Synthesis and reactive oxygen species scavenging activity of halogenated alkaloids from boldine

Article abstract of DOI:10.1007/s00044-011-9844-5

Six halogenated alkaloids have been semisynthesized from natural boldine as starting material. Their antioxidant activity toward reactive oxygen species (ROS) generation by neutrophils stimulated with N-formylmethionyl- leucyl-phenylalanine and in the hypoxanthine- xanthine oxidase system was evaluated. Most of the alkaloids synthesized inhibited ROS generation in both systems in a concentration-dependent manner. The alkaloids with phenolic substituents displayed more powerful anti-oxidative activity than those containing methoxylated groups. None of the alkaloids assayed had any effect on xanthine oxidase activity. Therefore, halogenated phenanthrene alkaloids can become in promising candidates for the development of novel and potent anti-inflammatory drugs. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9844-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3133–3139
DOI 10.1007/s00044-011-9844-5
ORIGINAL RESEARCH
Synthesis and reactive oxygen species scavenging activity
of halogenated alkaloids from boldine
•
Lara Milian Rafael Ballesteros Marıa Jesus Sanz
•
•
´
Marıa Amparo Blazquez
´
´
´
´
Received: 28 April 2011 / Accepted: 25 October 2011 / Published online: 11 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Six halogenated alkaloids have been semi-
synthesized from natural boldine as starting material. Their
antioxidant activity toward reactive oxygen species (ROS)
generation by neutrophils stimulated with N-formyl-
methionyl-leucyl-phenylalanine and in the hypoxanthine–
xanthine oxidase system was evaluated. Most of the alka-
loids synthesized inhibited ROS generation in both systems
in a concentration-dependent manner. The alkaloids with
phenolic substituents displayed more powerful anti-oxida-
tive activity than those containing methoxylated groups.
None of the alkaloids assayed had any effect on xanthine
oxidase activity. Therefore, halogenated phenanthrene
alkaloids can become in promising candidates for the
development of novel and potent anti-inflammatory drugs.
Introduction
The increasing recognition of the participation of free
radical-mediated oxidative events in the initiation and/or
progression of cardiovascular, tumoural, inflammatory, and
neurodegenerative disorders (Bauerova and Bezek, 1999;
Jaeschke, 1995; Zhao, 2009; Dinkova-Kostova, 2008;
Griendling et al., 2000), has given rise to the search for
new antioxidant molecules either natural or synthetic. In
fact, the decreased activity of a large number of enzymes
involved in reactive oxygen species (ROS) defence, par-
ticularly with aging process, can explain the main etio-
logical factor of acute or chronic disorders. In this regard,
boldine and other related aporphine alkaloids have been
shown to behave as potent antioxidants in a number of
´
experimental models (Speisky et al., 1991; Martınez et al.,
1992; Cederbaum et al., 1992; Jang et al., 2000; Youn
´
et al., 2002; Rancan et al., 2002; Milian et al., 2004; Yu
Keywords Halogenated phenanthrene alkaloids ꢀ
Halogenated aporphine alkaloids ꢀ Semi-synthesis ꢀ
Antioxidant activity
et al., 2009).
Some years ago our group isolated two phenanthrene
alkaloids from the roots of Dennettia tripetala, uvariop-
´
´
sine, and stephenanthrine (Lopez-Martın et al., 2002).
These alkaloids were able to inhibit Ang-II-induced leu-
´
kocyte-endothelial cell interactions in vivo (Estelles et al.,
´
´
L. Milian ꢀ M. A. Blazquez (&)
2003). This effect was partly mediated through inhibition
of the generation of ROS, down-regulation of P-selectin
expression on the endothelial cell and blockade of PAF-
induced responses by interacting with its receptor. Based
on their structural features, we semi-synthesized four
phenanthrene and one aporphine alkaloid from natural
boldine and evaluated their inhibitory effect on ROS gen-
Department of Pharmacology, Faculty of Pharmacy, University
´ ´
of Valencia, Av. Vicent Andres Estelles s/n, 46100 Burjassot,
Valencia, Spain
e-mail: amparo.blazquez@uv.es
R. Ballesteros
Department of Organic Chemistry, Faculty of Pharmacy,
´ ´
University of Valencia, Av. Vicent Andres Estelles s/n,
46100 Burjassot, Valencia, Spain
´
eration (Milian et al., 2004). The reasons for this semi-
synthetic process were two: first to increase the number of
aporphine and phenanthrene alkaloids to enhance their
ROS scavenging properties and second, to obtain enough
M. J. Sanz
Department of Pharmacology, Faculty of Medicine, University
´
of Valencia, Av. Blasco Iban
˜ez 15, 46013 Valencia, Spain
123

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Med Chem Res (2012) 21:3133–3139
quantities of the compounds to test their pharmacological
activity in different in vivo and in vitro models.
