents and conditions (a) 10 mol % of BF3ꢀSiO2, ethanol (a small
amount of acetic acid was added for a few reactions for the solubility of aldehyde),
stirring at 30–40 °C.
l
as hazardous and toxic to many normal cell lines even at lower
concentrations.17
The DPPHÅ scavenging activity was also performed at 0.5, 1, 2, 4,
6, 12 and 24 h intervals. The ABNs 8 and 10 (80%) showed higher
antioxidant activity compared to the positive control at a concen-
tration of 50 lM (Fig. 5).
Figure 2. Anisotropic displacement representation of ABN 1 (CCDC No.: 864527)13
with atoms represented with 30% probability ellipsoids. The ABN 1, C24H29NO5,
crystallized in a triclinic system under the space group P-1 with cell parameters,
Figure 4. DPPHÅ scavenging activity of ABNs 1–10 as a function of concentration.
The antioxidant activity is expressed in terms of IC50 value, which based on the
amount of compound required for a 50% decrease of the initial DPPHÅ concentration.
Statistically significant variances: (P <0.001).
a = 7.0116(2) Å, b = 10.3453(4) Å, c = 15.4906(6) Å,
= 104.450(2) and Z = 2.
a = 100.081(2), b = 97.603(2),
c

D. H. Park et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6362–6367
6365
Figure 5. DPPHÅ scavenging activity of ABNs 1–10 as function of time. (P <0.001).
695 nm, and the results are expressed as equivalents to 100 lM
vitamin C (Fig. 6). The lead ABNs 8 and 10 exhibited antioxidant
activity of 56.8% and 62.3%, respectively.
We performed the assay of potassium ferric cyanide reducing
model system19 to determine the power of reducing effect of ABNs.
The obtained results are compared with the percentage of vitamin
C equivalent in Figure 7. We found that ABNs 8 (116%) and 10
(163%) exhibit higher reducing effect than positive control and
thus ensured the result of radical scavenging method. In fact, the
reducing ability of a compound mainly depends on the presence
of reductones. Reductone is an ‘enediol’ structure that stabilized
by conjugation and hydrogen bonding with an adjacent carbonyl
group, thus exhibits antioxidative potential by breaking the free
radical chain by donating hydrogen atom. The surprising outcome
of ABNs 8 and 10 by this assay is probably being the impact of
amine group besides the predominant effect from reductone like
3,4-dihydroxy and curcumin like ortho-methoxyphenolic groups.
Reactive oxygen species (ROS) plays an important role in vari-
ous diseases by oxidative modification of DNA, carbohydrates, pro-
tein, lipids and small cellular molecules.20 ROS can easily damage
the DNA, and thus responsible for the neurodegenerative patho-
genesis such as Alzheimer and Parkinson. In order to evaluate
the ROS inhibition efficacy of ABN compounds, we performed FACS
assay21 for one of the lead molecule ABN 10.
Figure 6. Total antioxidant activity of ABNs 1–10, which determined by the
reduction of Mo (VI) to Mo (V). Statistically significant variances (P <0.0047).
The ROS were generated in RAW 264.7 cells using hydrogen
peroxide (H2O2). ROS content in the cells were measured by the
fluorescence intensity of H2DCF-DA excited at 488 nm (Fig. 8).
The fluorescence intensity is discussed here in terms of the ‘Geo
Mean (g)’ value of independent experiments measured using BD
software. A ten-fold enhancement of ROS generation (g = 206.38)
was observed in the presence of H2O2 in 30 min interval in RAW
264.7 cells compared to blank (g = 20.26). Subsequently, the gener-
ated ROS was significantly reduced in cells pre-incubated with ABN
10 at 50 lM (g = 35.28) and 100 lM (g = 30.78) concentrations. The
decrease of ‘g’ in treated ABN 10 towards blank indicates that ABN
10 significantly inhibited the production of ROS in dose-dependent
manner.
In conclusion, we synthesized a series of new curcumin-related
2,4-diaryl-3-azabicyco[3.3.1]nonan-9-ones with enhanced yield
using BF3ꢀSiO2 under mild conditions. All the azabicyclic ketones
(ABNs 1–10) exist in the twin-chair conformation with an equato-
rial orientation of the highly functionalized phenyl groups. The
target azabicyclic ketones that similar to curcumin units (ortho-
methoxyphenol in ABN 8 and ortho-dihydricphenol in ABN 10) reg-
Figure 7. Reducing power of ABNs 1–10 by potassium ferricyanide method.
