Antioxidant effects of the hydroxy groups in the simple phenolic carbazoles

Article abstract of DOI:10.3987/COM-15-13363

Antioxidant activities of the simple phenolic carbazoles 5-11 were evaluated by 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate)⁺ radical scavenging assays. The simple phenolic carbazoles 5-7, 9, and 11 exhibited stronger antioxidant activities than α-tocopherol, and similar antioxidant activities as phenolic carbazole alkaloids carazostatin (1), and carbazomadurins A (3) and B (4). Bond dissociation energies and highest occupied molecule orbital energy levels of a series of phenolic carbazoles including phenolic carbazole alkaloids were calculated. The reducing ability of the phenolic carbazole core could be important role for the antioxidant activity of carbazole alkaloids 1, 3, and 4.

Full text of DOI:10.3987/COM-15-13363

HETEROCYCLES, Vol. 92, No. 1, 2016, pp. -. © 2016 The Japan Institute of Heterocyclic Chemistry
Received, 9th November, 2015, Accepted, 26th November, 2015, Published online, 8th December, 2015
DOI: 10.3987/COM-15-13363
ANTIOXIDANT EFFECTS OF THE HYDROXY GROUPS IN THE
SIMPLE PHENOLIC CARBAZOLES
a
b
c
d
a
Yuhzo Hieda, Noriyuki Hatae, Makoto Anraku, Nobuyasu Matsuura,
e
a
a
Kazuhide Uemura, Satoshi Hibino, Tominari Choshi, * Hisao Tomida,
f
a
Osamu Hori, and Haruto Fujioka *
a
Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University,
b
Fukuyama, Hiroshima 729-0292, Japan School of Pharmaceutical Sciences,
Health Science University of Hokkaido, Ishikari, Tobetsu, Hokkaido 061-0293,
c
Japan Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto 860-0082,
d
Japan Department of Life Science, Okayama University of Science, Kita-ku,
e
Okayama 700-0005, Japan Department of Biomedical Sciences, College of Life
and Health Sciences, Chubu University, Kasugai, Aichi 487-8501, Japan
f
Department of Neuroanatomy, Kanazawa University, Graduate School of
Medical Sciences, Takaramachi, Kanazawa 920-8640, Japan. E-mail:
choshi@fupharm.fukuyama-u.ac.jp and fujioka@fupharm.fukuyama-u.ac.jp
Abstract – Antioxidant activities of the simple phenolic carbazoles 5-11 were
evaluated
by
2,2-diphenyl-1-picrylhydrazyl
and
+
2
,2’-azinobis-(3-ethylbenzthiazoline-6-sulfonate) radical scavenging assays. The
simple phenolic carbazoles 5-7, 9, and 11 exhibited stronger antioxidant activities
than α-tocopherol, and similar antioxidant activities as phenolic carbazole
alkaloids carazostatin (1), and carbazomadurins A (3) and B (4). Bond
dissociation energies and highest occupied molecule orbital energy levels of a
series of phenolic carbazoles including phenolic carbazole alkaloids were
calculated. The reducing ability of the phenolic carbazole core could be important
role for the antioxidant activity of carbazole alkaloids 1, 3, and 4.
Reactive oxygen species (ROS) are highly reactive small substances with an important role in the
initiation of various diseases such as carcinogenesis, drug-associated toxicity, inflammation, and
atherogenesis as well as aging in aerobic organisms. Natural antioxidants, including ascorbic acid,
α-tocopherol (VE), ubiquinol, uric acid, polyphenols and their analogs have been intensive investigation

1
for clinical applications. Edaravone (3-methyl-N-phenylpyrazolin-5-one) scavenges free radicals and
protects the brain against ischemic lesions after stroke, thus improving the clinical outcomes for patients
2
with acute ischemic damage. Synthetic and/or medicinal chemists are interested in obtaining novel
1
,2
antioxidant substances as an important approach to treating oxidative stress-related diseases.
3
4
Carbazole alkaloids are a well-known class of natural products possessing a benzo[b]indole ring system.
These heterocyclic natural products have a wide variety of bioactivities, such as antibacterial, antibiotic,
5
,6
7
8
antimalarial, antitumor,
cardiotonic, anti-inflammatory, free radical scavenging and neuronal
9
-13
cell-protecting
activities. Due to their interesting biologic effects, carbazole alkaloids and their related
compounds constitute an important class of natural products.