Results and discussion
In that study, boldine, secoboldine, and boldine methine
displayed more powerful ROS scavenger activity than
other semi-synthesized alkaloids or even uvariopsine and
stephenanthrine and the classic antioxidant ascorbic acid,
The synthesis of halogenated aporphinods (compounds
1–6) using boldine as starting material is shown in Scheme 1.
Several strategies have been employed for the synthesis of
halogenated compounds. Among the different methods
available for the formation of halogenated aporphine, the
treatment with the appropriate N-halosuccinimide in triflu-
oroacetic acid has been proved to be useful, notably in the
synthesis of S(?)-3-bromoboldine, S(?)-3,8-dibromobol-
´
used as control in the same assays (Milian et al., 2004).
Furthermore, boldine and the semi-synthesized secoboldine
and boldine methine could inhibit Ang-II-induced leuko-
cyte-endothelial cell interactions in vivo.
´
dine, and S(?)-3-chloroboldine (Martınez et al., 1999).
Secoboldine and boldine methine were also capable of
reducing neutrophil infiltration into the peritoneal cavity of
the rat, CXC chemokine and PAF release elicited by Ang-II
The reaction of boldine in TFA with N-bromosuccinimide
was carried out at room temperature. Under these conditions,
two reaction products were obtained, 3-bromoboldine and
3,8-dibromoboldine, which were characterized through
spectroscopic data. The crude was extracted with dichloro-
methane and the organic phase subjected to silica gel column
using EtOAc as mobile phase to afford 3-bromoboldine (1)
as a single product. First attempts to obtain 2-bromoseco-
boldine led to the synthesis of the protonated form. It was
revealed when synthesis of product 6 from 2 failed but it was
possible to obtain 2-bromo-3,7-dimethoxy-N,N-dimethyl-
secobodine iodide (3) when 2 was treated at room tempera-
ture with an excess of both methyl iodide and K2CO3. Mass
spectrometry of 2 was done revealing the presence of an extra
proton. To obtain 2-bromosecoboldine in its basic form,
basification of the crude of reaction was needed. The treat-
ment of 1 with aqueous NH4OAc and EtOH under reflux
during 48 h, led to the opening of the piperidinium ring to
give after crystallization compound 2 in 51% yield. Com-
pound 4 was simply prepared by methylation overnight at
room temperature of compound 1 with an excess of methyl
iodide (100% yield) in a mixture of CH3CN–MeOH (7.5:5).
Compound 5 was obtained in a more efficient synthesis
by treating compound 4 with aqueous ammonium as
described above for compound 2. The crude was concen-
trated and extracted with dichloromethane, then 2-bromo-
N-methylsecoboldine was obtained in 79% yield. Finally,
the treatment of compound 5 with methyl iodide at room
temperature gave compound 6 (29% yield).
´
in vivo (Estelles et al., 2005). The effects displayed by these
two alkaloids were partly mediated through inhibition of the
endothelial generation of ROS and interleukin-8 release after
Ang-II stimulation. The inhibition of ROS generation seems
to be the key mechanism by which these alkaloids inhibits
Ang-II-induced responses since P-selectin up-regulation and
inhibition of CXC chemokine synthesis are events linked to
this capability (Riaz et al., 2003; Gaboury et al., 1994;
Johnston et al., 1996). By contrast, the aporphine alkaloid
boldine although endowed with antioxidant properties did
not markedly affect the inflammatory activity elicited by
´
Ang-II (Estelles et al., 2005). In this way, the use of a
phenanthrene alkaloid such as boldine methine could con-
stitute an alternative therapy for the control of Ang-II-
induced neutrophilic inflammation in cardiovascular disease
states where Ang-II plays a critical role.
