Synthesis of novel derivatives of murrayafoline a and their inhibitory effect on LPS-stimulated production of pro-inflammatory cytokines in bone marrow-derived dendritic cells

Article abstract of DOI:10.1007/s12272-013-0100-z

A series of N-substituted-1,2,3-triazole murrayafoline A derivatives were successfully synthesized using click azide-alkyne Huisgen cycloaddition reaction between 1-methoxy-3-methyl-9-(3-azido)-propyl-9H-carbazole and substituted alkynes. Their chemical structures were confirmed by ¹H, ¹³C NMR and HR-ESI-MS spectral data. In addition, the interested effects on LPS-stimulated production of pro-inflammatory cytokines in bone marrow-derived dendritic cells of synthetic murrayafoline A derivatives were also investigated. Our results indicated that murrayafoline A derivatives containing 1,2,3-triazole nucleus potentially possessed anti-inflammatory action through inhibiting production of IL-6, IL-12 p40 and TNF-α.

Full text of DOI:10.1007/s12272-013-0100-z

Arch. Pharm. Res. (2013) 36:832–839
DOI 10.1007/s12272-013-0100-z
RESEARCH ARTICLE
Synthesis of novel derivatives of murrayafoline A and their
inhibitory effect on LPS-stimulated production of pro-
inflammatory cytokines in bone marrow-derived dendritic cells
•
Tran Thi Thu Thuy Nguyen Manh Cuong Tran Quoc Toan Ngo Ngoc Thang
•
•
•
•
•
•
Bui Huu Tai Nguyen Xuan Nhiem Hye-Jin Hong Sohyun Kim
•
•
Stephanie Legoupy Young Sang Koh Young Ho Kim
•
Received: 22 October 2012 / Accepted: 13 March 2013 / Published online: 6 April 2013
Ó The Pharmaceutical Society of Korea 2013
Abstract A series of N-substituted-1,2,3-triazole mur-
rayafoline A derivatives were successfully synthesized using
click azide–alkyne Huisgen cycloaddition reaction between
1-methoxy-3-methyl-9-(3-azido)-propyl-9H-carbazole and
substituted alkynes. Their chemical structures were con-
Introduction
The first isolation of naturally carbazole alkaloid murraya-
nine was reported in 1964 from the leaves of curry-leaf tree
(Murraya koenigii) (Burman et al. 1964). Since then a large
number of carbazole alkaloids have been isolated from
higher plants, fungi, microorganisms and recently from other
natural sources, for example, the blue–green algae (Knoelker
and Reddy 2002). This class of structure possesses a variety
of biological activities including anti-microbial, anti-fungal,
anti-tumor, anti-inflammatory, anti-platelet aggregation,
anti-oxidative and so on. (Ito et al. 2000; Itoigawa et al.
2000). The synthesis of carbazole derivatives has been a
strong interest of many research groups because of their
potentially useful biological activities. The anti-inflamma-
tory activity of carbazole derivatives has attracted wide-
spread attention. Among these derivatives (Fig. 1), 1-ethyl-
8-n-propyl-1,2,3,4-tetrahydrocarbazole-1-acetic acid (1)
was discovered as a novel anti-inflammatory agent.
6-Chloro-1,2,3,4-tetrahydrocarbazole-2-carboxylic acid (2)
was clinically active in the treatment of acute gout. Car-
profen (3), a cyclooxygenase inhibitor, was approved for use
in animals as an anti-inflammatory drug with weak ulcero-
genic activity. Murrayafoline A (4), isolated from the root of
several species of the genus Murraya (Itoigawa et al. 2000),
Glycosmis (Cuong et al. 2008) and Clausena (Rutaceae) (Cui
et al. 2002), exhibits strong fungicidal activity against
Cladosporium cucumerinum and growth inhibitory activity
on human fibrosarcoma HT-1080 cells, cell cycle M-phase
inhibitory, and apoptosis inducing activities on mouse
tsFT210 cells (Cui et al. 2002). Murrayafoline A was found
to attenuate the Wnt/b-catenin pathway by promoting the
degradation of intracellular b-catenin proteins (Choi et al.
2010). However, there are few of researches on the synthesis
and inflammatory activity of murrayafoline A derivatives
1
firmed by H, 13C NMR and HR-ESI–MS spectral data. In
addition, the interested effects on LPS-stimulated production
of pro-inflammatory cytokines in bone marrow-derived
dendritic cells of synthetic murrayafoline A derivatives were
also investigated. Our results indicated that murrayafoline A
derivatives containing 1,2,3-triazole nucleus potentially
possessed anti-inflammatory action through inhibiting pro-
duction of IL-6, IL-12 p40 and TNF-a.