HO
R4
R5
R3
R2
5
4
OR
3
OH
OR
6
7
9
2
N
1
N
N
H
N
H
8
R1
H
C H
7
HO
H
OH
15
1
2
3
4
5
7
8
9
: R = OH, R = R = R = R = H
2
1
3
4
5
: R = OH, R = R = R = R = H
R
3
1
2
4
5
: R = OH, R = R = R = R = H
4
1
2
3
5
1
2
: carazostatin (R= H)
3: carbazomadurin A (R=Me)
5: R= H
6: R= Et
10: R = OH, R = R = R = R = H
1
5
2
3
4
: O-ethylcarazostatin (R= Et) 4: carbazomadurin B (R= Et)
11: R = R = OH, R = R = R = H
Figure 1. Phenolic carbazole alkaloids and simple hydroxycarbazoles
In 1989, the 3-hydroxycarbazole alkaloid carazostatin (1) was isolated from Streptomyces chromofuscus
9
a
as a free radical-scavenging substance. This alkaloid exhibits strong inhibitory activity against free
9
b
radical–induced lipid peroxidation and stronger antioxidant activity in liposomal membranes than VE.
Furthermore, the antioxidant activity of 1 and 2-hydoxycarbazole (8) was assessed in the oxidation of
1
0
methyl linolate induced by aza radical initiator.
Few years later, 3,8-dihydroxycarbazoles
1
1
carbazomadurins A (3) and B (4) were isolated from Actinomadula madurae 2808-SV-1. The
antioxidative activity of these alkaloids was evaluated based on their inhibition of L-glutamate toxicity in
N18-RE-195 cells. In addition, the first example of carbazole-3,4-quinone alkaloid carquinostatin A was
1
2a
isolated in 1993 from Streptomyces exfoliates 2419-SVT2. This natural product is a potent neuronal
1
2b
cell-protecting substance and exhibits free radical scavenging activity.
On the other hand,
3
-hydroxycarbazole lipocarbazoles A1-A4, closely related to carazostatin, were found in Tsukamurella
1
3
pseudospumae Acta 1857. The antioxidant activity of lipocarbazole A3 was reported to be more active
1
3
than ascorbic acid in a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay.
These 3-hydroxycarbazole, 3,8-dihydroxycarbazole and carbazole-3,4-quinone alkaloids were
independently evaluated in a bioassay system during isolation, but have not yet been evaluated
comprehensively. We recently reported that 3-hydroxycarbazole carazostatin (1), 3,8-dihydroxycarbazole

alkaloids carbazomadurins A (3) and B (4), and related carbazoles, except carbazole-3,4-quinone alkaloid
carquinostatin A, exhibit strong antioxidant effects based on a comprehensive evaluation in several assay
1
4
systems using propyl galate (PG), VE and edaravone as standards. Recently, the radical scavenging
activities by antioxidants were rationalized based on their chemical properties using quantum chemical
calculations. The scavenging activities of free radicals by lipocarbazoles were well evaluated by the O-H
1
5
and N-H bond dissociation enthalpies. The glutathione peroxidase-like antioxidant activities of aliphatic
1
6
selenides were consistent with their highest occupied molecule orbital (HOMO) energy levels (EHOMO).
In the present paper, we describe the relationship between the calculated structure and antioxidant activity
of simple oxygenated carbazoles 5-11 in comparison with carazostatin (1) and carbazomadurins A (3) and
B (4) (Figure 1).
OEt
OEt
OEt
OEt
I
a
b, c
f
d
CHO
CHO
N
N
N
N
R
N
H
OMOM
OMOM
OH
SO2
SO2
SO2
Ph
Ph
Ph
1
1
5: R= SO Ph
6: R= H
12
13
14
2
6
e
g
OH
OH
OEt
OEt
i
h
j
N
H
N
H
N
H
N
H
OH
C H
7
C H
7 15
OTf
15
1
2
17
5
Scheme 1. Synthesis of carazostatin (1) and related carbazoles 2, 5 and 6
Reagents and conditions: (a) 2-ethoxyvinylstannane, PdCl (PPh , Et
4%; (b) ethynylmagnesium bromide, THF, 0 C, 1 h, 82%; (c) MOMCl, i-Pr
o
2
3
)
2
4
NCl, DMF, 80 C, 2 h,
o
8
2
2 2
NEt, CH Cl , 50
o
o
C, 12 h, 97%; (d) t-BuOK, t-BuOH, THF, 90 C, 15: 41%, 16: 43%; (e) 3M NaOH, MeOH,
o
o
THF, 80 C, 18 h, 98%; (f) TMSCI, NaI, MeCN, -20 C, 5 min, 63%; (g) Tf
2
O, pyridine, CH
2
2
Cl ,
o
o
0
C, 2 h, 85%; (h) 1-heptene, 9-BBN, 3M NaOH, PdCl
Cl , -78 C, 4 h, 90%; (j) (1) BBr , CH Cl , -78 C, 4 h; (2) LiAlH , THF, 0 C, 60% (2
2 2 3 2 2 4
2
(dppf), THF, 80 C, 1 h, 85%; (i) BBr ,
3
o
o
o
CH
steps)
To evaluate the antioxidant activities of 3-hydroxycarbazole alkaloid, carazostatin (1) was prepared by an
allene-mediated electrocyclic reaction involving the indole 2,3-bond according to the previously reported
1
7
procedure (Scheme 1).