On the other hand, both boldine and semi-synthetic
´
derivatives (Sobarzo-Sanchez et al., 2002) have attracted
the attention by the wide range of pharmacological activ-
ities. Thus, it has recently been reported that boldine dis-
plays anti-proliferative activity against glioma cells
(Gerhardt et al., 2009) opening the study of the potential
anticancer activities of boldine and its derivatives. Methi-
odides of boldine and derivatives showed neuronal nico-
´
tinic acetylcholine receptor blockers (Iturriaga-Vasquez
et al., 2007) or more recently boldine and derivatives can
be envisaged as new therapeutic candidates with novel
mechanisms of action for treating resistant forms of
tuberculosis (Guzman et al., 2010) or new phenanthrene
alkaloids semi-synthesized from boldine for the treatment
of S. pneumoniae infections resistant to other antibiotics
The effect of the halogenated aporphine and phenan-
threne alkaloids on free radical generation was investigated
by two different methods: measurement of ROS generation
in stimulated human PMNs (Schudt et al., 1991), and
measurement of ROS scavenging activity in a enzymatic
system (Sekiguchi and Nagamine, 1994). In the first assay,
all the alkaloids tested except compound 3 inhibited in a
concentration-dependent manner N-formyl-methionyl-leu-
cyl-phenylalanine (f-MLP)-induced ROS generation in
human PMNs although with different potency. The order of
potency, based on the calculated inhibitory concentration
50% (IC50) values (Table 1) showed that the phenanthrene
alkaloid with phenolic groups at C-3, C-7 (compound 2, 5,
´
(Garcıa et al., 2011). Based on these results, in this study
we have synthesized novel antioxidant agents. The alka-
loids synthesized have been halogenated in order to
investigate and perform further studies regarding its
structure–activity relationships searching for more power-
ful compounds than those previously investigated. There-
fore, they may become in the future promising candidates
for the development of novel and potent anti-inflammatory
drugs.
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Med Chem Res (2012) 21:3133–3139
3135
Br
CH3
N
CH3
N
Br
HO
Br
H
H
3CO
3CO
HO
CH3
HO
H
CH3
N
H
H
3CO
3CO
CH3
N
H
H
3CO
H3CO
CH3
b
a
H
3CO
c
3CO
OCH3
H3CO
OH
OH
1
2
3
OH
Boldine
d
Br
CH3
N
Br
Br
CH3
HO
HO
HO
N
CH3
CH3
CH3
CH3
N
H
3CO
H
H
3CO
CH3
H3CO
e
f
H3CO
3CO
H3CO
OH
OH
OH
6
1
4
5
Scheme 1 Synthesis of compounds 1–6. Reagents and conditions: (a) NBS; (b) NH4AcO, EtOH; (c) MeOH, CH3CN, K2CO3, CH3I; (d) MeOH,
CH3CN, CH3I; (e) NH4AcO, EtOH; (f) MeOH, CH3CN, CH3I
100
and 6) were more potent than their corresponding equiva-
lents with aporphine skeleton. Phenanthrene alkaloids
75
without phenolic group displayed lower activity than even
3-Bromoboldine
50
25
0
those with aporphine skeleton (3, Fig. 1). All the alkaloids
assayed were more potent than ascorbic acid (IC50
2-Bromosecoboldine
2-Bromo-N,N-dimethylsecoboldine iodide
2-Bromo-N-methylsecoboldine
3-Bromo-N-metilboldine iodide
´
99.6 lM), the reference drug previously employed (Milian
et al., 2004). In general, it seems that halogenation of the
alkaloid does not affect its antioxidant activity in this
system since most of the alkaloids assayed presented
similar IC50 values than their corresponding non-haloge-
-6
-5
Log M
-4
-25
Fig. 1 Effect of alkaloids on ROS generation in f-MLP-stimulated
PMNs
´
nated alkaloids (Milian et al., 2004).