Keywords Murrayafoline A Á 1,2,3-triazole Á TNF-a Á
IL-6 Á IL-12 p40
T. T. T. Thuy (&) Á N. M. Cuong Á T. Q. Toan Á N. N. Thang
Institute of Natural Products Chemistry, Vietnam Academy of
Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi,
Vietnam
e-mail: thuytran.inpc@gmail.com
B. H. Tai Á N. X. Nhiem Á Y. H. Kim (&)
College of Pharmacy, Chungnam National University, Daejeon
305-764, South Korea
e-mail: yhk@cnu.ac.kr
H.-J. Hong Á S. Kim Á Y. S. Koh
School of Medicine and Brain Korea 21 Program, and Institute
of Medical Science, Jeju National University, Jeju 690–756,
South Korea
S. Legoupy
´
´
LUNAM Universite, Universite d’Angers, CNRS UMR 6200,
Laboratoire MOLTECH-Anjou, 2 Bd Lavoisier, 49045 Angers
Cedex, France
123

Anti-inflammatory activity of N-substituted-1,2,3-triazole murrayafoline A derivatives
833
Isolation of murrayafoline A
Cl
CO2H
Dried powdered roots (3 kg) of Glycosmis stenocarpa were
extracted with MeOH, filtered and evaporated in vacuo.
The suspension of the methanol residue in MeOH/H2O
(1:1) was successively fractioned with n-hexane, chloro-
form, and butanol to give hexane (80 g), chloroform
(50 g), and butanol fractions. The hexane fraction (80 g)
was chromatographed on silica gel (350 g, Merck silica
80–120 mesh) using a gradient of hexane and EtOAc as
eluent to give 12 fractions. Fraction 4 was further rechro-
matographed on flash silica gel (hexane/EtOAc 10/1 as
eluent) to yield murrayafoline A (9 g, 3 %).
N
H
N
H
CO2H
2
1
Cl
N
H
N
H
OMe
OH
O
4
3
Synthesis of 1-methoxy-3-methyl-9-(3-bromo)-propyl-
9H-carbazole (5) and 1-methoxy-3-methyl-9-(propen-
2-yl)-9H-carbazole (6)
Fig. 1 Several carbazole derivatives possessing highly biological
activities
(Cuong et al. 2008). Otherwise, a wide variety of pharmaco-
logical properties have been shown to be associated with
1,2,3- and 1,2,4-triazole nucleus (Choi et al. 2010) which
including antibacterial (Prakash et al. 2004; Sztanke et al.
2006), antifungal (Lebouvier et al. 2007; Liu et al. 2008),
antioxidant (Kamotra et al. 2007), anticancer (Sztanke et al.
2008), anticonvulsant (Almasirad et al. 2004), analgesic
(Gilani et al. 2008), antiviral (De Clercq 2002; Dong et al.
2000), and anti-inflammatory (Buckler et al. 1978; Shafi et al.
2012) activities. Recently, the synthesis, anti-bacterial and
antifungal activities of imidazole and 1,2,4-triazole-based
N-substituted carbazole derivatives have been reported
(Zhang et al. 2010). However, their anti-inflammatory effects
of them have not been extensively studied. Therefore, we
describehereinthesynthesisandanti-inflammatoryactivityof
N-substituted-1,2,3-triazole murrayafoline A derivatives
(8a-h) which have not been reported in the literature.
A solution of murrayafoline A (2 g, 9.5 mmol) in anhy-
drous THF (50 mL), NaH (0.3 g, 11.9 mmol), and 1,3-
dibromopropane (6 mL, 28.5 mmol) were added into a
100 mL round bottom flask under inert gas. Reaction
mixture was stirred at room temperature for about 72 h
under inert gas until the starting carbazole had been com-
pletely consumed (by TLC monitoring). THF was removed
in vacuo and then the mixture was cooled. Water (50 mL)
was added and the solution was extracted with CH2Cl2
(3 9 40 mL). All the combined organic phases were
washed by water (30 mL), dried over Na2SO4 and then
evaporated under reduced pressure. The residue was sep-
arated with a silica gel column chromatography (n-hexane/
ethyl acetate, 6/1) to give 5 (2.6 g, 83 %) and 6 (0.26 g,
1
11 %) as white solids. 5: H NMR (500 MHz, CDCl3) dH
7.99 (d, J = 7.5 Hz, 1H), 7.48 (s, 1H), 7.43 (m, 2H), 7.18
(dd, J = 7.5, 1.5 Hz), 6.73 (s, 1H), 4.69 (t, J = 6.5 Hz,
2H), 3.96 (s, 3H), 3.37 (t, J = 6.5 Hz, 2H), 2.50 (s, 3H),
2.39 (quin., J = 6.5 Hz, 2H). 13C NMR (125 MHz,
CDCl3) dC 146.4, 140.9, 129.2, 127.7, 125.6, 125.0, 122.9,
120.3, 118.8, 112.7, 108.8, 108.7, 55.5, 43.6, 33.9, 30.9,
21.6. 6: 1H NMR (500 MHz, CDCl3) dH 8.01 (d,
J = 7.5 Hz, 1H), 7.49 (s, 1H), 7.40 (d, J = 7.5 Hz, 1H),
7.33 (d, J = 7.5 Hz, 1H), 7.18 (d, J = 7.5 Hz, 1H), 6.74 (s,
1H), 6.01 (m, 1H), 5.21 (d, J = 5.0 Hz, 2H), 5.06 (dd,
J = 10.0, 1.5 Hz, 1H), 4.93 (dd, J = 17.0, 1.5 Hz, 1H),
3.95 (s, 3H), 2.51 (s, 3H). 13C NMR (125 MHz, CDCl3) dC
146.6, 140.9, 134.4, 129.1, 128.0, 125.4, 124.9, 123.1,
120.2, 118.7, 115.6, 112.7, 109.2, 109.1, 55.8, 47.5, 21.7.
