Lycorine Derivatives Against Trichomonas vaginalis

Article abstract of DOI:10.1111/j.1747-0285.2012.01333.x

Six lycorine derivatives were prepared, characterized, and evaluated for their in vitro anti-Trichomonas vaginalis activity. Compounds bearing an acetyl (2), lauroyl (3), benzoyl (4 and 5), and p-nitrobenzoyl (6 and 7) groups were synthesized. The best activity was achieved with lycorine esterified at C-2 position with lauroyl group. Preliminary structure-activity relationship points that unprotected OH group at C-1 and C-2 is not necessary to the antiparasitic activity, and none of the derivative was less active than lycorine. The lycorine structural requisites required to kill this amitochondriate cell seem to be different in comparison with the derivatives most active against other parasites and tumor cell lines, both mitochondriated cells. This result is an important contribution with our ongoing studies regarding the mechanism of action of the Amaryllidaceae alkaloids on T. vaginalis cell death opening a new perspective to optimize this innovative pharmacological potential.

Full text of DOI:10.1111/j.1747-0285.2012.01333.x

Chem Biol Drug Des 2012; 80: 129–133
ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01333.x
Research Letter
Lycorine Derivatives Against Trichomonas
vaginalis
Raquel B. Giordani1, Celso O. R. Junior2,
Jean P. de Andrade3, Jaume Bastida3,
Jose A. S. Zuanazzi1, Tiana Tasca1 and
Mauro V. de Almeida2,*
(3), and the ability of trichomonads to produce subcutaneous
abscess lesions in mice (4). Complications related to trichomonosis
include infertility (5), cervical cancer (6), pelvic inflammatory disease
(7), birth outcomes, and increase in human immunodeficiency virus
(HIV) transmission and acquisition (8).
1Faculdade de Farmꢀcia, Universidade Federal do Rio Grande do
Sul, Av. Ipiranga 2752, 90610-000 Porto Alegre, RS, Brazil
2Departamento de Quꢁmica, Instituto de CiÞncias Exatas,
Universidade Federal de Juiz de Fora, Campus Martelos, 36036-900
Juiz de Fora, MG, Brazil
Metronidazole and tinidazole are two drugs of choice recommended
by Food and Drug Administration (FDA, USA) for the treatment of
human trichomonosis (9). However, its potential carcinogenicity in
rats and mutagenicity in bacteria, when administered at high doses
for protracted periods of time (10,11), have been harmful. Reports on
metronidazole-resistant isolates of T. vaginalis have increased,
besides common adverse reactions (12–14). To improve the current
chemotherapy of T. vaginalis infection, natural products as well as
synthetic compounds could be a source of new antiprotozoal drugs
with high activity, low toxicity, and alternative mechanisms of action.
3Departament de Productes Naturals Biologia Vegetal i Edafologia,
Facultat de Farmꢂcia, Universitat de Barcelona, Avda. Diagonal 643,
E-08028, Barcelona, Spain
*Corresponding author: Mauro V. de Almeida,
mauro.almeida@ufjf.edu.br
Six lycorine derivatives were prepared, character-
ized, and evaluated for their in vitro anti-Tricho-
monas vaginalis activity. Compounds bearing an
acetyl (2), lauroyl (3), benzoyl (4 and 5), and p-
nitrobenzoyl (6 and 7) groups were synthesized.
The best activity was achieved with lycorine esteri-
fied at C-2 position with lauroyl group. Preliminary
structure–activity relationship points that unpro-
tected OH group at C-1 and C-2 is not necessary to
the antiparasitic activity, and none of the deriva-
tive was less active than lycorine. The lycorine
structural requisites required to kill this amitoc-
hondriate cell seem to be different in comparison
with the derivatives most active against other para-
sites and tumor cell lines, both mitochondriated
cells. This result is an important contribution with
our ongoing studies regarding the mechanism of
action of the Amaryllidaceae alkaloids on T. vagi-
nalis cell death opening a new perspective to opti-
mize this innovative pharmacological potential.
Our ongoing studies regarding the anti-T. vaginalis activity of natu-
ral products, specially on Amaryllidaceae alkaloids, have showed
interesting results. Crude extracts of alkaloids and isolated com-
pounds from several Amaryllidaceae species showed antitrichomon-
as activity (15). The investigation of the mechanism of action
implicated, using the alkaloids lycorine and candimine, demon-
strated that the extracellular ATP and adenosine levels could be
modulated by these compounds, and it could be relevant in increas-
ing susceptibility of T. vaginalis to host immune response in the
presence of these alkaloids (16). Additionally, we verified that cell
death induced by lycorine and candimine involves arrest of the par-
asite cell cycle and displays an unprecedented group of effects that
failed to fulfill the criteria for apoptosis and apoptosis-like death
already reported in trichomonads, suggesting some similarities to
paraptotic cell death described for multicellular organisms (17,18).