Since most of these compounds may exert this action
through their free radical scavenging activity, they were
also assayed on a cell-free ROS generation system. All the
alkaloids tested were capable of scavenging ROS generated
by the hypoxanthine–xanthine oxidase system in a con-
centration-dependent manner (Fig. 2).
more active than the classic antioxidant ascorbic acid (IC50
167.8 lM). In this assay, a similar profile of structure–
activity relationship to that encountered in ROS generated
in f-MLP-stimulated neutrophils was observed. Indeed,
phenanthrene alkaloids with phenolic substituents were the
most active, followed by the group of the aporphine alka-
loids with phenolic groups. Only, compound 6, 2-bromo-
The order of potency based on the calculated IC50 values
(Table 1) revealed that all the synthesised compounds were
Table 1 IC50 of the alkaloids tested for ROS generation by f-MLP-stimulated human neutrophils or hypoxanthine–xanthine oxidase system
Alkaloid
f-MLP-stimulated human
neutrophils IC50 (lM)
Hypoxanthine–xanthine
oxidase system IC50 (lM)
3-Bromoboldine (1)
0.81 0.03**
0.55 0.10**
0.16 0.10**
3.01 0.05**
2.00 0.06**
0.19 0.03**
2.11 0.08**
2-Bromosecoboldine (2)
0.64 0.03**
2-Bromo-3,7-dimethoxy-N,N-dimethylsecoboldine iodide (3)
3-Bromo-N-methylboldine iodide (4)
2-Bromo-N-methylsecoboldine (5)
2-Bromo-N,N-dimethylsecoboldine iodide (6)
A 100 lM 62% inhibition
1.27 0.02**
0.78 0.12**
0.74 0.07**
Data are means SEM; statistical significance ** P \ 0.01
123

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Med Chem Res (2012) 21:3133–3139
100
Experimental section
75
50
25
0
3-Bromoboldine
General experimental procedure
2-Bromosecoboldine
2-Bromo-N,N-dimethylsecoboldine iodide
Melting points were determined on a Fisher-John apparatus
and are uncorrected. Optical rotation measurements were
determined on a Perkin-Elmer 241 polarimeter at room
temperature in MeOH solution. UV spectra were taken on a
Shimadzu 2101 UV/VIS spectrophotometer in MeOH
solution. MS were performed using a VG Auto Spec Fisons
Spectrometer. NMR measurements (data reported in d)
were run on a Varian Unity-400 instrument. The chemical
shift are referenced to solvent (Cl3CH, Cl3CD) signals at
7.25 and 77.0 ppm, respectively. Multiplicities of 13C
NMR resonances were determined by distortion less
enhancement by polarization transfer. Standard pulse
sequences were employed for magnitude correlated spec-
troscopy. HMQC (heteronuclear multiple quantum coher-
ence) and HMBC (heteronuclear multiple bond
connectivity) experiments to establish correlations were
carried out. Thin-layer chromatography was run on Merck
precoated silica gel F254 type 60 plates with detection by
UV light and Munnier’ reagent.
2-Bromo-N-methylsecoboldine
3-Bromo N-methylboldine iodide
2-Bromo-3,7-dimethoxy-N,N-
dimethylsecoboldine iodide
-8
-7
-6
-5
-4
Log M
Fig. 2 Effect of alkaloids on ROS generation by hypoxanthine–
xanthine oxidase system
N,N-dimethylsecoboldine iodide, was slightly less potent
than its corresponding alkaloid with aporphine skeleton
(compound 4). Similarly, the phenanthrene alkaloid with-
out phenolic groups showed less potency than any of the
alkaloids tested in this system regardless its phenanthrene
or aporphine skeleton. When they were compared with
´
their corresponding non-halogenated alkaloids (Milian
et al., 2004), the halogenated alkaloids were either more
potent than the non-halogenated group or exerted similar
potency.