Materials and methods
General experimental and procedures
Melting points were recorded on Buchi B-540 melting
point appraratus. 1H NMR and 13C NMR were recorded on
a Bruker AV 500 using TMS as an internal standard at 500
and 125 MHz respectively. NMR spectra were recorded in
CDCl3 or CD3OD at room temperature. J values refer to
coupling constants, and signal splitting patterns are
described as singlet (s), doublet (d), triplet (t), quartet (q),
quintet (quin.), multiplet (m), or combination thereof. The
HR-ESI–MS were obtained from an Agilent 6530 Accu-
rate-Mass Q-TOF LC/MS system. TLC was performed on
silica gel plates 60 F254 (0.25 mm, Merck) and column
chromatography using silica gel (230–400 mesh, Merck).
Synthesis of 1-methoxy-3-methyl-9-(3-azido)-propyl-
9H-carbazole (7)
A solution of 5 (2.5 g, 7.5 mmol) in acetonitrile (80 mL),
sodium azide (0.75 g, 11.25 mmol) was added into a
123

834
T. T. T. Thuy et al.
250 mL round bottom flask. The mixture was heated under
reflux conditions for 72 h until the starting material (5) had
been completely consumed (by TLC monitoring). The
mixture was cooled and ethyl acetate (50 mL) was added.
The obtained mixture was washed with Na2CO3 saturated
solution (3 9 50 mL), brine (40 mL) and water (50 mL).
The combined organic layers were then dried with Na2SO4
and evaporated under reduced pressure. The residue was
purified on a silica gel column chromatography (n-hexane/
ethyl acetate, 5:1) to give 7 (2.11 g, 96 %) as white solid.
1H NMR (500 MHz, CDCl3) dH 8.00 (d, J = 8.0 Hz, 1H),
7.48 (s, 1H), 7.42 (dd, J = 8.0, 7.5 Hz, 1H), 7.37 (d,
J = 8 Hz, 1H), 7.18 (dd, J = 8.0, 7.5 Hz, 1H), 6.73 (s,
1H), 4.64 (t, J = 6.5 Hz, 2H), 3.97 (s, 3H), 3.25 (t,
J = 6.5 Hz, 2H), 2.51 (s, 3H), 2.10 (quin., J = 6.5 Hz,
2H). 13C NMR (125 MHz, CDCl3) dC 146.4, 140.9, 129.2,
127.7, 125.6, 124.9, 122.9, 120.3, 118.8, 112.7, 108.7,
108.6, 55.5, 48.9, 42.3, 29.9, 21.6.
temperature. After the reaction came to the end (by TLC
monitoring), solution of NH4OH (5 mL) was added and the
mixture was extracted with ethyl acetate (2 9 4 mL). The
combined organic layers were washed by water (5 mL),
dried with Na2SO4, and evaporated under reduced pressure.
The residue was purified with a silica gel column chro-
matography (n-hexane/ethyl acetate) to give 8a, 8b, 8d,
8f-h (86–94 %).
1-(3-(1-Methoxy-3-methyl-9H-carbazol-9-yl)propyl)-1H-
1,2,3-triazole-4-carboxylic acid (8a)
1H NMR (500 MHz, CDCl3) dH 8.01 (d, J = 7.5 Hz, 1H),
7.70 (s, 1H), 7.49 (s, 1H), 7.41 (m, 2H), 7.25 (d,
J = 7.5 Hz, 1H), 7.19 (d, J = 7.0 Hz, 1H), 6.74 (s, 1H)
4.69 (t, J = 7.0 Hz, 2H), 4.37 (t, J = 7.0 Hz, 2H), 3.91 (s,
3H), 2.51 (overlapped, 5H). 13C NMR (125 MHz, CDCl3)
dC 146.3, 140.6, 129.5, 127.6, 125.7, 125.0, 123.5, 123.1,
120.4, 119.0, 112.8, 108.8, 108.6, 55.5, 47.9, 42.3, 30.9,
21.7.
