Reactive oxygen species (ROS) generation inhibited by aporphine and phenanthrene alkaloids semi-synthesized from natural boldine

Article abstract of DOI:10.1248/cpb.52.696

Four phenanthrene and one aporphine alkaloids semi-synthesized from boldine were evaluated for their inhibitory effect on reactive oxygen species (ROS) generation. ROS generation by neutrophils stimulated with N-formyl-methionyl- leucyl-phenylalanine was inhibited in a concentration dependent manner. Alkaloids exerted similar inhibitory effect in the hypoxanthine-xanthine oxidase system than in stimulated neutrophils, which could be attributed to a direct ROS scavenging activity. None of the alkaloids assayed had any effect on xanthine oxidase activity. Therefore the synthesized alkaloids might constitute an alternative therapy in inflammation disorders in which ROS generation is involved.

Full text of DOI:10.1248/cpb.52.696

696
Chem. Pharm. Bull. 52(6) 696—699 (2004)
Vol. 52, No. 6
Reactive Oxygen Species (ROS) Generation Inhibited by Aporphine and
Phenanthrene Alkaloids Semi-Synthesized from Natural Boldine
Lara MILIÁN,a Rossana ESTELLÉS,b Belén ABARCA,c Rafael BALLESTEROS,c María Jesús SANZ, b and
María Amparo BLÁZQUEZ*,a
a Department of Pharmacology, Faculty of Pharmacy, University of Valencia; b Department of Pharmacology, Faculty of
Medicine, University of Valencia; and c Department of Organic Chemistry, Faculty of Pharmacy, University of Valencia;
Avda Vicent Andrés Estellés s/n 46100, Burjassot, Valencia, Spain.
Received December 22, 2003; accepted March 11, 2004
Four phenanthrene and one aporphine alkaloids semi-synthesized from boldine were evaluated for their in-
hibitory effect on reactive oxygen species (ROS) generation. ROS generation by neutrophils stimulated with N-
formyl-methionyl-leucyl-phenylalanine was inhibited in a concentration dependent manner. Alkaloids exerted
similar inhibitory effect in the hypoxanthine–xanthine oxidase system than in stimulated neutrophils, which
could be attributed to a direct ROS scavenging activity. None of the alkaloids assayed had any effect on xanthine
oxidase activity. Therefore the synthesized alkaloids might constitute an alternative therapy in inflammation dis-
orders in which ROS generation is involved.
Key words phenanthrene alkaloid; aporphine alkaloid; semi-synthesis; antioxidant activity
tion and vessel wall reactivity,8,9) and we have recently found
A generalized inflammatory response is associated to the
that phenanthrene alkaloids can exert antiinflammatory activ-
ity through inhibition of ROS generation,10) we have evalu-
ated the possible antioxidant activity of these compounds by
two different methods. Thus, they may have a potential use as
antiinflammatory agents.
pathogenesis of various circulatory and neurodegenerative
disorders such as thrombosis, atherosclerosis1) and
Alzheimer’s disease,2) which is now thought to have an in-
flammatory base.3) The development of antiinflammatory
agents which prevent the accumulation of leucocytes, platelet
activation or lost of function of the vessel wall, could consti-
tute new therapeutic strategies for the control of these and
others inflammatory diseases.
Interestingly aporphines and phenanthrene alkaloids have
been shown to exert a strong antiplatelet and vasorelaxing ef-
fects.4,5) In this context, the aporphine boldine can inhibit the
aggregation of rabbit platelets induced by arachidonic acid
and collagen but it did not affect to the platelet aggregation
induced by platelet activating factor (PAF), thrombin or a
tromboxane analog U46619.6) Conversely, its corresponding
phenanthrene alkaloid secoboldine, in addition to arachi-
donic acid and collagen, it can also inhibit the platelet aggre-
gation induced by PAF or U46619.
Experimental
Reagents All chemicals are of the highest commercially available pu-
rity. Boldine, BSA, DMSO, f-MLP, gelatin, glucose, hypoxanthine, luminol,
microperoxidase and xanthine oxidase were purchased from Sigma Chemi-
cal Co. (St. Louis, MO, U.S.A.). All other reagents are purchased from
Aldrich-Chemie (Steinheim, Germany).