To advance in our study on cytotoxicity of Amaryllidaceae alkaloids
against T. vaginalis, in this work, we report the preparation of six
lycorine derivatives, their antiprotozoal activity, and some prelimin-
ary conclusions about structure–activity relationships (SAR).
Key words: alkaloid, Amaryllidaceae, Hippeastrum, lycorine deriva-
tives, Trichomonas vaginalis
Received 22 June 2011, revised 1 December 2011 and accepted for
publication 8 January 2012
Materials and Methods
Chemical experimental information
Trichomonas vaginalis is a flagellate protozoan that infects the
human urogenital tract and causes trichomonosis, the most common
non-viral sexually transmitted disease (1). The factors considered to
be involved in the pathogenicity of T. vaginalis include the ability of
trichomonads to adhere to vaginal epithelial cells (2), the cytotoxic
effect of the pathogen on host cells, trichomonad proteinase activity
All reagents and solvents were purchased from commercial sources.
Silica gel GF254 was used for thin-layer chromatography and Silica
1
gel 60 G for column chromatography. The H NMR (300 MHz) and
13C NMR (75 MHz) spectra were recorded on a Bruker DRX spec-
trometer using tetramethylsilane as the internal standard and CDCl3
as the solvent; the chemical shifts are reported in ppm (d).
129

Giordani et al.
Lycorine (1) was obtained as the main alkaloid from the bulbs of
Hippeastrum santacatarina (Traub) Dutilh (19). 1H NMR (CDCl3,
300 MHz): d 6.80 (1H, s, H-7); 6.57 (1H, s, H-10); 5.40 (1H, s, H-3);
5.90 (2H, s, OCH2O); 4.37 (1H, s, H-1); 4.15 (1H, s, H-2); 4.10 (1H, d,
J 14.1 Hz, H-6); 3.30 (1H, d, J 14.1 Hz, H-6); 3.25 (1H, m, H-12);
2.79 (1H, d, J 10.2 Hz, H-4a); 2.65 (1H, d, J 10.1 Hz, H-10b); 2.55
(2H, m, H-11); 2.35 (1H, m, H-12). 13C NMR (CDCl3, 75 MHz): d
149.7, 148.1, 137.9, 130.6, 125.7, 122.9, 108.8, 106.4, 102.8, 71.9,
70.1, 61.8, 54.2, 55.1, 38.2, 30.3. The lycorine derivatives were pre-
pared using the synthetic strategy described in Scheme 1.
dichlorometane (1 mL) and pyridine (3 mL) in the presence of cata-
lytic amount of DMAP. The reaction mixture was stirred for 5 days
at room temperature. The solvent was evaporated under reduced
pressure and extracted with chloroform and water. After evapora-
tion at reduced pressure of the organic layer, the residue was puri-
fied by column chromatography using dichlorometane ⁄ methanol as
eluent furnishing 3 as a pale yellow oil in 40% yield (18 mg,
25
0.04 mmol); [a]D + 85.0 (c 0.4, CHCl3); IR mmax ⁄ cm: 3421 (O-H); 2853–
1
2959 (C-H aliphatic chain); 1732 (C=O). H NMR (CDCl3, 300 MHz): d
6.80 (1H, s, H-7); 6.61 (1H, s, H-10); 5.92 (2H, s, OCH2O); 4.50 (1H, s,
H-1); 5.31 (1H, s, H-3); 5.48 (1H, s, H-2); 4.12 (1H, d, J 14.3 Hz, H-6);
3.68 (1H, d, J 14.3 Hz, H-6); 3.37 (1H, m, H-12); 2.70 (4H, m, H-4a,
H-10b and H-11); 2.32 (1H, m, H-12), 1.24 (C12H25). 13C NMR (CDCl3,
75 MHz): d 173.5, 147.0, 146.6, 145.2, 130.7, 129.3, 114.5, 107.9,
104.9, 101.2, 73.4, 69.1, 60.6, 56.5, 53.9, 41.2, 29.7, 22.8, 14.3.
1,2-di-O-acetyllycorine (2) was obtained by reaction of lycorine
(0.105 mmol) with acetic anhydride (0.53 mmol) in pyridine (3 mL) in
the presence of catalytic amount of dimethylaminopyridine (DMAP).