Protection of propargyl amine
Propargyl amine (0.4 g, 7.26 mmol) and (Boc)2O (1.58 g,
7.26 mmol) were dissolved in THF (8 mL). Distilled water
(8 mL) were added and the mixture was stirred at r.t. in
48 h. Ethyl acetate (3 9 10 mL) was added and the
organic layer was separated. The combined organic layers
were washed with 5 % NaHCO3 solution (10 mL), water
(2 9 10 mL), dried with Na2SO4, and concentrated under
reduced pressure. The residue was purified with a column
of silica gel (n-hexane/EtOAc, 95:5) to give a white solid
(94 %).
t-Butyl 1-(3-(1-methoxy-3-methyl-9H-carbazol-9-
yl)propyl)-1H-1,2,3-triazol-4-ylcarbamate (8b)
1H NMR (500 MHz, CDCl3) dH 8.01 (d, J = 7.5 Hz, 1H),
7.49 (s, 1H), 7.41 (t, J = 7.5 Hz, 1H), 7.32 (br, 1H), 7.26
(d, J = 7.5 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 6.74 (s, 1H),
4.69 (t, J = 7 Hz, 2H), 4.35 (d, J = 6 Hz, 2H), 4.30
(t, J = 7 Hz, 2H), 3.93 (s, 3H), 2.51 (s, 3H), 2.49 (quin.,
J = 7.0 Hz, 2H), 1.43 (s, 9H). 13C NMR (125 MHz,
CDCl3) dC 146.3, 145.3, 140.6, 129.5, 127.6, 125.67,
125.0, 123.0, 121.8, 120.4, 119.0, 112.8, 108.8, 108.6,
55.5, 48.1, 42.3, 36.1, 30.8, 28.4, 21.6. HR-ESI–MS:
484.2143 [M?Cl]- (calcd. 484.2115 for C25H31N5O3Cl).
Protection of propargyl alcohol
A solution of propargyl alcohol (0.9 mmol) in DMF
(0.5 mL), t-butyldiphenylsilyl chloride (0.3 mL) and
imidazole (74 mg) were added into a 10 mL round bottom
flask. The mixture was stirred overnight at room temper-
ature and water (0.5 mL) was added. After extraction with
diethyl ether (4 9 1 mL), the combined organic extracts
were dried (Na2SO4), the solvent was removed under
reduced pressure and the residue was purified by column
chromatography (n-hexane/ethyl acetate; 95:5) to give a
colorless oil (96 %).
1-(3-(1-Methoxy-3-methyl-9H-carbazol-9-yl)propyl)-1H-
1,2,3-triazol-4-amine (8c)
Solution of 8b (150 mg, 0.34 mmol) in CH2Cl2 (1 mL) and
TFA (1 mL) was added into a 10 mL round bottom flask.
The mixture was stirred in 2 h at room temperature and
evaporated under reduced pressure. EtOAc (3 mL) were
added to dissolve the residue and the solution obtained was
washed with 0.3 N NaOH solution (2 mL), brine (2 mL)
and water (3 mL). The organic layer was dried with
Na2SO4 and evaporated of EtOAc. The residue was puri-
fied with a column of silica gel (n-hexane : EtOAc, 4:1) to
give 8c as colorless oil (75 %). 1H NMR (500 MHz,
CD3OD) dH 8.00 (d, J = 8.0 Hz, 1H), 7.76 (s, 1H), 7.48
(s, 1H), 7.40 (m, 2H), 7.16 (m, 1H), 6.83 (s, 1H), 4.72
(t, J = 7.0 Hz, 2H), 4.44 (t, J = 7.0 Hz, 2H), 3.98 (s, 2H),
Click reaction using for synthesis of 1,2,3-triazole
derivatives of murrayafoline A (8a, 8b, 8d, 8f-h)
A solution of azide 7 (1 eq, 0.1 g), alkyne (1.2 eq) in
t-BuOH (2 mL), solution of sodium ascorbate (200 mol%)
in water (1 mL) and CuSO4Á5H2O (20 mol%) in water
(1 mL) were added into a 10 mL round bottom flask under
inert gas. The mixture was stirred overnight at room
3.95 (s, 3H), 2.50 (s, 3H), 2.46 (quin., J = 7.0 Hz, 2H). 13
C
NMR (125 MHz, CD3OD) dC 147.8, 142.1, 130.7, 128.9,
123

Anti-inflammatory activity of N-substituted-1,2,3-triazole murrayafoline A derivatives
835
126.6, 126.2, 124.3, 124.0, 121.1, 119.9, 113.5, 109.9,
109.8, 56.1, 43.4, 36.8, 31.9, 21.7.