In order to confirm that the ROS scavenging activity
elicited by aporphine and phenanthrene alkaloids was not
due to xanthine oxidase inhibition, another set of experi-
ments was carried out to investigate such possibility. None
of the alkaloids assayed affected xanthine oxidase activity
(Table 2). Therefore, we have synthesized a potent new
class of halogenated phenanthrene alkaloids with ROS
scavenger activity.
Commercial chemicals
All chemical are of the highest commercially available
purity. Boldine, bovine serum albumin (BSA), dimethyl-
sulfoxide (DMSO), f-MLP, gelatine, glucose, hypoxan-
thine, luminol, microperoxidase, and xanthine oxidase
were purchased from Sigma Chemical Co. (St. Louis, MO).
All other reagents were purchased from Aldrich-Chemie
(Steinheim, Germany).
The analysis of the structure–activity relationship
showed that the presence of free phenolic groups is
essential for a potent antioxidant activity. In general, the
alkaloids with phenanthrene skeleton resulted in more
active compounds than their respective alkaloids with
aporphine skeleton. The higher activity reported for phe-
nolic-phenanthrenic relative to phenolic-aporphinic mole-
cules may reside on the existence of a third benzylic ring
which may confer a greater capacity to delocalize the
phenoxy-free radical to the former.
Chemistry
3-Bromoboldine (1)
A solution of boldine (2 g, 6.1 mmol) in TFA (15 ml) was
treated with NBS (1.086 g, 6.1 mmol) at room tempera-
ture. After 3 h stirring the mixture was poured into cold
water (50 ml) and the aqueous solution was adjusted to pH
8–9 with aqueous NH4OH 25%. The filtrate was extracted
with CH2Cl2. The combined organic phases were dried and
evaporated under vacuum, and the resulting residue was
purified by silica gel column chromatography (EtOAc) to
give 1 as brown solid, yield 35.9%; mp 181–183°C;
[aD] = ?22.02°; UV (MeOH): kmax (log e) = 285 (2.10),
311 (2.02) nm; 1H NMR (DMSO-d6) d: 2.27 (dd, 1H,
J = J00 = 13.3 Hz, H-7b), 2.30 (m, 1H, H-5b), 2.40 (s, 3H,
N–CH3), 2.68 (m, 2H, H-4a,b), 2.80 (dd, 1H, J = 13.4,
3.9 Hz, H-6), 2.95 (dd, 1H, J = 13.4, 3.9 Hz, H-7a), 2.97
(dd, 1H, J = 3.9, 13.9 Hz, H-5a), 3.49 (s, 3H, OCH3-1),
Table 2 Effect of alkaloids on isoxantopterine formation from
pterine by measuring enzyme activity (lmol/min)
Control
3.65 0.25
4.09 0.42
3.45 0.07
3.98 0.11
Control
3.25 0.15
2.82 0.57
3.77 0.52
3.60 0.33
1
4
5
2
3
6
Data are means SEM; no statistical significance P \ 0.05 from
control was observed
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Med Chem Res (2012) 21:3133–3139
3137
3-Bromo-N-methylboldine iodide (4)
3.78 (s, 3H, OCH3-10), 6.74 (s, 1H, H-8), 7.80 (s, 1H,
H-11). 13C NMR (DMSO-d6) d: 30.1 (C-4), 33.3 (C-7),
43.5 (NCH3), 52.6 (C-5), 55.7 (OCH3, C-10), 59.8 (OCH3,
C-1), 62.3 (C-6a), 110.2 (C-3), 111.5 (C-11), 115.2 (C-8),
121.8 (C-11a), 125.4 (C-1b), 127.3 (C-1a), 129.5 (C-7a),
143.1 (C-1), 146.2 (C-9), 146.3 (C-10), 146.6 (C-2).
[HMQC and HMBC data were used to assign proton and
carbon correlations] HR-EI-MS: m/z = 405.0552 calc.
mass 405.0576 C19H20BrNO4: EI-MS: m/z (rel. int.) = 407
(63), 406 (46), 405 (85), 404 (36), 390 (16), 326 (5), 307
(100), 289 (50).