General Experimental Procedure Melting points were determined on
a Fisher-John apparatus and are uncorrected. UV spectra were taken on a
Shimadzu 2101 UV/Vis spectrophotometer in MeOH solution. MS were per-
formed using a VG Auto Spec Fisons Spectrometer. NMR measurements
(data reported in d) were run on a Varian Unity-400 instrument. The chemi-
cal shift are referenced to solvent signals at 7.25 and 77.0 ppm respectively.
Multiplicities of 13C-NMR resonances were determined by distortionless en-
hancement by polarization transfer (DEPT). Standard pulse sequences were
employed for magnitude COSY (correlated spectroscopy). HMQC (het-
Furthermore, some aporphine and phenanthrene alkaloids eronuclear multiple quantum coherence) and HMBC (heteronuclear multiple
have been shown to inhibit PAF binding to its receptor.7) In
this study it was suggested that the piperidine ring (ring B) of
bond connectivity) experiments to establish correlations were carried out.
Thin-layer chromatography (TLC) was run on Merck precoated silica gel
F254 type 60 plates with detection by UV light and Munnier’s reagent.
the aporphine skeleton may not be important for PAF antago-
nisms from the established structure–activity relationship.
On the other hand, studies investigating the potential va-
sorelaxing activity of these type of alkaloids demonstrated
that phenanthrene alkaloids with a tertiary amine or N-oxide
and two methoxy groups or a methylenedioxy group at C-3
and C-4 (ring A), were the most potent vasorelaxing agent. In
contrast, when the substituent group at C-6 and C-7 in ring C
are both methoxy groups vasorelaxing action was reduced.4)
Therefore, the aim of the present study is to synthesize
phenanthrene alkaloids with secondary, tertiary or quaternary
amine from boldine as starting material with oxygenated
substituents in C-3 and C-4 (ring A) and with or without both
methoxy groups at C-6, C-7 (ring C). Despite this class of
compounds may inhibit platelet aggregation or behave as va-
sorelaxing agents, little is known about their antioxidant
Synthesized Compound 1: A mixture of boldine (1 g, 3.05 mmol) and
2 M aqueous ammonium acetate (7.5 ml) and ethanol (7.5 ml) was refluxed in
a bath at 103 °C during 48 h. After it was cooled at room temperature a
white amorphous powder was precipitated (887.9 mg). The solid was filtered
under vacuum and washed with ethanol to give 111) as a white crystal, yield
88.8%; mp 163—165 °C. UV (MeOH): lmax (log e)ꢀ265 (1.64), 320 (0.35)
nm. 1H-NMR (DMSO-d6) d: 2.38 (3H, s, N–CH3), 2.84 (2H, m, CH2a), 3.13
(2H, m, CH2b), 3.79 (3H, s, OCH3-4), 3.93 (3H, s, OCH3-6), 7.07 (1H, s, H-
2), 7.19 (1H, s, H-8), 7.44 (1H, d, Jꢀ8.8 Hz, H-9), 7.69 (1H, d, Jꢀ8.8 Hz,
H-10), 9.04 (1H, s, H-5). 13C-NMR (DMSO-d6) d: 32.04 (CH2b), 34.84
(NCH3), 51.65 (CH2a), 55.30 (OCH3, C-6), 59.16 (OCH3, C-4), 108.50 (C-
5), 111.57 (C-8), 117.96 (C-2), 120.61 (C-10), 122.68 (C-9), 123.30 (C-5a),
123.82 (C-4a), 124.37 (C-10a), 128.22 (C-8a), 132.43 (C-1), 142.23 (C-4),
146.60 (C-7), 147.70 (C-6), 148.12 (C-3). [HMQC data were used to assign
proton and carbon correlations] HR-EI-MS: m/zꢀ327.146326 calc. mass
327.147058 C19H21NO4: EI-MS: m/z (rel. int.)ꢀ327 (20), 284 (100), 269
(8.2), 240 (10), 84 (6.5), 58 (2.8).