1H), 6.72 (s, 1H), 4.73 (t, J = 7.0 Hz, 2H), 4.34 (t,
J = 7.0 Hz, 2H), 3.89 (s, 3H), 2.56 (quin., J = 7.0 Hz,
2H), 2.49 (s, 3H). 13C NMR (125 MHz, CDCl3) dC 146.3,
140.6, 131.5, 131.4, 131.2, 129.6, 129.3, 128.7, 127.6,
125.7, 125.1, 125.0, 124.7, 124.6, 123.1, 123.0, 122.4,
122.4, 120.5, 119.1, 112.8, 108.9, 108.6, 55.5, 48.2, 42.2,
30.7, 21.6. HR-ESI–MS: 499.1535 [M?Cl]- (calcd.
499.1512 for C26H23F3N4OCl).
9-(4((tert-Butyldiphenylsilyloxy)methyl)-1H-1,2,3-triazol-
1-yl)propyl)-1-methoxy-3-methyl-9H-carbazole (8d)
8d was prepared by click reaction and directly used to the
deprotection without any purification.
(1-(3-(1-Methoxy-3-methyl-9H-carbazol-9-yl)propyl)-1H-
9-(3-(4-((4-(2,5-Difluorobenzyl)piperazin-1-yl)methyl)-1H-
1,2,3-triazol-1-yl)propyl)-1-methoxy-3-methyl-9H-
carbazole (8h)
1,2,3-triazol-4-yl)methanol (8e)
The solutions of silylated alcohol 8d (0.4 mmol) in THF
(4 mL), and TBAF 1 M in THF (800 lL) were added into
a 10 mL round bottom flask. The reaction mixture was
stirred at room temperature for 30 min then saturated
NH4Cl and ethyl acetate were added. The organic layer was
separated, dried over Na2SO4 and concentrated under
reduced pressure. The residue was purified by column
chromatography with dichloromethane/methanol to give 8e
1H NMR (500 MHz, CDCl3) dH 8.01 (d, J = 7.5 Hz, 1H),
7.49 (s, 1H), 7.40 (t, J = 7.5 Hz, 1H), 7.33 (s, 1H), 7.25 (d,
J = 7.5 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 7.09 (m, 1H),
6.95 (ddd, J = 9.0, 9.0, 4.5 Hz, 1H), 6.89 (m, 1H), 6.74 (s,
1H), 4.70 (t, J = 7.0 Hz, 2H), 4.32 (t, J = 7.0 Hz, 2H),
3.94 (s, 3H), 3.67 (s, 2H), 3.54 (s, 2H), 2.49 (s, 3H), 2.48
(m, overlapped, 10H). 13C NMR (125 MHz, CDCl3) dC
158.5 (d, JC–F = 12.6 Hz), 156.5 (d, JC–F = 13.0 Hz),
146.3, 140.6, 132.6, 129.5, 127.7, 126.8, 125.7, 125.0,
123.1, 122.7, 120.4, 119.0, 117.5, 116.3, 115.0, 112.8,
108.9, 108.6, 55.6, 54.8, 53.2, 52.8, 52.8, 52.8, 52.8, 52.6,
48.0, 42.3, 30.9, 21.6.
1
as colorless oil. H NMR (500 MHz, CDCl3) dH 8.00 (d,
J = 7.5 Hz, 1H), 7.47 (s, 1H), 7.39 (t, J = 7.5 Hz, 1H),
7.31 (s, 1H), 7.23 (d, J = 7.5 Hz, 1H), 7.17 (t, J = 7.5 Hz,
1H), 6.72 (s, 1H), 4.73 (s, 2H), 4.65 (t, J = 7.0 Hz, 2H),
4.28 (t, J = 7.0 Hz, 2H), 3.90 (s, 3H), 2.53 (br, 1H), 2.50
(s, 3H), 2.46 (quin., J = 7.0 Hz, 2H). 13C NMR (125 MHz,
CDCl3) dC 147.6, 146.2, 140.6, 129.5, 127.6, 125.6, 125.0,
123.0, 121.6, 120.4, 119.0, 112.7, 108.9, 108.5, 56.4, 55.5,
48.0, 42.2, 30.7, 21.6; HR-ESI–MS: 385.1071 [M?Cl]-
(calcd. 385.1431 for C20H22N4O2Cl).