-iodoindole-2-carbaldehyde 12 in three steps, with t-BuOK afforded tri-substituted carbazole 15.
Cleavage of the phenylsulfonyl group of 15 with NaOH, followed by treatment of TMS-I afforded the
-hydroxy-3-ethoxycarbazole 6. Treatment of 6 with Tf O gave the O-triflate 17, which was subjected to
a Pd-catalyzed cross-coupling reaction with 9-hexyl-9-BBN to give the 1-hexylcarbazole 2. Subsequent
treatment of 2 with BBr provided carazostatin (1). In addition, 1,3-dihydroxycarbazole 5 was prepared in
two steps from 17. Carazostatin (1), 1-hexyl-3-ethoxycarbazole 2, 1,3-dihydroxycarbazole 5, and
-hydroxy-3-ethoxycarbazole 6 were used to evaluate antioxidant activities. 3,8-Dihydroxycarbazole
Treatment of the indole-2-propargyl ether 14, derived from
3
1
2
3
1

alkaloids carbazomadurins A (3) and B (4) were prepared according to our reported procedure and used in
1
8
radical scavenging assays.
R3
R2
R3
R2
R3
R2
R2
R3
b
c
a
NH2
Br
N
N
N
R1
R1
R1
R1
H
H
H
1
2
3
1
2
3
1
2
3
1
2
3
1
1
8a: R = OMe, R = H 19a: R = H
20a: R = OMe, R = R = H 21a: R = OMe, R = R = H
7: R = OH, R = R = H
1
2
3
1
3
2
1
3
2
2
1
3
8b: R = H, R = OMe 19b: R = OMe 20b: R = R = H, R = OMe 21b: R = R = H, R = OMe
9: R = OH, R = R = H
1
3
2
1
3
2
1
3
2
2
0c: R = R = OMe, R = H 21c: R = R = OMe, R = H
11: R = R = OH, R = H
Scheme 2. Synthesis of simple hydroxycarbazoles 7, 9 and 11
Reagents and conditions: (a) Pd(OAc) (0.04 equiv.), BINAP (0.04 equiv.), K
toluene, 12 h, 150 C, 20a: 98%, 20b: 83%, 20c: 79%; (b) Pd(OAc) (1.2 equiv.), AcOH, 130 C,
, -78 C to rt, 5 h, 7: 65%, 9: 60%, 11:
2
2 3
CO (1.4 equiv.),
o
o
2
o
1
3 2 2
5 h, 21a: 61%, 21b: 31%, 21c: 29%; (c) BBr , CH Cl
4
9%
1
9
Simple hydroxycarbazoles 7, 9 and 11 were prepared by the reported procedures as follows (Scheme 2).
Buchwald-Hartwig amination of bromobenzene (19a) with o-anisidine (18a) or p-anisidine (18b) gave
the diphenylamine 20a and 20b, respectively. Similarly, amination of 4-bromoanisole (19b) with
o-anisidine (18a) afforded the diphenylamine 20c. Diphenylamines 20a, 20b and 20c were subjected to a
2
0
21
palladium(II)-catalyzed oxidative cyclization to produce corresponding methoxycarbazoles 21a, 21b
2
2
and 21c, which were treated with BBr
3
to provide three hydroxycarbazoles 7, 9 and 11, respectively.
2
0
23
1
-Hydroxycarbazole (7), 3-hydroxycarbazole (9) and 1,6-dihydroxycarbazole (11) were used in our
2
4
assays. Commercially available 2-hydroxycarbazole (8) and 4-hydroxycarbazole (10) were used.
The structures of all synthesized compounds (Figure 1), except commercially available 8 and 10, were
1
13
25
elucidated on the basis of H and C NMR spectra. Antioxidant activities against DPPH, and
+
26
2
,2’azinobis-(3-ethylbenzthiazoline-6-sulfonate) cations (ABTS ) were measured.
Evaluation of DPPH radical scavenging activity is a rapid and convenient method for screening the
2
5
antioxidant activity. In the DPPH radical scavenging assay (Table 1), hydroxycarbazoles 1, 3, 4, 5, 6, 7,
and 11 exhibited higher radical-scavenging activities than VE (15.4) and edaravone (19.7). Among
them, simple 1-hydroxycarbazole (7) (10.4) and 3-hydroxycarbazole (9) (9.4) displayed the same potent
9
1
4
scavenging activity as carbazomadurins A (3) (9.9) and B (4) (9.8) as reported previously. Compared to
carazostatin (1), 3-hydroxycarbazole (9) exhibited the more potent radical scavenging activity. In addition,
simple 1,6-dihydroxycarbazole (11) (8.0), the core structure of 3,8-dihydroxycarbazole carbazomadurins
A (3) and (B) (4), exhibited the strongest radical scavenging activity of all tested compounds compared
with PG (6.1). Furthermore, 1,3-dihydroxycarbazole 5 (13.7) and 1-hydroxy-3-ethoxycarbazole 6 (13.2)
exhibited better radical scavenging activity than VE or edaravone. O-Ethylcarazostatin (2) and

Table 1. Radical scavenging activities of carbazole alkaloids and hydroxycarbazoles
Data are expressed as the mean ±S.E. of three experiments.
a
b
PG = propyl gallate, VE = α-tocopherol
2
-hydroxycarbazole (8), however, exhibited weak antioxidant activities in this assay because they could
not be transformed to a suitable iminoquinone form.