Compound 2: To a solution of 1 (100 mg, 0.305 mmol) in a mixture of
methanol (5 ml) and acetonitrile (7.5 ml) was added CH3I (0.019 ml,
properties. Since ROS are involved in both platelet aggrega- 0.305 mmol). The resulting mixture was stirred overnight at room tempera-
∗ To whom correspondence should be addressed. e-mail: amparo.blázquez@uv.es © 2004 Pharmaceutical Society of Japan

June 2004
697
ture. At the same time a solution of 1 (100 mg, 0.305 mmol) in the above
conditions was treated during 48 h with an excess of CH3I (0.5 ml,
8.034 mmol). In both experiments compound 2 after crystalization with ace-
tone was obtained as yellow needles, combined yield 95%; mp 237—
239 °C. UV (MeOH): lmax (log e)ꢀ265 (1.57), 320 (0.33) nm. 1H-NMR
(DMSO-d6) d: 3.22 (9H, s, 3ꢁN–CH3), 3.44 (2H, m, CH2a), 3.57 (2H, m,
CH2b), 3.79 (3H, s, OCH3-4), 3.95 (3H, s, OCH3-6), 7.17 (1H, s, H-2), 7.23
(1H, s, H-8), 7.50 (1H, d, Jꢀ9.2 Hz, H-9), 7.68 (1H, d, Jꢀ9.2 Hz, H-10),
9.04 (1H, s, H-5). 13C-NMR (DMSO-d6) d: 25.99 (CH2b), 52.29 (NCH3),
55.32 (OCH3, C-6), 59.29 (OCH3, C-4), 65.32 (CH2a), 108.39 (C-5), 111.62
(C-8), 118.40 (C-2), 120.20 (C-10), 122.54 (C-5a), 123.84 (C-4a), 123.95
(C-9), 124.47 (C-10a), 128.21 (C-8a), 129.13 (C-1), 142.86 (C-4), 146.60
(C-7), 147.85 (C-6), 148.17 (C-3). EI-MS 356 C19H21NO4 m/z (rel.
int.)ꢀ341 (5), 327 (17), 285 (17), 284 (100), 269 (51), 268 (11), 240 (11),
127 (15), 58 (44).
of luminol 5 mM, CaCl2 1 mM and N-formyl-methionyl-leucyl-phenylalanine
(f-MLP) 100 nM was added sequentially to each well, except in the blank
group. Experiments with the appropriate DMSO concentration were also
carried out (1—0.01%). Chemiluminescence was recorded at 4 s intervals
over a 100 s period per well and the area under the curve (AUC’s) was inte-
grated. Drug-induced reduction was expressed as % inhibition. Inhibitory
concentration 50% (IC50) values were then calculated from the concentra-
tion–inhibition curves by non-linear regression analysis.
Measurement of ROS Generation by Hypoxanthine–Xanthine Oxi-
dase The ROS was generated by hypoxanthine–xanthine oxidase system
and detected by luminol-enhanced chemiluminescence using a modified
method.17) Assay 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 synthe-
sized alkaloids (final concentration in 200 ml, 100—0.01 mM) 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 ml
of luminol 5 mM, CaCl2 1 mM and xanthine oxidase 0.02 u/ml was added se-
quentially to each well, except in the blank group. Experiments with the ap-
propriate DMSO concentration were also carried out (1—0.01%). Chemilu-
minescence was recorded at 4 s intervals over a 100 s period per well and the
area under the curve (AUC’s) was integrated. Drug-induced reduction was
expressed as % inhibition. Inhibitory concentration 50% (IC50) values were
then calculated from the concentration–inhibition curves by non-linear re-
gression analysis.
Measurement of the Effect on Xanthine Oxidase Activity by Isoxan-
topterine 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 wave-
length at 390 nm) following the previously described method.18)
Statistical Analysis The IC50 values were calculated from non-linear re-
gression by a software of Prisma 3.0 (Graph Pad Software, San Diego, Cali-
fornia, U.S.A.). All values are shown as meanꢂS.E.M. The difference be-
tween two values was determined by use of unpaired Student’s t-test. The
differences were considered statistically significant if the p-value was less
than 0.05.
Compound 3: The compound 3 was prepared according to the method de-
scribed for 2 by employing a solution of boldine (100 mg, 0.305 mmol) and
CH3I (0.019 ml, 0.305 mmol) to afford compound 312) as a white crystal,
yield 98%; mp 168—170 °C. UV (MeOH): lmax (log e)ꢀ284.9 (0.93), 305
1
(1.02) nm. H-NMR (DMSO-d6) d: 2.79 (1H, dd, Jꢀ14.2, 13.2 Hz, H-7a),
2.90 (1H, m, H-4a), 2.94 (3H, s, N–CH3), 3.13 (1H, m, H-4b), 3.21 (1H, dd,
Jꢀ13.2, 3.6 Hz, H-7b), 3.35 (3H, s, N–CH3), 3.61 (3H, s, OCH3-1), 3.66
(1H, m, H-5a), 3.75 (1H, m, H-5b), 3.77 (3H, s, OCH3-10), 4.55 (1H, dd,
Jꢀ14.2, 3.6 Hz, H-6a), 6.66 (1H, s, H-3), 6,79 (1H, s, H-8), 7.84 (1H, s, H-
11). 13C-NMR (DMSO-d6) d: 23.03 (C-4), 28.09 (C-7), 42.99—52.97
(2ꢁNCH3, C-6), 55.74 (OCH3, C-10), 59.54 (OCH3, C-1), 60.18 (C-5),
68.02 (C-6a), 111.88 (C-11), 114.24 (C-3), 115.23 (C-8), 118.09 (C-3a),
121.76 (C-7a), 124.94 (C-1a), 125.89 (C-11a), 143.81 (C-1), 146.50 (C-9),
146.73 (C-10), 150.91 (C-2). [HMQC data were used to assign correlations
and HMBC for quaternary carbons]. EI-MS 342 C20H24NO4 m/z (rel.