Statistically significant variances: (P <0.001).
The quantitative antioxidant capacity18 of ABN compounds was
performed by measuring the green phospho-Mo (V) complex at

6366
D. H. Park et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6362–6367
Negative Blank:
(b)
Blank :
(a)
(c)
O
Without H2DCF-DA
With H2DCF-DA
HO
HO
OH
OH
N
H
ABN 10
Control:
H2O2 +
H2DCF-DA
ABN 10
ABN 10
(d)
(e)
(100µM) +
(50µM)+
H2O +
O
H2
+
2
2
H2DCF-DA
H2DCF-DA
Figure 8. The inhibition effect of ABN 10 on intracellular ROS generation in Raw 264.7 macrophage cells. (a) Negative blank:without H2DCF-DA; (b) Blank:with H2DCF-DA; (c)
Control:H2DCF-DA with H2O2 (ABN 10 is 0 M); (d) and (e) 50 and 100 M concentrations of ABN 10.
l
l
(d, 2H, J = 2.5 Hz), 3.92 (s, 6H), 3.90 (s, 6H), 2.86 (m, 1H), 2.45 (br s, 2H), 1.97
(dd, 2H, J = 14.0, 5.0 Hz), 1.76–1.68 (m, 2H), 1.65 (br s, 1H), 1.41 (quint, 1H);
Mass (APCI, positive mode): 412.2 (M+H); 2,4-Bis(3,4,5-trimethoxyphenyl)-3-
azabicyclo[3.3.1]nonan-9-one (ABN 3): Yield: 59%; 1H NMR (500 MHz, CDCl3):
6.83 (s, 4H), 4.35 (d, 2H, J = 2.5 Hz), 3.837 and 3.830 (18H), 2.87 (m, 1H), 2.49
(br s, 2H), 1.93 (dd, 2H, J = 14.0, 5.0 Hz), 1.74–1.67 (m, 2H), 1.60 (br s, 1H), 1.44
(quint, 1H); CHN Calcd (found): 66.22 (66.30), 7.05 (7.04), 2.97 (3.00); Mass
(APCI, positive mode): 472.3 (M+H); 2,4-Bis(4-ethoxy-3-methoxyphenyl)-3-
azabicyclo[3.3.1]nonan-9-one (ABN 4): Yield: 64%; 1H NMR (500 MHz, CDCl3):
7.13 (dd, 2H, J = 8.0, 1.5 Hz), 7.02 (d, 2H, J = 1.5 Hz), 6.91 (d, 2H, J = 8.0 Hz), 4.34
(d, 2H, J = 2.50 Hz), 4.12 (q, 4H), 3.90 (s, 6H), 2.86 (m, 1H), 2.45 (br s, 2H), 1.97
(dd, 2H, J = 14.0, 5.0 Hz), 1.91 (br s, 1H), 1.75–1.68 (m, 2H), 1.48 (t, 6H,
J = 6.3 Hz), 1.42 (quint, 1H); CHN Calcd (found): 71.05 (71.00), 7.57 (7.60), 3.19
(3.20); Mass (APCI, positive mode): 440.3 (M+H); 2,4-Bis(4-(benzyloxy)-3-
methoxyphenyl)-3-azabicyclo[3.3.1]nonan-9-one (ABN 5): Yield: 61%; 1H NMR
(500 MHz, CDCl3): 7.43–7.29 (m, 10H), 7.07 (dd, 2H, J = 8.0, 2.0 Hz), 7.02 (d, 2H,
J = 2.0 Hz), 6.91 (d, 2H, J = 8.0 Hz), 5.15 (s, 4H), 4.31 (d, 2H, J = 2.50 Hz), 3.90 (s,
6H), 2.83 (m, 1H), 2.43 (br s, 2H), 1.94 (dd, 2H, J = 14.5, 5.0 Hz), 1.90 (br s, 1H),
1.73–1.66 (m, 2H), 1.38 (quint, 1H); CHN Calcd (found): 76.71 (76.40), 6.62
(6.60), 2.48 (2.48); Mass (APCI, positive mode): 564.3 (M+H); 2,4-Bis(4-