+
The ABTS radical assay is a conventional and excellent model for assessing the antioxidant activities of
+
hydrogen-donation and chain-breaking antioxidants. In this assay, ABTS radicals were produced by
2
6
reacting ABTS with potassium persulfate in sodium phosphate buffer solution. As shown in Table 1, all
oxygenated carbazoles 1, 3-7, and 9-11, except O-ethylcarazostatin (2) and 2-hydroxycarbazole (8),
+
exhibited better ABTS radical scavenging activities than the strong antioxidants PG (188) with better
IC50 values.
Among them, seven hydroxycarbazoles 3 (112), 4 (116), 5 (116), 6 (121), 7 (118), 10 (116), and 11 (102)
+
exhibited stronger ABTS radical scavenging activities than PG. Simple 1,3-dihydroxycarbazole 5,
1
3
1
-hydroxycarbazole (7), and 4-hydroxycarbazole (10) possessed strong antioxidant activities similar to
,6-dihydroxycarbazole alkaloids carbazomadurins A (3) and B (4). Based on this assay, the simple
,6-dihydroxycarbazole (11) (102), the core structure of carbazomadurins A (3) and B (4), had the
strongest antioxidant activity of all tested compounds. Although O-ethylcarazostatin (2) displayed weak
+
DPPH radical scavenging activity, the ABTS radical scavenging activity of 2 (196) was between that of
+
VE (211) and PG (188). The simple 2-hydroxycarbazole (8) exhibited the lowest ABTS radical
scavenging activity in this assay.
For scavenging activity of the free radicals by hydroxycarbazoles, the O-H and N-H groups provided
2
7-29
efficient DPPH scavenging activity.
Both O-H and N-H bond dissociation energies, examined by
density functional theory at ωB97X-D/6-31+G(d), were calculated for all of the tested compounds (Table

2
). Among all of the carbazoles, except compound 2, the bond dissociation energies of the O-H groups
were lower than those of the N-H group.
Table 2. Bond dissociaction (BD) energy values and HOMO energy levels
of carbazole alkaloids and hydroxycarbazoles
a
BD energies are indicated bond dissociation energies, which were calculated by a density
functional theory (DFT) at the ωB97X-D/6-31+G(d).
b
O-H radicals (1) mean the hydroxy group with lower substitution number than another one.
O-H radicals (2) mean the hydroxy group with higher substitution number than another one.
c
d
EHOMO (HOMO energy levels) were calculated by DFT at the ωB97X-D/6-31+G(d).
In conclusion, the simple phenolic carbazoles 5-11 were subjected to the evaluation of antioxidant
+
activities by DPPH radical and ABTS radical scavenging assays in comparison with potent antioxidant
alkaloids carazostatin (1), and carbazomadurins A (3) and B (4). Among them, the simple phenolic
carbazoles 5-7, 9, and 11 exhibited stronger antioxidant activities than α-tocopherol, and similar
antioxidant activities as phenolic carbazole alkaloids 1, 3, and 4. As a result, three simple phenolic
carbazoles 7, 9, and 11 exhibited the strong antioxidant activities similar to the potent antioxidant
alkaloids 3 and 4. 1-Hydroxycarbazole (7), 3-hydroxycarbazole (9) and 1,6-dihydroxycarbazole (11) are
the minimum core structures of alkaloids 1, 3 and 4, respectively. Consequently, the existence of one or
two hydroxy groups at least at the 1-, 3-, 6-, or 8-positions of the carbazole ring is essential to the
iminoquinone form. By contrast, it is considered that the weak antioxidant activities of
O-ethylcarazostatin (2) and 2-hydroxycarbazole (8) cannot be transformed to a suitable iminoquinone
form through a radical scavenging process. Furthermore, the antioxidant activity by hydroxy groups in
simple phenolic carbazoles 7-10 could be correlated with their EHOMO levels, but not their bond
dissociation energies. Based on these results, the reducing ability of the phenolic carbazole core could be
important role for the ability of carbazole alkaloids 1, 3, and 4.

EXPERIMENTAL
All non-aqueous reactions were carried out under nitrogen in dried glassware, unless otherwise noted.