Synthesis of 3-(1-methoxy-3-methyl-9H-carbazol-9-
yl)propane-1,2-diol (9)
A solution of alkene 6 (45 mg, 0.18 mmol) in a THF/H2O
mixture (10/1, 1 mL), NMP (63 mg, 3 eq) and 4 % aque-
ous solution of OsO4 (23 lL, 0.36 mmol) were added into
a 10 mL round bottom flask. The mixture was stirred for
2 h at 30 °C. 20 % solution of NaHSO3 was added and the
solution was extracted with EtOAc, and washed with brine.
The combined organic layers were dried over Na2SO4 and
evaporated under reduced pressure. The residue was puri-
fied with a silica gel column chromatography (CH2Cl2/
9-(3-(4-(3,5-Difluorophenyl)-1H-1,2,3-triazol-1-yl)propyl)-
1-methoxy-3-methyl-9H-carbazole (8f)
1H NMR (500 MHz, CDCl3) dH 8.00 (d, J = 7.5 Hz, 1H),
7.47 (s, 1H), 7.42 (s, 1H), 7.40 (t, J = 7.5 Hz, 1H), 7.27
(m, 3H), 7.19 (t, J = 7.5 Hz, 1H), 6.76 (m, 1H), 6.72 (s,
1H), 4.73 (t, J = 7.0 Hz, 2H), 4.33 (t, J = 7.0 Hz, 2H),
3.89 (s, 3H), 2.56 (quin., J = 7.0 Hz, 2H), 2.50 (s, 3H). 13
C
MeOH, 9:1) to give 9 as white solid (36 mg, 70 %). H
1
NMR (125 MHz, CDCl3) dC 164.4 (d, JC–F = 12.6 Hz),
162.4 (d, JC–F = 13.1 Hz), 146.3, 140.6, 129.6, 127.6,
125.7, 125.0, 123.1, 120.5, 119.1, 112.8, 108.9, 108.6,
108.5, 108.4, 108.3, 103.5, 103.3, 103.1, 55.5, 48.2, 42.2,
30.6, 21.6. MS: 432.1762. HR-ESI–MS: 467.1472
[M?Cl]- (calcd. 467.1450 for C25H22F2N4OCl).
NMR (500 MHz, CD3OD) dH 7.98 (d, J = 8.0 Hz, 1H),
7.54 (d, J = 8.0 Hz, 1H), 7.48 (s, 1H), 7.40 (dd, J = 7.0,
1.0 Hz, 1H), 7.15 (dd, J = 7.0, 0.5 Hz), 6.84 (s, 1H), 4.72
(dd, J = 6.0, 14.5 Hz, 1H), 4.53 (dd, J = 7.0, 14.5 Hz,
1H), 4.15 (m, 1H), 4.00 (s, 3H), 3.54 (m, 2H), 2.50 (s, 3H).
13C NMR (125 MHz, CD3OD) dC 147.9, 142.9, 130.4,
126.4, 126.3, 124.1, 120.8, 119.7, 113.5, 110.6, 109.9,
73.7, 65.4, 56.1, 49.3, 21.7. HR-ESI–MS 320.0710
[M?Cl]- (calcd. C17H19NO3Cl for 320.1053).
1-Methoxy-3-methyl-9-(3-(4-(3-(trifluoromethyl)phenyl)-
1H-1,2,3-triazol-1-yl)propyl)-9H-carbazole (8g)
1H NMR (500 MHz, CDCl3) dH 8.00 (d, J = 8.0 Hz, 1H),
7.98 (s, 1H), 7.95 (d, J = 7.5 Hz, 1H), 7.57 (d, J = 7.5 Hz,
1H), 7.50 (m, 2H), 7.46 (s, 1H), 7.40 (t, J = 8.0 Hz, 1H),
7.28 (s, 1H), 7.25 (d, J = 8.0 Hz, 1H), 7.19 (t, J = 8.0 Hz,
Cell cultures and measurement of cytokine production
Bone marrow-derived dendritic cells (BMDCs) were
grown from wild-type C57BL/6 mice (Taconic Farm, NY,
123

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T. T. T. Thuy et al.
USA). Briefly, bone marrow from tibia and femur was
obtained by flushing with DMEM, and bone marrow cells
were cultured in RPMI 1640 medium containing 10 %
heat-inactivated fetal bovine serum (FBS) (Gibco, NY,
USA), 50 lM of 2-ME, and 2 mM of glutamine supple-
mented with 3 % J558L hybridoma cell culture supernatant
containing granulocyte–macrophage colony-stimulating
factor (GM-CSF). The culture medium containing GM-
CSF was replaced every other day. At day 6 of culture,
nonadherent cells and loosely adherent DC aggregates
were harvested, washed, and resuspended in RPMI 1640
supplemented with 5 % FBS. DCs were incubated in
48-well plates at a density of 2 9 105 cells/mL, and then
treated with the isolated compounds at the concentration of
0–50 lM for 1 h before stimulation with 10 ng/mL of LPS
from Salmonella minnesota (Alexis, NY, USA). Superna-
tants were harvested 16 h after stimulation. Concentrations
of murine IL-12 p40, IL-6, and TNF-a in the culture
supernatants were determined by ELISA (BD PharMingen,
CA, USA) according to the manufacture’s instructions. All
experiments were performed at least three times. The data
were presented as mean standard deviation (SD) of three
independent experiments in triplicate.