hydroxy-3,5-dimethoxyphenyl)-3-azabicyclo[3.3.1]nonan-9-one (ABN 6):
Yield: 46%; 1H NMR (500 MHz, CDCl3): 6.81 (s, 4H), 4.34 (d, 2H, J = 2.5 Hz),
3.84 (12H), 2.87 (m, 1H), 2.48 (br s, 2H), 1.93 (dd, 2H, J = 14.5, 5.0 Hz), 1.74–
1.67 (m, 2H), 1.66 (br s, 1H), 1.43 (quint, 1H); CHN Calc. (found): 65.00 (65.12),
6.59 (7.01), 3.16 (3.16); Mass (APCI, positive mode): 444.2 (M+H); 2,4-Bis(3-
bromo-4-hydroxy-5-methoxyphenyl)-3-azabicyclo[3.3.1]nonan-9-one (ABN
7): Yield: 62%; 1H NMR (500 MHz, CDCl3): 7.35 (d, 2H, J = 1.5 Hz), 6.88 (d,
2H, J = 1.5 Hz), 5.89 (br s, 2H), 4.29 (d, 2H, J = 2.5 Hz), 3.93 (s, 6H), 2.76 (m, 1H),
2.43 (br s, 2H), 1.96 (dd, 2H, J = 14.0, 4.5 Hz), 1.77–1.70 (m, 2H), 1.64 (br s, 1H),
1.44 (quint, 1H); CHN Calcd (found): 48.82 (48.78), 4.28 (4.29), 2.59 (2.58);
Mass (APCI, positive mode): 540.0 (M+H); 2,4-Bis(4-hydroxy-3-
methoxyphenyl)-3-azabicyclo[3.3.1]nonan-9-one (ABN 8): Yield: 53%; 1H
NMR (500 MHz, CDCl3): 7.12 (dd, 2H, J = 8.5, 2.0 Hz), 7.03 (d, 2H, J = 2.0 Hz),
6.93 (d, 2H, J = 8.5 Hz), 4.33 (d, 2H, J = 2.5 Hz), 3.92 (s, 6H), 2.86 (m, 1H), 2.45
(br s, 2H), 1.96 (dd, 2H, J = 14.5, 5.0 Hz), 1.76–1.68 (m, 3H), 1.42 (quint, 1H);
CHN Calcd (found): 68.91 (68.80), 6.57 (6.61), 3.65 (3.66); Mass (APCI, positive
mode): 384.2 (M+H); 2,4-Bis(3-ethoxy-4-hydroxyphenyl)-3-azabicyclo[3.3.1]
nonan-9-one (ABN 9): Yield: 43%; 1H NMR (500 MHz, CDCl3): 7.10 (dd, 2H,
J = 8.5, 2.0 Hz), 7.02 (d, 2H, J = 2.0 Hz), 6.93 (d, 2H, J = 8.5 Hz), 6.01 (br s, 2H),
4.34 (d, 2H, J = 2.5 Hz), 4.12 (q, 4H), 2.86 (m, 1H), 2.45 (br s, 2H), 1.97 (dd, 2H,
J = 14.5, 5.0 Hz), 1.77–1.69 (m, 2H), 1.63 (br s, 1H), 1.47 (t, 6H, J = 6.4 Hz), 1.41
(quint, 1H); CHN Calcd (found): 70.05 (70.03), 7.10 (7.07), 3.40 (3.42);
Mass (APCI, positive mode): 412.2 (M+H); 2,4-Bis(3,4-dihydroxyphenyl)-3-
azabicyclo[3.3.1]nonan-9-one (ABN 10): Yield: 41% (high yield obtained by
demethoxylation of ABN 2 by BBr3); 1H NMR (500 MHz, DMSO-d6): 7.01 (dd,
2H, J = 8.0, 2.5 Hz), 7.02 (d, 2H, J = 2.0 Hz), 6.80 (d, 2H, J = 8.0 Hz), 4.37 (d, 2H,
J = 2.5 Hz), 2.97 (m, 1H), 2.45 (br s, 2H), 1.77 (dd, 2H, J = 14.0, 5.0 Hz), 1.64–1.55
(m, 3H), 1.09 (quint, 1H); CHN Calc. (found): 67.59 (67.60), 5.96 (5.99), 3.94
(3.92);Mass (APCI, positive mode): 356.2 (M+H).