Solvents were dried and distilled according to standard protocols. Analytical thin layer chromatography
was performed with silica gel 60PF254 (Merck). Silica gel column chromatography was performed with
silica gel 60N (63-210 µm, Kanto Chemical Co., Inc.). All melting points were determined with a
Yanagimoto micro melting point apparatus. The ultraviolet (UV) spectra were measured by a Shimadzu
1
UV-2550 spectrophotometer. The proton nuclear magnetic resonance ( H NMR) spectroscopic data were
recorded with a JEOL AL-300 at 300 MHz and a JEOL JMN-LA500 at 500 MHz. Chemical shifts are
4 3
reported relative to Me Si (δ = 0.00 ppm). The NMR spectroscopic data were recorded using CDCl ,
unless otherwise noted. Multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), m
1
3
(
multiplet), and br. (broad). The carbon nuclear magnetic resonance ( C NMR) spectroscopic data were
recorded with a JEOL AL-300 at 75 MHz and JEOL JMN-LA500 at 125 MHz. Chemical shifts are
reported relative to CDCl (δ = 77.0 ppm) and DMSO-d (δ = 39.7 ppm). Infrared spectra were recorded
3
6
using the attenuated total reflectance (ATR) method with a Shimadzu FTIR-8000 spectrophotometer and
Technologies DuraScop. Low and high resolution mass spectra were recorded with JEOL JMS-700
spectrometers by using a direct inlet system.
Synthesis of carbazoles
1
7
1
,3-Dihydroxy-2-methylcarbazole (5): To a stirred solution of O-triflate-carbazole 17 (100 mg, 0.29
o
mmol) in CH
2
Cl
2
(4 mL) was added BBr
3
(85 µL, 0.87 mmol) at -78 C. After stirring at an ambient
temperature for 12 h, the reaction mixture was poured into an ice water. The mixture was extracted with
EtOAc. The EtOAc phase was washed with water and brine, dried with Na SO , and concentrated under
2
4
reduced pressure. The residue was used for the next step without further purification. To a solution of the
o
residue in THF (2 mL) was added LiAlH
4
(33.0 mg, 0.87 mmol) at -78 C. After stirring at rt for 5 h, the
reaction mixture was quenched with EtOAc and water. The mixture was filtered off using Celite pad, and
then the Celite pad washed with EtOAc. The filtrate was extracted with EtOAc. The EtOAc phase was
2 4
washed with water and brine, dried with Na SO , and concentrated under reduced pressure. The residue
was purified by column chromatography (silica gel, 20 g; EtOAc/hexane, 1/4 v/v) to give the
o
1
,3-dihydroxycarbazole (5) (37 mg, 0.17 mmol, 60%) as a dark solid, mp 196-198 C (EtOAc-hexane).
-
1 1
IR (ATR): 3456, 3221 cm . H NMR (300 MHz, DMSO-d
6
): δ 2.16 (s, 3H), 6.97 (s, 1H), 7.01 (t, J = 7.7
Hz, 1H), 7.24 (t, J = 7.5 Hz, 1H), 7.40 (d, J = 7.7 Hz, 1H), 7.83 (d, J = 7.7 Hz, 1H), 8.76 (br. s, 1H), 8.79
1
3
(
br. s, 1H), 10.33 (br. s, 1H). C NMR (75 MHz, DMSO-d
6
): δ 9.91, 96.35, 110.18, 111.45, 118.15,
+
1
20.01, 120.93, 123.16, 124.73, 124.95, 139.74, 140.64, 149.76. MS (EI): m/z 213[M] . HRMS (EI):
calcd. for C13
2
H11NO 213.0790; found 213.0754.
General procedure for the synthesis of N-phenylanilines

2
-Methoxy-N-phenylaniline (20a): To a stirred solution of o-anisidine (18a) (1 g, 8.12 mmol) in dry
toluene (10 mL) was added to a solution of bromobenzene (19a) (1.27 g, 8.12 mmol), Pd(OAc) (73 mg,
(1.57 g, 11.37 mmol) at rt, and then the
2
0
2 3
.32 mmol), rac-BINAP (202 mg, 0.32 mmol) and K CO
o
mixture was heated at 140 C for 12 h. After cooling to an ambient temperature, the mixture was
quenched with water. The resulting mixture was extracted with EtOAc. The EtOAc phase was washed
2 4
with water and brine, dried with Na SO , and concentrated under reduced pressure. The residue was
purified by column chromatography (silica gel, 20 g; EtOAc/hexane, 1/19 v/v) to give the
-
1 1
2
-methoxy-N-phenylaniline (20a) (1.60 g, 8.03 mmol, 99%) as a colorless oil. IR (ATR): 3406 cm . H
NMR (300 MHz, CDCl ): δ 3.90 (s, 3H), 6.15 (br. s, 1H), 6.82-6.97 (m, 4H), 7.14-7.17 (m, 2H),
3
1
3
7
.25-7.32 (m, 3H). C NMR (75 MHz, CDCl
3
): δ 55.59, 110.50, 114.65, 118.55, 119.86, 120.78, 121.11,
+
1
29.24, 132.96, 142.69, 148.25. MS (EI): m/z 199 [M] . HRMS (EI): calcd. for C13
H
13NO: 199.0997;
found 197.0951.