int.)ꢀ341 (11), 283 (3), 128 (6), 59 (3), 58 (100).
Compound 4: A solution of 3 (53 mg, 0.155 mmol) in 1 M aqueous ammo-
nium acetate (0.4 ml) and ethanol (0.4 ml) was refluxed in a bath at 104 °C
for 24 h. The ethanol was evaporated under vacuum and the aqueous layer
was extracted with dichloromethane. The combined organic extracts were
dried over anhydrous Na2SO4 and the solvent was evaporated to give 413,14)
as a yellow crystal, combined yield 50 mg (94%); mp 227—229 °C. UV
1
(MeOH): lmax (log e)ꢀ265 (1.10), 320 (0.25) nm. H-NMR (DMSO-d6) d:
Results and Discussion
2.46 (6H, s, 2ꢁN–CH3), 2.78 (2H, m, CH2a), 3.16 (2H, m, CH2b), 3.79 (3H,
s, OCH3-4), 3.95 (3H, s, OCH3-6), 7.09 (1H, s, H-2), 7.19 (1H, s, H-8), 7.42
(1H, d, Jꢀ9.2 Hz, H-9), 7.66 (1H, d, Jꢀ9.2 Hz, H-10), 9.05 (1H, s, H-5).
13C-NMR (DMSO-d6) d: 29.81 (CH2b), 44.04 (2ꢁNCH3), 55.26 (OCH3, C-
6), 59.13 (OCH3, C-4), 59.20 (CH2a), 108.45 (C-5), 111.54 (C-8), 117.95
(C-2), 120.39 (C-10), 122.69 (C-5a), 123.42 (C-9), 123.76 (C-4a), 124.36
(C-10a), 128.17 (C-8a), 131.97 (C-1), 142.27 (C-4), 146.53 (C-7), 147.68
(C-6), 148.05 (C-3). HR-EI-MS: m/zꢀ341.161528 calc. mass 341.162708
C20H23NO4. EI-MS: m/z (rel. int.)ꢀ341 (50), 283 (15), 240 (12), 58 (100).
Compound 5: This compounds was prepared in three diferents conditions.
K2CO3 (200 mg, 1.449 mmol) was added to three solutions of boldine
(100 mg, 0.305 mmol) in acetonitrile (7.5 ml) and methanol (5 ml). After ad-
dition of K2CO3 was completed the solutions were treated with 2 equivalents
(0.038 ml, 0.610 mmol), 3 equivalents (0.057 ml, 0.915 mmol) or an excess
of CH3I (0.1 ml, 1.607 mmol). The mixtures were stirred at room tempera-
ture for 48 h. The methanol/acetonitrile was evaporated under vacuum and
extracted with dichloromethane. The combined organic extracts were puri-
fied by addition of hexane to give in all reactions compound 515) as a yellow
crystal, combined yield 75%; mp 361—363 °C. UV (MeOH): lmax
(log e)ꢀ265 (0.85), 310 (0.24) nm. 1H-NMR (DMSO-d6) d: 3.26 (9H, s,
3ꢁN–CH3), 3.56 (2H, m, CH2a), 3.63 (2H, m, CH2b), 3.79 (3H, s, OCH3-4),
3.95 (3H, s, OCH3-6), 7.47 (1H, s, H-2), 7.53 (1H, s, H-8), 7.70 (1H, d,
Jꢀ9.2 Hz, H-9), 7.79 (1H, d, Jꢀ9.2 Hz, H-10), 9.11 (1H, s, H-5). 13C-NMR
(DMSO-d6) d: 26.19 (CH2b), 52.31 (3ꢁNCH3), 55.22—59.52 (4ꢁOCH3),
65.24 (CH2a), 108.31 (C-5), 108.40 (C-2), 115.27 (C-8), 120.16 (C-10),
125.24 (C-9). EI-MS 384 C23H30NO4 m/z (rel. int.)ꢀ324(11), 277 (12), 58
(100).