istered excellent antioxidant properties by DPPH, total antioxidant,
reducing power and FACS assays. Between the active molecules 8
and 10, ABN 10 registered one-fold (IC50 = 26.1
scavenging activity over standard -tocopherol (59.0
ter activity than vitamin C and curcumin (35.1 M). These findings
l
M) superior DPPHÅ
a
lM), and bet-
l
of chemical methods sustained by its intracellular ROS inhibition
ability as well, and thus present study ensures the reliability of fur-
ther developments.
Acknowledgment
This work was supported by the National Research Foundation
research Grant 2010.
References and notes
1. (a) Katritzky, A. R.; Fan, W. J. Org. Chem. 1990, 55, 3205. and references cited
therein; (b) Silverman, R. B. In Organic Chemistry of Drug Design and Drug Action;
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&id=44&Itemid=68.
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S. W., Joshi, B. S., Eds.; Wiley: New york, 1991; p 297; (b) Hardick, D. J.;
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Park, K. S.; Jeong, Y. T. Monatsh. Chem. 2010, 141, 79; (f) Parthiban, P.; Rathika,
P.; Ramkumar, V.; Son, S. M.; Jeong, Y. T. Bioorg. Med. Chem. Lett. 2010, 20, 1642;
(g) Parthiban, P.; Subalakshmi, V.; Balasubramanian, K.; Islam, M. N.; Choi, J. S.;
Jeong, Y. T. Bioorg. Med. Chem. Lett. 2011, 21, 2287.
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Food Chem. 2002, 50, 3122.
8. Halliwell, B.; Gutteridge, J. M. C. Free Radical Biol. Med. 1989, 1, 331.
9. Dindulkar, S. D.; Parthiban, P.; Jeong, Y. T. Monatsh. Chem. 2012, 143, 113.
10. 2,4-Bis(2,5-dimethoxyphenyl)-3-azabicyclo[3.3.1]nonan-9-one (ABN 1): Yield:
64%; 1H NMR (500 MHz, CDCl3): 7.54 (d, 2H, J = 2.5 Hz), 6.78 (d, 4H, J = 4.0 Hz),
4.74 (d, 2H, J = 2.5 Hz), 3.837 and 3.832 (6H), 3.768 (s, 6H), 2.86 (m, 1H), 2.65
(br s, 2H), 1.93 (dd, 2H, J = 13.75, 4.75 Hz), 1.74–1.66 (m, 2H), 1.46 (br s, 1H),
1.32 (quint, 1H); CHN Calcd (found): 70.05 (70.03), 7.10 (7.11), 3.40 (3.41);
Mass (APCI, positive mode): 412.2 (M+H); 2,4-Bis(3,4-dimethoxyphenyl)-3-
azabicyclo[3.3.1]nonan-9-one (ABN 2): Yield: 63%; 1H NMR (500 MHz, CDCl3):
7.17 (dd, 2H, J = 8.5, 2.0 Hz), 7.02 (d, 2H, J = 2.0 Hz), 6.92 (d, 2H, J = 8.5 Hz), 4.35
11. Nardelli, M. Acta Crystallogr. 1983, C39, 1141.
12. Cremer, D.; Pople, J. A. J. Am. Chem. Soc. 1975, 97, 1354.
13. Single-crystal XRD data of ABN
1 was deposited in CCDC (No. 864527).
Supplementary crystallographic data can be obtained free of charge at
www.ccdc.cam.ac.uk/conts/retrieving.html.
14. (a) Bondet, V.; Brand-Williams, W.; Berset, C. LWT-Food Sci. Technol. 1997, 30,
609. and references cited therein; (b) http://www.medallionlabs.com/
Downloads/Antiox_acti_.pdf.; (c) http://www.terranostrachocolate.com/files/
Comparative_and_General_Antioxidant_Information.pdf.; (d) http://www.phar-
mainfo.net/reviews/vitro-models-antioxidant-activity-evaluation-review.