-Methoxy-N-phenylaniline (20b): The same procedure as above was carried out using p-anisidine
18b) (500 mg, 4.06 mmol), bromobenzene (19a) (637 mg, 4.06 mmol), Pd(OAc) (36 mg, 0.16 mmol),
4
(
2
rac-BINAP (101 mg, 0.16 mmol) and K
2
CO
3
(786 mg, 5.68 mmol) to give the
o
4
-methoxy-N-phenylaniline (20b) (670 mg, 3.36 mmol, 83%) as a white solid, mp 105-107 C
3
0
o
-1 1
(
2 3
Et O-hexane) (Lit., mp 104-106 C). IR (ATR): 3386 cm . H NMR (300 MHz, CDCl ): δ 3.85 (s, 3H),
1
3
5
3
.54 (br. s, 1H), 6.86-6.97 (m, 5H), 7.10-7.15 (m, 2H), 7.24-7.30 (m, 2H). C NMR (75 MHz, CDCl ): δ
+
5
5.56, 114.64, 115.61, 119.54, 122.18, 129.28, 135.70, 145.14, 155.25. MS (EI): m/z 199 [M] . HRMS
(
EI): calcd. for C13
H
13NO: 199.0997; found 197.0971.
-Methoxy-N-(4-methoxyphenyl)aniline (20c): The same procedure as above was carried out using
(36 mg,
(786 mg, 5.68 mmol) to give the
2
p-anisidine (18b) (500 mg, 4.06 mmol), o-bromoanisole (19b) (759 mg, 4.06 mmol), Pd(OAc)
2
0
.16 mmol), rac-BINAP (101 mg, 0.16 mmol) and K
2 3
CO
o
2
-methoxy-N-(4-methoxyphenyl)aniline (20c) (720 mg, 3.21 mmol, 79%) as a white solid, mp 70-71 C
3
1
o
-1 1
(Et
2
O-hexane) (Lit., mp 70-71 C). IR (ATR): 3379 cm . H NMR (300 MHz, CDCl
3
): δ 3.81 (s, 3H),
3
.91 (s, 3H), 5.98 (br. s, 1H), 6.75-6.91 (m, 5H), 7.05 (dd, J = 1.8, 9.0 Hz, 1H), 7.13 (d, J = 9.0 Hz, 2H).
1
3
C NMR (75 MHz, CDCl
3
): δ 55.52(×2), 110.16, 112.55, 114.50, 118.50, 120.88, 122.71, 135.00, 135.37,
+
1
47.33, 155.25. MS (EI): m/z 229 [M] . HRMS (EI): caclcd. for C14
H
15NO
2
: 229.1103; found 229.1112.
General procedure for the synthesis of carbazoles
-Methoxycarbazole (21a): To a stirred solution of 2-methoxy-N-phenylaniline (20a) (200 mg, 1.00
1
2
mmol) in acetic acid (5 mL) was added to a solution of Pd(OAc) (270 mg, 1.21 mmol) at rt, and then the
o
mixture was heated at 130 C for 12 h. After cooling the reaction to an ambient temperature, the mixture
was quenched with water. The resulting mixture was extracted with EtOAc. The EtOAc phase was
2 4
washed with water and brine, dried with Na SO , and concentrated under reduced pressure. The residue

was purified by column chromatography (silica gel, 20 g; EtOAc/hexane, 3/97 v/v) to give the
o
1
-methoxycarbazole (21a) (120 mg, 0.61 mmol, 61%) as a white solid, mp 71-72 C (Et
2
O-hexane)
2
0
o
-1 1
(
Lit., mp 70-72 C). IR (ATR): 3413 cm . H NMR (300 MHz, CDCl
3
): δ 4.00 (s, 3H), 6.90 (d, J = 7.9
Hz, 1H), 7.17 (t, J = 7.9 Hz, 1H), 7.21-7.27 (m, 1H), 7.39-7.46 (m, 2H), 7.70 (d, J = 7.9 Hz, 1H), 8.07 (d,
1
3
J = 7.9 Hz, 1H), 8.28 (br. s, 1H). C NMR (75 MHz, CDCl
3
): δ 55.46, 105.89, 110.89, 112.83, 119.32,
+
1
19.70, 120.51, 123.61, 124.29, 125.64, 129.75, 139.14, 145.63. MS (EI): m/z 197 [M] . HRMS (EI):
caclcd. for C13
H11NO: 197.0841; found 199.0817.