the main bromide compound 5 (83 %), the side-product 6
(11 %) was also obtained due to the elimination reaction in
small proportion. Dihydroxylation (Marsac et al. 2005) of N-
allyl-murrayafoline A (6) by OsO4/NMP in the mixture of
THF/H2O gave the dihydroxyl compound 9 with isolated
yield of 70 %. The azide 7 was obtained with good yield
(96 %) by treatment of 5 with NaN3 in acetonitrile under
reflux conditions (Scheme 1). The cycloaddition reactions
between azide 7 and various terminal alkynes (Scheme 2)
under standard conditions (cat. CuSO4Á5H2O, sodium
ascorbate, mixture t-BuOH/H2O: 1/1, inert gas) proceeded in
good yield (86–94 %) to afford the 1,2,3-triazole derivatives
of murrayafoline A (8a, 8b, 8d, 8f-h) (Table 1). In case of
propargyl amine (in 8c) and propargyl alcohol (in 8e), pro-
tections of amino and alcohol group were required before
this cycloaddition reaction. Protecting groups (Boc in 8b and
TBDPS in 8d) were removed by using TFA and TBAF
respectively after click reaction. The structures of these new
1
compounds were confirmed by HR-MS, H and 13C NMR
spectra (See ‘‘Experimental’’ section). To evaluate these
synthesized compounds for anti-inflammatory activity, all of
products, intermediate compounds, and initial material were
tested for their effects in the inflammatory response on
BMDCs. Particularly, each compound was examined for
their effects to the interleukin-6 (IL-6), IL-12 p40, and tumor
necrosis factor-a (TNF-a) production in BMDCs since they
were well known as one of the important chemical mediators
of inflammation (Kim et al. 2010). Briefly, TNF-a, a cyto-
kine involved in systemic inflammation, plays a central role
in the inflammatory response and implicates in the patho-
genesis of both acute and chronic inflammatory diseases
(Beutler and Cerami 1986). IL-6 has both pro- and anti-
inflammatory effects. It was a key player in chronic inflam-
mation, and IL-6 production levels are important factors in
inflammatory diseases in humans (Cem 2006). IL-12, a pro-
inflammatory cytokine, stimulates the production of inter-
feron-gamma (IFN-c), TNF-a. It is rapidly produced by
Results and discussion
The key step to introduce the 1,2,3-triazolyl groups into the
murrayafoline A molecule was Cu(I)-catalyzed 1,3-dipolar
cycloaddition reaction (click reaction) (Rostovtsev et al.
2002) between azide derivatives and terminal alkynes. The
1,2,3-triazole-based murrayafoline A derivatives were syn-
thesized as in the synthetic route showed in Scheme 1. The
starting material, murrayafoline A, was isolated and purified
from the roots of Glycosmis stenocarpa (see ‘‘Experimental’’
section). Murrayafoline A bromide 5 was prepared according
to the method described by Zhang (Zhang et al. 2010). Beside
Scheme 1 Synthesis of
derivatives 5–9
OMe
OMe
OMe
a
NH
N
N
Br
5 (83%)
4
6 (11%)
c
b 96%
70%
Reagents and conditions:
(a) Br-C3H6-Br, NaH, THF,
r.t., 72h; (b) NaN3, CH3CN,
reflux, 72h; (c) NMP,
OMe
OMe
OH
N
N3
N
OH
OsO4, THF/H2O, 300C, 2h.
7
9
123

Anti-inflammatory activity of N-substituted-1,2,3-triazole murrayafoline A derivatives
837
OMe
OMe
CuSO4.5H2O (20 mol%)
NaAsc (200 mol%)
N
N
N
N
N3
R
N
R
tBuOH/H2O 1/1, r.t., 12h
7
8a, 8b, 8d, 8f-h
Scheme 2 Click azide–alkyne Huisgen cycloaddition reaction
120
80
40
0
Table 1 Structure of 1,2,3 triazole N-substituted murrayafoline A
derivatives and their reaction yields
*
OMe
N
N
N
N
*
R
*
No.