D. H. Park et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6362–6367
6367
15. Je, J.-Y.; Park, P.-J.; Kim, S.-K. Eur. Food Res. Technol. 2004, 219, 60. Briefly, the
desired concentrations of ABN compounds prepared by DMSO (1 mL) were
added to methanolic solution of DPPH (1.5 ꢂ 10ꢁ4 M, 1 mL). The mixture was
vigorously shaken for 10 s and left to stand at room temperature. The
scavenging activity of ABN compounds on DPPH was determined by the
absorbance at 520 nm. The percent scavenging activity of ABN compounds was
21. (a) MTT assay: The cytotoxicity of ABN 10 was measured using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and it did not
exhibit cytotoxicity against RAW 264.7 macrophage cells up to 100 lM. RAW
264.7 macrophage cells were obtained from the American Type Culture
Collection (Rockville, MD). The cells were cultured in Dulbecco’s Modified
Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM
calculated according to the following equation:
%
Scavenging activity =
glutamine, 100 lg/mL of penicillin, and 100 lg/mL of streptomycin. The cells
Ablank ꢁ Asample/Ablank ꢂ 100.
were incubated at 37 °C in a humidified atmosphere (5% CO2). The cells were
grown in 24-well plates at a density of 1 ꢂ 104 cells/well. After 24 h, the cells
were treated with control medium and medium supplemented with different
concentrations of ABNs. After incubation for 24 h, MTT solution (1 mg/mL) was
added and incubated for another 4 h. Finally, 1 mL of DMSO was added to
solubilize the formed formazan crystals. The amount of formazan crystals was
determined by measuring the absorbance at 540 nm using GENiosÒ microplate
reader (Tecan Austria GmbH, Austria).; (b) FACS assay: The culture medium
16. Somparn, P.; Phisalaphong, C.; Nakornchai, S.; Unchern, S.; Morales, N. P. Biol.
Pharm. Bull. 2007, 30, 74.
17. Van Den Heuvel, R.; Leppens, H.; Schoeters, G. Cell Biol. Toxicol. 1999, 15, 101.
18. Smirnoff, N.; Cumbes, Q. J. Phytochem. 1989, 28, 1057. Briefly, the assay is
based on the reduction of Mo(VI) to Mo(V) by ABNs and subsequent formation
of a green phosphate/Mo (V) complex at acidic pH. The tubes containing ABNs
and reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM
ammonium molybdate) were incubated at 95 °C for 90 min. After the mixture
had cooled to room temperature, the absorbance of each solution was
measured at 695 nm against a blank. The antioxidant capacity was expressed
was replaced with 1ꢂ Hank’s Balanced Salt Solution (HBSS) containing 20
lM
of 20,70-dichlorodihydrofluorescein diacetate (H2DCF-DA) and incubated for
30 min. The H2DCF-DA containing medium was replaced by 1ꢂ HBSS
as vitamin C equivalent (100
19. Wang, J.; Zhang, Q.; Zhang, Z.; Li, Z. Int. J. Biol. Macromol. 2008, 42, 127. Briefly,
1 mL of reaction mixture, containing 250 l of different sample concentration
in phosphate buffer (0.2 M, pH 6.6), was incubated with 250 L of potassium
ferricyanide (1% w/v) at 50 °C for 20 min. The reaction was terminated by
250 l of trichloroacetic acid (TCA) solution (10% w/v). The solution was then
mixed with 250 L of ferric chloride (0.1% w/v) solution and the absorbance
was measured at 700 nm. The result was expressed as a percentage of the
activity with 100 M vitamin C.
20. Cai, H.; Harrison, D. G. Circul. Res. 2000, 87, 840.
lM).
containing different concentrations of ABN 10 (50 and 100 lM), and the cells
were incubated for 30 min at 37 °C in the dark. From this step, light must be
limited to avoid photo bleaching of the fluorescent probe. Further, cells were
treated with H2O2 and incubated for 30 min. Scraper was used to remove the
Raw 264.7 cells from culture plates; suspended cells in 1 mL of 1ꢂ HBSS and
l
l
l
gently passed through a 40 lm nylon mesh filter for flow cytometric analysis.
l
ROS content in the cells was measured by the fluorescence intensity of H2DCF-
DA, excited at 488 nm, using the standard operational procedure for BD
FACSCalibur.
l