To a solution of 1 (200 mg, 0.492 mmol) in a mixture of
methanol (5 ml) and acetonitrile (7.5 ml) was added CH3I
(0.031 ml, 0.492 mmol). The resulting mixture was stirred
overnight at room temperature. Compound 4 after crys-
tallization with MeOH/DCM was obtained as yellow nee-
dles, yield 100%; mp 185–187°C; [aD] = ?1.45°; UV
(MeOH): kmax (log e) = 288 (0.65), 307 (0.7) nm. 1H
NMR (DMSO-d6) d: 2.95 (s, 3H, N–CH3), 2.99 (m, 2H,
H-4a,b), 3.37 (s, 3H, N–CH3), 3.53 (s, 3H, OCH3-1), 3.66
(dd, 1H, J = 13.4, 3.5 Hz, H-5b), 3.77 (s, 3H, OCH3-10),
3.86 (m, 1H, H-5a), 4.61 (m, 1H, H-6), 6.84 (s, 1H, H-8),
7.79 (s, 1H, H-11). 13C NMR (DMSO-d6) d: 25.26 (C-4),
27.82 (C-7), 43.08 (NCH3), 53.05 (NCH3), 56.79 (OCH3,
C-10), 59.87 (C-5), 60.20 (OCH3, C-1), 67.74 (C-6a),
111.40 (C-11), 115.30 (C-8). FAB-MS: m/z = 420.0798
calc. mass 420.0810 C20H23BrNO4: FAB-MS (M ? 1): m/
z (rel. int.) = 421 (97), 420 (33), 419 (100), 392 (10), 329
(33), 175 (98).
2-Bromosecoboldine (2)
A mixture of 3-bromoboldine (200 mg, 0.492 mmol) and
2 M aqueous ammonium acetate and ethanol (1:1) was
refluxed in a bath at 103°C during 48 h. After it was cooled
at room temperature, the mixture was basified with NH4OH
25% until pH 9 and extracted with EtOAc to give 2 after
crystallization with MeOH/DCM, yield 51%; mp
174–175°C. UV (MeOH): kmax (log e) = 268 (2.17), 284
(1.21), 320 (0.31) nm. 1H NMR (DMSO-d6) d: 2.43 (s, 3H,
N–CH3), 2.76 (m, 2H, CH2a), 3.44 (m, 2H, CH2b), 3.75 (s,
3H, OCH3-4), 3.94 (s, 3H, OCH3-6), 7.22 (s, 1H, H-8), 7.47
(d, 1H, J = 9.2 Hz, H-9), 7.81 (d, 1H, J = 9.2 Hz, H-10),
8.94 (s, 1H, H-5). 13C NMR (DMSO-d6) d: 31.82 (CH2b),
34.74 (NCH3), 49.89 (CH2a), 55.27 (OCH3, C-6), 59.64
(OCH3, C-4), 108.19 (C-5), 111.57 (C-8), 120.88 (C-10),
124.45 (C-9). HR-EI-MS: m/z = 405.0544 calc. mass
405.0576 C19H20BrNO4: EI-MS: m/z (rel. int.) = 407 (8),
405 (10), 390 (4), 364 (100), 362 (97), 326 (43), 296 (16).