The reaction mixture was stirred for 24 h at room temperature. The
solvent was evaporated and extracted with chloroform and water.
The organic layer was evaporated under reduced pressure, and the
residue was purified by column chromatography using dichlorome-
tane ⁄ methanol as eluent furnishing 2 as a white crystal in 62%
1,2-O-dibenzoyllycorine (4) and 2-O-benzoyllycorine (5) were
obtained by reaction of lycorine (0.105 mmol) with benzoyl chloride
(0.21 mmol) in pyridine (2 mL) and in the presence of catalytic
amount of DMAP. The reaction mixture was stirred for 2 days at
room temperature. The solvent was evaporated under reduced pres-
sure followed by extraction with chloroform and saturated aqueous
solution of sodium bicarbonate. The organic layer was concentrated,
and the residue was purified by column chromatography using
25
yield (25 mg, 0.065 mmol). m.p. 217–218 ꢀC; [a]D + 10.0 (c 0.2,
CHCl3); [lit.(20) m.p. 216–217 ꢀC; [a]D25)31.1 (c 1.1, CHCl3]; IR
mmax ⁄ cm: 2731–2954,7 (C-H aliphatic chain); 1728 (C=O). 1H NMR
(CDCl3, 300 MHz): d 6.73 (1H, s, H-7); 6.56 (1H, s, H-10); 5.90 (2H,
s, OCH2O); 5.72 (1H, s, H-1); 5.52 (1H, s, H-3); 5.24 (1H, s, H-2);
4.17 (1H, d, J 12.4 Hz, H-6); 3.53 (1H, d, J 12.4 Hz, H-6); 3.36 (1H,
m, H-12); 2.87 (1H, d, J 10.3 Hz, H-10b); 2.79 (1H, d, J 10.3 Hz, H-
4a); 2.64 (2H, m, H-11); 2.41 (1H, m, H-12), 2.07 (3H, s, OCOCH3);
1.94 (3H, s, OCOCH3). 13C NMR (CDCl3, 75 MHz): d 170.1, 169.9,
146.6, 146.5, 146.1, 129.4, 126.7, 114.0, 107.5, 105.2, 101.1, 71.0,
69.4, 61.3, 56.9, 53.7, 40.5, 28.8, 21.3, 21.0.
dichlorometane ⁄ methanol as eluent yielding compounds
(0.046 mmol, 23 mg, 47%) and 5 (0.023 mmol, 9 mg, 23%).
4
Compound 4 was obtained as a white crystal; m.p. 90–93 ꢀC;
25
[a]D + 14.3 (c 0.14, CHCl3); IR mmax ⁄ cm: 3036–3037 (O-H); 2775–
1
2924 (C-H aliphatic chain); 1718 (C=O). H NMR (CDCl3, 300 MHz):
d 8.06 (2H, d, J 7.2 Hz, H-2¢, H-6¢), 7.90 (2H, d, J 7.2 Hz, H-2¢, H-
6¢), 7.62 (4H, m, H-3¢, H-5¢), 7.30 (2H, m, H-4¢), 6.57 (1H, s, H-10);
6.86 (1H, s, H-7); 6.14 (1H, s, H-2); 5.68 (2H, s, H-1, H-3); 5.85 (2H,
2-O-lauroyllycorine (3) was obtained by reaction of lycorine
(0.105 mmol) with lauroyl chloride (0.800 mmol) in a mixture of
Scheme 1: Reagents and
conditions: (a) Ac2O, DMAP, py; (b)
CH3(CH2)10COCl, DMAP, py; (c)
C6H5COCl, DMAP, dichlorometane,
py; (d) p-NO2C6H4COCl, DMAP,
dichlorometane, py. DMAP,
dimethylaminopyridine
130
Chem Biol Drug Des 2012; 80: 129–133

Lycorine Derivatives Against Trichomonas vaginalis
s, OCH2O); 4.21 (1H, d, J 14.2 Hz, H-6); 3.60 (1H, d, J 14.2 Hz, H-6);
3.44 (1H, m, H-12); 3.17 (1H, d, J 10.3 Hz, H-4a); 3.04 (1H, d, J
10.3 Hz, H-10b); 2.63 (2H, m, H-11); 2.53 (1H, m, H-12). 13C NMR
(CDCl3, 75 MHz): d 165.5, 165.4, 146.7, 146.5, 146.4, 133.3, 131.3,
130.0, 128.5, 128.5, 114.2, 107.5, 105.3, 101.1, 71.2, 69.8, 61.8,
57.0, 53.8, 41.1, 29.8.