3
4
3
-Methoxycarbazole (21b): The same procedure as above was carried out using
-methoxy-N-phenylaniline (20b) (300 mg, 1.51 mmol), Pd(OAc)
2
(405 mg, 1.81 mmol) to give the
o
-methoxycarbazole (21b) (93 mg, 0.47 mmol, 31%) as a white solid, mp 151-153 C (EtOAc-hexane)
2
1
o
-1 1
(
Lit., mp 146-148 C). IR (ATR): 3421 cm . H NMR (300 MHz, CDCl
3
): δ 7.07 (dd, J = 2.5, 7.9 Hz,
1
H), 7.18-7.24 (m, 1H), 7.34 (d, J = 7.9 Hz, 1H), 7.40 (d, J = 2.5 Hz, 1H), 7.56 (d, J = 2.5 Hz, 1H), 7.92
1
3
(
br. s, 1H), 8.03 (d, J = 7.9 Hz, 1H). C NMR (75 MHz, CDCl
3
): δ 56.07, 103.18, 110.72, 111.27, 115.05,
+
1
19.04, 120.24, 123.35, 123.79, 125.78, 134.36, 140.27, 153.89. MS (EI): m/z 197 [M] . HRMS (EI):
calcd. for C13
H11NO: 197.0841; found 199.0860.
1
,6-Dimethoxycarbazole (21c): The same procedure as above was carried out using
4
-methoxy-N-(2-methoxyphenyl)aniline (20c) ( (300 mg, 1.31 mmol), Pd(OAc)
2
(352 mg, 1.57 mmol) to
o
give the 1,6-dimethoxycarbazole (21c) (147 mg, 0.65 mmol, 49%) as a white solid, mp 120-121 C
2
3
o
-1 1
(
EtOAc-hexane) (Lit., mp 118-120 C). IR (ATR): 3410 cm . H NMR (300 MHz, CDCl
3
): δ 3.90 (s,
3
H), 4.01 (s, 3H), 6.89 (d, J = 7.9 Hz, 1H), 7.06 (dd, J = 2.5, 7.9 Hz, 1H), 7.13 (t, J = 7.9 Hz, 1H), 7.36 (d,
1
3
J = 7.9 Hz, 1H), 7.52 (d, J = 2.5 Hz, 1H), 7.63 (d, J = 7.9 Hz, 1H), 8.12 (br. s, 1H). C NMR (75 MHz,
CDCl
3
): δ 103.18, 105.74, 111.62, 112.75, 115.08, 119.32, 124.05, 124.27, 130.61, 134.11, 145.75,
+
1
53.81. MS (EI): m/z 227 [M] . HRMS (EI): calcd. for C14
H13NO
2
: 227.0946; found 227.0980.
General procedure for the synthesis of hydroxycarbazoles
1
2 2
-Hydroxycarbazole (7): To a solution of 1-methoxycarbazole (21a) (85 mg, 0.43 mmol) in CH Cl (2
o
mL) was added BBr
3
(169 µL, 1.72 mmol) at -78 C. After stirring at rt for 5 h, the reaction mixture was
poured into ice water. The mixture was extracted with EtOAc. The EtOAc phase was washed with water
and brine, dried with Na SO , and concentrated under reduced pressure. The residue was purified by
column chromatography (silica gel, 20 g; EtOAc/hexane, 1/4 v/v) to give the 1-hydroxycarbazole (7) (51
2
4
o
20
o
mg, 0.28 mmol, 65%) as a white solid, mp 165-167 C (CHCl
3
) (Lit., mp 163-165 C). IR (ATR): 3429,
): δ 4.99 (br. s, 1H), 6.83 (d, J = 7.9 Hz, 1H), 7.08 (t, J = 7.9 Hz,
H), 7.21-7.26 (m, 1H), 7.40-7.48 (m, 2H), 7.69 (d, J = 7.9 Hz, 1H), 8.06 (d, J = 7.9 Hz, 1H), 8.24 (br. s,
-
1 1
3
1
1
1
197 cm . H NMR (300 MHz, CDCl
3
1
3
H). C NMR (75 MHz, CDCl
3
): δ 110.63, 110.94, 113.25, 119.44, 119.68, 120.59, 123.57, 125.34,
+
25.93, 128.95, 139.41, 141.00. MS (EI): m/z 183 [M] . HRMS (EI): calcd. for C12
H
9
NO: 183.0684;

found 183.0706.
-Hydroxycarbazole (9): The same procedure as above was carried out using 3-methoxycarbazole (21b)
90 mg, 0.46 mmol), BBr (179 µL, 1.83 mmol) to give the 3-hydroxycarbazole (9) (50 mg, 0.27 mmol,
3
(
3
o
23
o
6
0%) as a brown solid, mp 234-236 C (EtOAc-hexane) (Lit., mp 260-261 C). IR (ATR): 3398, 3151
-
1 1
3
cm . H NMR (300 MHz, CDCl ): δ 6.87 (dd, J = 2.6, 7.8 Hz, 1H), 7.05 (t, J = 7.8 Hz, 1H), 7.25-7.32 (m,
1
3
2
H), 7.37-7.40 (m, 2H), 7.96 (d, J = 7.8 Hz, 1H), 8.90 (br. s, 1H), 10.85 (br. s, 1H). C NMR (75 MHz,
CDCl
3
), δ 104.83, 110.81, 111.28, 114.99, 117.70, 120.06, 122.27, 123.03, 125.18, 133.74, 140.39,
+
1
1
1
1
50.37. MS (EI): m/z 183 [M] . HRMS (EI): calcd. for C12
H
9
NO: 183.0684; found 183.0666.