2-Bromo-N-methylsecoboldine (5)
A solution of 4 (270 mg, 0.493 mmol) in 2 M aqueous
ammonium acetate and ethanol (1:1) was refluxed in a bath
a 104°C for 48 h. The ethanol was evaporated under vac-
uum and the aqueous layer was basified with NH4OH 25%
until pH 8–9 and extracted with DCM. The combined
organic extract were dried over anhydrous Na2SO4 and
compound 5 after crystallization with MeOH/CH2Cl2/
Hexane mixtures was obtained a brown stars crystal, yield
79.1%; mp 125–128°C; UV (MeOH): kmax (log e) = 268
1
(0.59), 284 (0.33), 320 (0.7) nm. H NMR (DMSO-d6) d:
2-Bromo-3,7-dimethoxy-N,N-dimethylsecoboldine iodide
(3)
2.29 (s, 6H, N–(CH3)2), 2.43 (m, 2H, CH2a), 3.37 (m, 2H,
CH2b), 3.74 (s, 3H, OCH3-4), 3.95 (s, 3H, OCH3-6), 7.22
(s, 1H, H-8), 7.52 (d, 1H, J = 9.3 Hz, H-9), 7.71 (d, 1H,
J = 9.3 Hz, H-10), 8.93 (s, 1H, H-5). 13C-NMR (DMSO-
d6) d: 30.41 (CH2b), 44.95 (N–(CH3)2), 58.31 (CH2a),
55.29 (OCH3, C-6), 59.77 (OCH3, C-4), 108.12 (C-5),
111.57 (C-8), 113.92 (C-2), 120.68 (C-10), 121.89 (C-5a),
122.90 (C-10a), 124.16 (C-4a), 124.70 (C-9), 128.08 (C-
8a), 132.39 (C-1), 142.69 (C-4), 145.46 (C-3), 146.95 (C-
7), 148.10 (C-6). [HMQC and HMBC data were used to
assign proton and carbon correlations]. HR-EI-MS: m/
z = 419.3450 calc. mass 419.3510 C20H22Br NO4: EI-MS:
m/z (rel. int.) = 421 (85), 420 (35), 419 (100), 418 (17),
339 (27), 281 (5), 253 (6), 238 (60).
To a stirred solution of 2-bromosecoboldine (98.58 mg,
0.212 mmol) in acetonitrile (7.5 ml) and methanol (5 ml)
was added K2CO3 (200 mg, 1.449 mmol) and an excess of
CH3I (0.1 ml, 0.610 mmol) at room temperature during
48 h. The methanol/acetonitrile was evaporated under
vacuum and the residue extracted with acetone to give 3 as
white solid, yield 34.8%; UV (MeOH): kmax (log e) = 269
(1.35), 320 (0.13) nm. 1H NMR (DMSO-d6) d: 3.30 (s, 9H,
N–(CH3)3), 3.49 (m, 2H, CH2a), 3.75 (m, 2H, CH2b), 3.96
(s, 12H, (49 OCH3), 7.55 (s, 1H, H-8), 7.88 (d, 1H,
J = 9.2 Hz, H-9), 7.91 (d, 1H, J = 9.2 Hz, H-10), 9.04 (s,
1H, H-5). 13C NMR (DMSO-d6) d: 26.70 (CH2b), 52.30
(N–(CH3)3), 63.11 (CH2a), 55.29, 55.49, 60.28, 60.84
(OCH3, C-3,4,6,7), 107.93 (C-5), 108.45 (C-8), 127.81
(C-9,10). FAB-MS m/z = 462.1274 calc. mass 462.1280
C23H29BrNO4: FAB-MS (M ? 1): m/z (rel. int.) = 463
(29), 462 (100), 461 (31), 383(3), 368 (4), 310 (7), 295 (5),
267 (3).
2-Bromo-N,N-dimethylsecoboldine iodide (6)
A solution of methyl iodide was slowly added to a mag-
netically stirred solution of 2-bromo-N-methylsecoboldine
(5) (155 mg, 0.369 mmol) at room temperature. The
123

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Med Chem Res (2012) 21:3133–3139
following treatment with MeOH/Hexane gave analytically
pure 6, yield 29%; UV (MeOH): kmax (log e) = 268 (0.54),
284 (0.37), 320 (0.06) nm. 1H NMR (DMSO-d6) d: 3.28 (s,
9H, N–(CH3)3), 3.45 (m, 2H, CH2a), 3.72 (m, 2H, CH2b),
3.76 (s, 3H, OCH3-4), 3.95 (s, 3H, OCH3-6), 7.27 (s, 1H,
H-8), 7.58 (d, 1H, J = 9.2 Hz, H-9), 7.77 (d, 1H,
J = 9.2 Hz, H-10), 8.93 (s, 1H, H-5). 13C NMR (DMSO-
d6) d: 26.54 (CH2b), 52.25 (N–(CH3)3), 55.31 (OCH3, C-6),
59.82 (OCH3, C-4), 63.25 (CH2a), 108.04 (C-5), 111.65 (C-
8), 120.42 (C-10), 125.29 (C-9). FAB?: m/z (rel.
int.) = 436 (100), 435 (39), 434 (98).
the blank group. Experiments with the appropriate DMSO
concentration were also carried out (1–0.01%). Chemilu-
minescence was recorded at 4 s intervals over 100 s period
per well and the AUCs was integrated. Drug-induced
reduction was expressed as % inhibition. IC50 values were
then calculated from the concentration-inhibition curves by
non-linear regression analysis.