cally in vitro (21) and incubated at 37 ꢀC (€0.5). Organisms in the
logarithmic phase of growth and exhibiting motility and normal mor-
phology were harvested, centrifuged, and resuspended in new cul-
ture medium for cytotoxic assays. Parasites with cellular density of
5.0 · 105 trophozoites ⁄ mL were treated with lycorine and their
derivatives at final concentrations of 125 and 250 lM. Importantly,
DMSO at 0.6% (final percentage) was used for solubilization of the
samples according to the previous studies (22). All the results were
expressed as the percentage of living organisms compared to
untreated parasites after 24 h of incubation, considering motility,
normal morphology, and exclusion of trypan blue dye. All experi-
ments were performed in triplicate and with at least four indepen-
dent cultures (n = 4).
Compound 5 was obtained as a white crystal; m.p. 96 ꢀC;
25
[a]D + 100.0 (c 0.1, CHCl3); IR mmax ⁄ cm: 3412 (O-H); 2851–2957 (C-
H aliphatic chain); 1713 (C=O). 1H NMR (CDCl3, 300 MHz): d 8.02
(2H, d, J 7.0 Hz, H-2¢, H-6¢), 7.46 (1H, m, H-4¢), 7.43 (2H, m, H-3¢,
and H-5¢), 6.84 (1H, s, H-7); 6.64 (1H, s, H-10); 5.88 (2H, s, OCH2O);
5.65 (1H, s, H-2); 5.62 (1H, s, H-3); 4.71 (1H, s, H-1); 4.22 (1H, d, J
13.8 Hz, H-6); 3.78 (1H, d, J 13.8 Hz, H-6); 3.48 (1H, m, H-12); 3.22
(1H, d, J 10.3 Hz, H-4a); 2.97 (1H, d, J 10.3 Hz, H-10b); 2.76 (3H, m,
H-11 and H-12). 13C NMR (CDCl3, 75 MHz): d 171.0, 146.8, 145.3,
133.4, 133.1, 130.2, 130.0, 128.6, 128.4, 114.6, 108.0, 105.0, 101.3,
73.9, 69.2, 60.5, 56.2, 53.7, 41.1, 29.1.
Statistical analysis
Results were expressed as means (SD). Statistical analysis was
conducted using one-way analysis of variance (ANOVA) followed by
the Tukey post hoc test. Statistical significance was considered at
p £ 0.05.
2-O-p-nitrobenzoyllycorine (6) and 1-O-p-nitrobenzoyllycorine (7)
were obtained by reaction of lycorine (0.105 mmol) with p-nitro-
benzoyl chloride (0.30 mmol) in pyridine (2 mL) and in the presence
of catalytic amount of DMAP. The reaction mixture was stirred for
4 days at room temperature. The solvent was evaporated under
reduced pressure followed by extraction with chloroform and satu-
rated aqueous solution of sodium bicarbonate. The organic layer
was concentrated, and the residue was purified by column chroma-
tography using dichlorometane ⁄ methanol as eluent yielding com-
pounds 6 (0.016 mmol, 7 mg, 17%) and 7 (0.017 mmol, 8 mg,
18%).
Results and Discussion
The aromatic and aliphatic ester derivatives of lycorine (1), com-
pounds 2–7, were obtained by treatment of 1 with acetic anhy-
dride or acid chlorides, in the presence of pyridine and DMAP
(Scheme 1). To our knowledge, compounds 3–7 are new lycorine
derivatives, so far. Previous studies in this regard have highlighted
the difficulties attending the differential functionalization of the
hydroxyl groups present within the lycorine series (23) because it is
known that ring C of lycorine is aromatized by light, oxygen, or heat
(24). However, C-1 and C-2 positions could be considered a key
point to the discussion on lycorine SAR enabling comparisons with
several activities previously investigated.
Compound 6 was obtained as a colorless solid; m.p. 85–87 ꢀC;
[a]D25-1520.0 (c 0.1, CHCl3); IR mmax ⁄ cm: 3425 (O-H); 2851–2956 (C-
H aliphatic chain); 1724 (C=O). 1H NMR (CDCl3, 300 MHz): d 8.22
(4H, m, H-2¢, H-3¢, H-5¢ and H-6¢), 6.77 (1H, s, H-7); 6.58 (1H, s, H-
10); 5.89 (2H, s, OCH2O); 5.59 (2H, s, H-2; H-3); 4.65 (1H, s, H-1);
4.16 (1H, d, J 14.5 Hz, H-6); 3.55 (1H, d, J 14.5 Hz, H-6); 3.37 (1H,
m, H-12); 2.83 (2H, m, H-11); 2.66 (1H, d, J 10.5 Hz, H-4a and H-
10b); 2.40 (1H, m, H-12). 13C NMR (CDCl3, 75 MHz): d 163.9, 150.9,
147.5, 146.8, 136.3, 131.2, 131.1, 123.8, 123.7, 114.2, 107.7, 104.9,
101.2, 72.0, 70.7, 61.7, 57.0, 53.8, 41.1, 29.9.