R
Product
Yield (%)
1
2
3
4
5
6
–COOH
8a
8b
8c
8d
8e
8f
86
93
75
94
61
94
–CH2–NHBoc
–CH2–NH2
–CH2–OTBDPS
–CH2–OH
F
4
5
6
7
8a 8b 8c 8e 8f 8g 8h
9
Compounds (25 μM)
Fig. 2 Initial single point concentration screening and selection for
candidate compounds in synthetic compounds series. BMDCs were
pre-treated with indicated compounds for 1 h, and then stimulated
with LPS (10 ng/ml) for 16 h. The cytokine IL-12 p40 was measured
by ELISA. The compounds were diluted in DMSO. The candidate
compounds 5, 8c, and 8e are indicated with asterisk (*)
F
7
8
8g
8h
88
91
CF3
to the manufacture’s instructions. SB203580, an inhibitor of
cytokine suppressive binding protein/p38 kinase, was used
as a positive control. The preliminary results on the inhibi-
tory effects on cytokine IL-12 p40 production showed that at
a concentration of 25 lM, compounds 5, 8c, and 8e highly
inhibited the production of cytokine IL-12 p40 in DCs
induced by LPS stimulator with the inhibitory values 72.3,
100 and 61.8 %, respectively. Other compounds weakly
demonstrated inhibitory effects (inhibitory values lower than
30 %) (Fig. 2). Therefore, compounds 5, 8c, 8e were then
subjected for further studies. As showed in Fig. 3, com-
pounds 8c, 8e exhibited strong inhibitory effects in all IL-12
p40, IL-6 and TNF-a production meanwhile compound 5 did
not significantly inhibited the production of TNF-a. All of
them significantly inhibited higher than positive control with
the exception of TNF-a inhibitory activity of 5. Especially, at
concentration of 12.5 lM, compound 8c potentially inhib-
ited the production of IL-12 p40, IL-6 and TNF-a with
inhibitory values of 100, 88.7 and 79.3 %, respectively. It
also should be noted that, with the exception of compounds
8a, 8f, and 8g, all of synthetic compounds showed higher
inhibitory activity in compared with initial material (4)
F
N
N
F
activated inflammatory cell and playsan important role in the
activities of cellular immunity. It also involved in the dif-
ferentiation of naive T cell into type-1 helper T cell and
hence leads to generation of inflammatory disease (Trin-
chieri et al. 2003). As our study, we first used a colorimetric
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay (Sigma, MO, USA) to confirm that these
compounds have no or little effect on the cell viability (data
not shown). Next, the synthetic compounds were tested their
effects on the production TNF-a, IL-6, and IL-12 p40 in
BMDCs induced by LPS stimulator. In brief, BMDCs were
incubated in 48-well plates at a density of 2 9 105 cells/mL.
After that, they were treated for 1 h with the compounds at
the various concentrations, and then stimulated with LPS
(10 ng/mL). Supernatants were harvested 16 h after stimu-
lation. The IL-12 p40, IL-6 and TNF-a production were
determined by ELISA (BD PharMigen, CA, USA) according
123

838
T. T. T. Thuy et al.
120
80
40
0
A
B 100
80
60
40
20
0
0
5
0
0
5
25 50
.5
0
5
0
5
0
5
50
25
6. 12.
25 50
25
6. 12
25 50
25
6. 12.
50
25
6. 12.
25 50
25
6. 12.
25 50
25
6. 12.
25 50
Pos.
Comp. 5
Comp. 8c
Comp. 8e
Pos.
Comp. 5
Comp. 8c
Comp. 8e
μ
μ
Concentration ( M)
Concentration ( M)
120
80
40
0
C
ND
0
5
0
5
0
5
50
25
6. 12.
25 50
25
6. 12.
25 50
25
6. 12.
25 50
Pos.
Comp. 5
Concentration (μM)
Comp. 8c
Comp. 8e
Fig. 3 Effect of compounds 5, 8c, and 8e on IL-12 p40 (a), IL-6 (b),
and TNF-a (c) production by LPS-stimulated BMDCs. DCs were
treated with the compounds (0, 6.25, 12.5, 25, and 50 lM) for 1 h
before stimulation with LPS (10 ng/mL). Supernatants were harvested
16 h after stimulation. Concentrations of murine IL-12 p40, IL-6, and
TNF-a in the culture supernatants were determined by ELISA. The
data were presented as inhibition rate (%) compared to the value of
vehicle-treated DCs. SB203580 was used as positive control (pos.),
ND not detected
Foundation of Korea (NRF) funded by the Ministry of Education,
Science and Technology (2009-0093815), Republic of Korea.
(Fig. 2). This results warranted that carbazole alkaloids,
particularly murrayafoline A derivatives, containing 1,2,3-
triazole nucleus may be potent anti-inflammatory action and
further studies should be needed to elucidate the mechanism
of anti-inflammatory action of those compounds.
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123