The synthesis of the compounds 1—5 was performed
starting from boldine, according to the pathways shown in
Chart 1. A variety of synthetic methods to obtain secoboldine
1, have been reported in the literature, ranging from Von
Braun reaction of boldine by treatment with BrCN in CHCl3
under reflux, followed by alkaline hydrolysis,11) photolysis19)
and via solvolysis of boldine with NH4OAc in EtOH under
reflux.20,21) Among these reactions, we have selected this last
strategy since this method seemed to be a straightforward
one-pot reaction. In this method the participation of a pheno-
lic group and the temperature control were essential for satis-
factory yields. After several trials the bath temperature of
103—104 °C was the best range to obtain our purpose. At
bath temperature under 98 °C the reaction did not take place.
Boldine was refluxed 48 h in a bath at 103 °C with aqueous
NH4OAc and EtOH (1 : 1) to give after crystallization com-
pound 122) in 89% yield.
Modification of the methylamino-dimethylene side chain
of 1 was performed to reduce lipophilicity by treating with
methyl iodide in excess at room temperature during 48 h. The
crystallization as needles with acetone led us analytically
pure product 2 for biological assays.
The phenanthrene compound boldine methine 4, was pro-
vided as shown in Chart 1 in two steps. First, the N-methyl-
boldine 3,13) was synthesized using a simple reaction in
which the starting boldine was stirred overnight at room tem-
perature with an excess of methyl iodide (98% yield) in a
mixture of CH3CN–MeOH (7.5 : 5). The ring openning of the
piperidinium ring in compound 3 with aqueous ammonium,
Measurement of ROS Generation from Human PMNs The forma-
tion of ROS by human PMNs was assessed by luminol-enhanced chemilu-
minescence with a modified method.16) Assay was carried out in opaque 96
well plates, 105 cells per well were suspended in an assay volume of 180 ml
of Krebs-HEPES buffer containing glucose 5.6 mM, bovine serum albumin
(BSA) 0.05% (w/v), microperoxidase 2 mM with gelatine 0.1% pH 7.4, alone
or in combination with alkaloids (final concentration in 200 ml, 100—
0.01 mM) for 30 min at 37 °C. All the assays were performed in duplicate.
Plates were placed in a Wallac 1420 Victor2 Multilabel Counter. Then 20 ml

698
Vol. 52, No. 6
(a) NH4AcO, EtOH; (b) MeOH, CH3CN, CH3I; (c) MeOH, CH3CN, CH3I; (d) NH4AcO, EtOH; (e) MeOH, CH3CN, K2CO3, CH3I.
Chart 1
Table 1. IC50 of the Alkaloids Tested for ROS Generation by f-MLP Stim-
ulated Human Neutrophils or Hypoxanthine–Xanthine Oxidase System
f-MLP stimulated
human neutrophils
IC50 (mM)
Hypoxanthine–
xanthine oxidase
system IC50 (mM)
Alkaloid
Ascorbic acid
Boldine
99.60ꢂ0.2
167.80ꢂ13.5
0.27ꢂ0.17**
0.16ꢂ0.07**
0.15ꢂ0.09**
0.47ꢂ0.11**
0.23ꢂ0.08**
6.12ꢂ0.10**
1.39ꢂ0.18**
0.50ꢂ0.13**
0.62ꢂ0.22**
1.27ꢂ0.10**
0.15ꢂ0.07**
18.83ꢂ0.35**
1
2
3
4
5
Statistical significance from positive control ∗∗ pꢃ0.01
as described above, was carried out in the final step. The
crude was concentrated and extracted with dichloromethane.
Boldine methine14) was obtained in 94% yield.