Anti-T. vaginalis bioactivity assay was performed on lycorine (1) and
six ester derivatives (2–7), at 125 and 250 lM (Table 1). Lycorine
(1) did not display a significant difference at both concentrations
tested, and around 60% of the parasites remained viable after
24 h. The ester derivatives 2, 4, and 5 at both concentrations and
7 at 125 lM presented anti-T. vaginalis activity equivalent to 1.
The results from the diesterified compounds 2 and 4 showed that
no matter the aliphatic or aromatic nature of the substituent and
even C-1 and C-2, lycorine-substituted derivatives are active.
Compound 7 was obtained as a colorless solid; m.p. 107–108 ꢀC;
25
[a]D + 31.3 (c 0.1, CHCl3); IR mmax ⁄ cm: 3431 (O-H); 2850–2960 (C-
H aliphatic chain); 1728 (C=O). 1H NMR (CDCl3, 300 MHz): d 8.20
(4H, m, H-2¢, H-3¢, H-5¢ and H-6¢), 6.77 (1H, s, H-7); 6.58 (1H, s, H-
10); 5.90 (1H, s, H-1); 5.89 (2H, s, OCH2O); 5.62 (1H, s, H-3); 4.35
(1H, s, H-2); 4.23 (1H, d, J 13.3 Hz, H-6); 3.62 (1H, d, J 13.3 Hz, H-
6); 3.43 (1H, m, H-12); 3.08 (1H, d, J 10.7 Hz, H-4a); 2.97 (3H, d, J
10.7 Hz, H-10b, H-11); 2.52 (1H, m, H-12). 13C NMR (CDCl3,
75 MHz): d 165.5, 165.4, 146.7, 146.5, 146.4, 133.3, 131.3, 130.0,
128.5, 128.5, 114.2, 107.5, 105.3, 101.1, 71.2, 69.8, 61.8, 57.0, 53.8,
41.1, 29.8.
Compound 7 at 250 lM showed a discreet improve on activity: Just
40% of the trophozoites remain viable. Compound 6 showed a good
activity where at 125 lM was equally active than 7 and at 250 lM
was observed just 21% of viable trophozoites. In comparison, both
the p-nitrobenzoyl esters (C-1 or C-2 substituted) were more active
than lycorine, and C-2 unsubstituted seems it to be favorable to
antiprotozoal activity. Nitroimidazoles are the drugs of choice to
treat T. vaginalis, and they possess a nitro group that is involved
on the generation of free radicals and on the effectiveness of the
drug. We could hypothesize that similar event could be occur on
lycorine nitrobenzoyl derivatives as an additional cytotoxic requisite
of the molecule. It is reinforced by observing the results of the ben-
Biological experimental information
The T. vaginalis isolate 30236 (from the American Type Culture Col-
lection) was used in this study. Trichomonads were cultured axeni-
Chem Biol Drug Des 2012; 80: 129–133
131

Giordani et al.
Table 1: Anti-Trichomonas vaginalis activity of lycorine (1) and
their ester derivatives
synthetic difficulties related to lycorine. Finally, the findings
reported here are in agreement with our previous results because
the structural requirements necessary to induce apoptosis in tumor
cell lines were not accomplished by the most active anti-T. vaginalis
derivative.
OR
2
R O
1
H
N
Anti-T. vaginalis
activity (% viable
trophozoites)
O
O
H
Acknowledgments
We thank the CNPq (Brazil) for financial support:
a grant
[476616 ⁄ 2006-9] awarded to J.A.S.Z. and a grant [477348 ⁄ 2008-4]
awarded to T.T; R.B.G. gratefully acknowledges CAPES for fellow-
ship.
Compound
R1
R2
H
125 lM
250 lM
1
2
3
4
5
6
7
H
70a
77a
60b
81b
3b
Acetyl
H
Benzoyl
H
Acetyl
Lauroyl
Benzoyl
Benzoyl
p-Nitrobenzoyl
H
16*a
68
66b
65
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51*a
40*a
80*a
H
21b
42b
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