Measurement of the effect on xanthine oxidase activity
by isoxantopterine formation from pterine
A direct inhibitory effect on xanthine oxidase activity was
tested by measuring isoxantopterine formation from pterine
by fluorimetry (excitation wavelength at 345 nm and
emission wavelength at 390 nm) following the previously
described method (Beckman et al., 1989).
Pharmacology
Measurement of ROS generation from human PMNs
The formation of ROS by human PMNs was assessed by
luminol-enhanced chemiluminescence with a modified
method (Martinez et al., 1999). Assay was carried out in
opaque 96-well plates, 105 cells per well suspended in an
assay volume of 180 ll of Krebs-HEPES buffer containing
glucose 5.6 mM, BSA 0.05% (w/v), microperoxidase 2 lM
with gelatine 0.1% pH 7.4, alone or in combination with
alkaloids (final concentration in 200 ll, 100–0.01 lM) for
30 min at 37°C. All the assays were performed in dupli-
cate. Plates were placed in a Wallac 1420 Victor2 Multi-
label Counter. Then 20 ll of luminol 5 lM, CaCl2 1 mM
and f-MLP 100 nM was added sequentially to each well,
except in the blank group. Experiments with the appro-
priate DMSO concentration were also carried out
(1–0.01%). Chemiluminescence was recorded at 4 s inter-
vals over a 100 s period per well and the area under the
curve (AUCs) was integrated. Drug-induced reduction was
expressed as % inhibition. IC50 values were then calculated
from the concentration-inhibition curves by non-linear
regression analysis.
Statistical analysis
The IC50 values were calculated from non-linear regression
by a software of Prisma 3.0 (Graph Pad Software, San
Diego, California, USA). All values are shown as
mean SEM. The difference between two values was
determined by use of unpaired Student’s t test. The dif-
ferences were considered statistically significant if the
P value was less than 0.05.
Conclusion
The halogenation of some aporphine and phenantrene
´
alkaloids previously synthesized (Milian et al., 2004)
weakly increases its antioxidant activity in cell free sys-
tems. Therefore, halogenated phenanthrene alkaloids may
be suitable for being tested in vivo as anti-inflammatory
agents since they have shown potent ROS scavenging
activity. Thus, they can become promising candidates for
the development of novel and potent anti-inflammatory
drugs.
Measurement of ROS generation by hypoxanthine–
xanthine oxidase
The ROS was generated by hypoxanthine–xanthine oxidase
system and detected by luminol-enhance chemilumines-
cence using a modified method (Sekiguchi and Nagamine,
1994). Assays was carried out in opaque 96-well plates.
One hundred and eighty microliters of Krebs-HEPES
buffer containing hypoxanthine 0.1 mM, glucose 5.6 mM,
gelatine 0.1% pH 7.4, alone or in combination with syn-
thesized alkaloids (final concentration in 200 ll,
100–0.01 lM) were added to the wells for 5 min at 37°C.
All the assays were performed in duplicate. Plates were
placed in a Wallac 1420 Victor2 Multilabel Counter. Then
20 ll of luminol 5 lM, CaCl2 1 mM, and xanthine oxidase
0.02 l/ml was added sequentially to each well, except in
Acknowledgment This study was supported by grants SAF2008-
03477 and Project CONSOLIDER-INGENIO SUPRAMED CSD
2010-00065 and Generalitat Valenciana PROMETEO/2011/008,
Spanish Ministry of Science and Innovation and RIER RD08/0075/
0016, Carlos III Health Institute, Spanish Ministry of Health. L. Mil-
´
ian was supported by a grant from the Spanish Ministry of Science
and Innovation.
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