Finally, the treatment of boldine at room temperature,
using 3, 4 equivalents or an excess of methyl iodide and an
excess of K2CO3, gave in all the experiments the 1-phenan-
threne ethanaminium 3,4,6,7-tetramethoxy-N-trimethyl io-
dide 5.15)
Fig. 1. Effect of Alkaloids on ROS Generation in f-MLP Stimulated
PMNs
PMNs were incubated in the absence or presence of alkaloids (100—0.01 mM) at
37 °C as described in Experimental. Thirty minutes later f-MLP 100 nM was added to
each well and chemiluminiscence recorded at 4 s intervals over a 100 s period. Results
are expressed as percentage inhibition of ROS generation in PMNs stimulated with f-
The effect of aporphine and phenanthrene alkaloids on MLP. Results are represented as meanꢂS.E.M. of nꢀ5—7 preparations.
free radical generation was investigated by two different
methods: measurement of ROS generation in stimulated aporphine skeleton (Fig. 1). All the alkaloids assayed were
human PMNs16) and measurement of ROS scavenging activ- more potent than ascorbic acid (IC50 99.6 mM), the reference
ity in a enzymatic system.17) In the first test, all the alkaloids drug employed in this test. Since most of these compounds
tested inhibited in a concentration-dependent manner f-MLP may exert this action, as occurs with other types of com-
induced ROS generation in human PMNs although with dif- pounds such a-tocopherol or ascorbic acid, through their free
ferent potency. The order of potency, based on the calculated radical scavenging activity, they were tested on a cell free
IC50 values (Table 1) showed that the phenanthrene alkaloid ROS generation system. All the alkaloids assayed were capa-
with phenolic groups at C-3, C-7 (compounds 1, 2, 4) were ble of scavenging ROS generated by the hypoxanthine–xan-
more potent than their corresponding equivalents with apor- thine oxidase system in a concentration-dependent manner
phine skeleton. Phenanthrene alkaloids without phenolic (Fig. 2). The order of potency based on the calculated IC50
group displayed lower activity than even though those with values (Table 1) revealed that all the synthesized compounds

June 2004
699
The preliminary studies of structure–activity relationship
with these class of compounds revealed that the presence of
phenolic groups is essential for a strong antioxidant activity
and show that with the same substituents the alkaloids with
phenanthrene skeleton are more active than their respective
alkaloids with aporphine skeleton. In this sense phenanthrene
alkaloids may become promising candidates for the develop-
ment of antiinflammatory agents ought to their potent ROS
scavenging activity.
Acknowledgments The present study has been supported by grants
SAF 2002-01482 and PB 98-1422 from CICYT, Spanish Ministerio de
Ciencia y Tecnologia and has been awarded with the 2002 prize of the Span-
ish Pharmacological Society and Almirall-Prodesfarma laboratories. L. Mil-
ian was supported by a grant from Spanish, Ministerio de Educacion, Cul-
tura y Deporte.
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Fig. 2. Effect of Alkaloids on ROS Generation by Hypoxanthine–Xan-
thine Oxidase System
Hypoxanthine was incubated in the absence or presence of alkaloids (100—0.01 mM)
at 37 °C as described in Experimental. Five minutes later xanthine oxidase was added to
each well and chemiluminiscence recorded at 4 s intervals over a 100 s period. Results
are expressed as percentage inhibition of ROS generation in hypoxanthine–xanthine ox-
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Table 2. Effect of Alkaloids on Isoxantopterine Formation from Pterine by
Measuring Enzyme Activity (mmol/min)
Control
Boldine
4.827ꢂ0.247
5.001ꢂ0.320
4.858ꢂ0.072
5.160ꢂ0.110
Control
3.928ꢂ0.648
3.820ꢂ0.300
4.280ꢂ0.130
3.770ꢂ0.520
2
3
5
1
4
11) Lee S. S., Lin Y. J., Chen M. Z., Wu Y. C., Chen C. H., Tetrahedron
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(1983).
Data are meansꢂS.E.M. No statistical significance pꢃ0.05 from control was ob-
served.
were more active than the classic antioxidant ascorbic acid
(IC50 167.8 mM), employed as control in this test. In this
assay, a similar profile of structure–activity relationship to
that encountered in ROS generated in f-MLP stimulated neu-
trophils was observed. Indeed, phenanthrene alkaloids with
phenolic substituents were the most active, followed by the
group of the aporphine alkaloids with phenolic groups and
phenanthrene alkaloid without phenolic groups.
To confirm that aporphine and phenantrene alkaloids exert
ROS scavenging activity and that this effect was not due to
xanthine oxidase inhibition, another set of experiments were
carried out to investigated such possibility. None of the alka-
loids assayed affected xanthine oxidase activity (Table 2), in-
dicating a clear ROS scavenging activity of these com-
pounds. Therefore a potent new class of phenanthrene alka-
loids with ROS scavenger activity have been synthesized.
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