,6-Dihydroxycarbazole (11): The same procedure as above was carried out using
,6-dimethoxycarbazole (21c) (147 mg, 0.65 mmol), BBr
3
(253 µL, 2.59 mmol) to give the
o
,6-dihydroxycarbazole (11) (63 mg, 0.28 mmol, 49%) as a dark soliid, mp 200-202 C (EtOAc-hexane).
-
1 1
IR (ATR): 3413, 3278 cm . H NMR (300 MHz, DMSO-d
6
): δ 6.74 (d, J = 7.5 Hz, 1H): 6.81-6.88 (m,
2
H), 7.25 (d, J = 7.5 Hz, 1H), 7.31 (d, J = 2.0 Hz, 1H), 7.39 (d, J = 7.5 Hz, 1H), 8.84 (br. s, 1H), 9.62 (br.
1
3
s, 1H), 10.61 (br. s, 1H). C NMR (75 MHz, DMSO-d
6
): δ 105.47, 110.56, 112.12, 112.67, 115.59,
+
1
19.57, 124.22, 124.55, 130.75, 134.45, 143.48, 150.50. MS (EI): m/z 199 [M] . HRMS (EI): calcd. for
C
12
H
9
NO
2
: 199.0633; found 199.0627.
Radical scavenging assays
DPPH (047-04051) and ABTS (018-10311) were purchased from Wako Pure Chemical Industries, Ltd.
All the data were statistically analyzed with student’s test, and the data, whose association was calculated
less than 0.01 of p score, took significantly difference each other. Inhibition concentrations at which 50%
of the radicals were scavenged (IC50 values) were calculated to evaluate the antioxidant activity.
2
5
DPPH radical scavenging assay: Stock solutions were prepared for DPPH (200 µM) and the test
samples (100 µM) in MeOH. All solutions were kept cool and in the dark until use. For each sample
different aliquots of the stock solutions were taken and the volume was adjusted to 3 mL in MeOH. The
reaction was started by adding 1 mL of 200 µM DPPH stock solution (final DPPH concentration 50 µM).
After shaking for 30 min at ambient temperature in the dark, the absorbance was measured at 517 nm in
MeOH using a Shimadzu UV-2550 spectrophotometer. DPPH radical scavenging capacity was expressed
as µM, the results were expressed as means ± S.E. (n = 3 or higher, and the results of one of at least three
similar experiments are shown). DPPH radical scavenging (capacity) (%) = (Acontrol –Asample)/ Acontrol
x
100%, where Acontrol is the absorbance of the control (DPPH radical without test sample) and Asample is the
absorbance of the test sample (DPPH radical with test sample). The control contain all reagents except the
scavenger.
+
26
+
ABTS radical scavenging assay: The procedure to prepare the ABTS radical stock solution was
2
6
modified slightly. Sufficient amounts of the diammonium salts of ABTS and K
2
S2
O8 were dissolved in

5
.00 mL H
2
O to achieve concentrations of 7.00 and 1.40 mM, respectively. This solution was kept in the
+
dark for at least 16 h to form ABTS radical, then diluted to 100 mL with MeOH so that the solution had
an absorbance or Acontrol of ~0.80 at 734 nm using a Shimadzu UV-2550 spectrophotometer. Various
+
concentrations of carbazoles were added to ABTS radical solution at ambient temperature to reach a
+
stable absorbance (Acontrol). Then, the percentage of ABTS radical scavenged was calculated as follows.
+
ABTS radical scavenging (capacity) (%) = Acontrol –Asample)/ Acontrol x 100%, where Acontrol is the
+
absorbance of the control (ABTS radical without test sample) and Asample is the absorbance of the test
+
sample (ABTS radical with test sample). The control contain all reagents except the scavenger. The
+
concentration-percentage of scavenged ABTS radical relationships for each reagent, which were derived
from their IC50 values, were fitted to a four-parameter logistic equation using a non-linear curve-fitting
program (KaleidaGraph; Synergy Software, Reading, PA). Where appropriate, the results were expressed
as means ± S.E. (n = 3 or higher, and the results of one of at least three similar experiments are shown).
3
5
Bond dissociation (BD) energy and HOMO energy
Carbazoles and their corresponding radicals were calculated by density functional theory (DFT) at
ωB97X-D/6-31+G(d). The bond dissociation energies (BD energies) were calculated as the following
energies: BD energies = Energy of antioxidant radical + Energy of H radical - Energy of antioxidant.
ACKNOWLEDGEMENTS
This work was partly supported by a Grant-in Aid for Scientific Research (C) (No. 17590025) of the
Japan Society for the Promotion of Science (JSPS).
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