The synthesis and the in vitro cytotoxicity studies of bisnaphthalimidopropyl polyamine derivatives against colon cancer cells and parasite Leishmania infantum

Article abstract of DOI:10.1016/j.bmc.2006.09.031

Bisnaphthalimidopropyl derivatives (BNIPSpd, BNIPDaoct, BNIPDanon, BNIPDadec, BNIPDpta and BNIPDeta) were synthesised in yields ranging from 50% to 70% and their cytotoxicity against colon cancer cells (Caco-2) and the parasite Leishmania infantum determi

Full text of DOI:10.1016/j.bmc.2006.09.031

Bioorganic & Medicinal Chemistry 15 (2007) 541–545
The synthesis and the in vitro cytotoxicity studies of
bisnaphthalimidopropyl polyamine derivatives against colon cancer
cells and parasite Leishmania infantum
Joao Oliveira,a Lynda Ralton,a Joana Tavares,c Anabela Codeiro-da-Silva,c
˜
Charles S. Bestwick,b Anne McPhersona and Paul Kong Thoo Lina,*
aThe Robert Gordon University, School of Life Sciences, St. Andrew Street, Aberdeen AB25 1HG, Scotland, UK
bThe Rowett Research Institute, Greenburn Road, Aberdeen AB29 9SB, Scotland, UK
cLaboratorio de Bioquimica, Faculdade de Farmacia da Universidade do Porto, Rua Anibal Cunha 164, 4050-047 Porto, Portugal
Received 29 June 2006; revised 11 September 2006; accepted 15 September 2006
Available online 28 September 2006
Abstract—Bisnaphthalimidopropyl derivatives (BNIPSpd, BNIPDaoct, BNIPDanon, BNIPDadec, BNIPDpta and BNIPDeta)
were synthesised in yields ranging from 50% to 70% and their cytotoxicity against colon cancer cells (Caco-2) and the parasite Leish-
mania infantum determined using the MTT assay. Cytotoxicity within Caco-2 cells was manifested with IC50 values between 0.3 and
22 lM. Compounds with the central longer alkyl chains exhibited the highest cytotoxicity. Against L. infantum, IC50 values were
encompassed within a narrower concentration range of 0.47–1.54 lM. In the parasites, the presence of nitrogen in the central chain
and the length of the central alkyl chains did not especially enhance cytotoxicity. This may be due to the way these compounds are
transported in the cells.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
increased DNA binding and cleavage. For example, ace-
naphthalimide was introduced into the naphthalimide
chromophore to increase the solubility of the bisnaph-
thalimide compounds.5,6 Furan heterocycles were added
to the naphthalimide chromophore and those com-
pounds exhibited strong DNA binding properties with
toxicity to CEM leukaemia cells in the nanomolar con-
centration.7 Pyrazine heterocycles have also recently
been fused to naphthalimides and those pyrazino-naph-
thalimides exhibited in vitro toxicity with IC50 values
ranging from 0.002 to 7.8 lM after 72 h treatment in
cancer HT 29, HeLa and PC 3 cells.8
Naphthalimido derivatives exhibit considerable poten-
tial as cytotoxic agents for cancer chemotherapy.1,2 We
previously reported the synthesis and biological activi-
ties of a novel series of bisnaphthalimidopropyl poly-
amines.3 Subsequent work revealed the presence of the
bisnaphthalimidopropyl functionality to be essential
for optimum biological activity since the presence of
an oxygen atom in the a-position of the naphthalimido
ring tends to reduce activity.4 Majority research tradi-
tionally focused on the modification of the naphthalim-
ido rings to enhance anticancer activities through
However, in our laboratory we have developed bisnaph-
thalimidopropyl fragments linked to natural polyamines
such as putrescine, spermidine, and spermine. The sper-
midine and spermine derivatives exhibited enhanced
aqueous solubility while maintaining good biological
activity.9 In MCF 7 breast cancer cells, compounds were
observed within the cell nuclei after 6 and 12 h drug expo-
sure, with transport being potentially energy dependent.
Within MCF7 cells, the bisnaphthalimidopropyl com-
pounds inflicted significant quantitative DNA damage.10
Brana et al. reported similar qualitative observations of
DNA damage in response to their bisnaphthalimidoethyl
Abbreviations: BNIPSpd, bisnaphthalimidopropylspermidine; BNIP-
Daoct, bisnaphthalimidopropyldiamino-octane; BNIPDanon, bis-
naphthalimidopropyldiaminononane; BNIPDadec, bisnaphthalimido-
propyldiaminodecane; BNIPDpta, bisnaphthalimidopropyldipropyl-
triamine; BNIPDeta, bisnaphthalimidopropyldiethyltriamine; DMF,
dimethylformamide; THF, tetrahydrofuran; MTT, 3-(4,5-dimethylthi-
azol-2-yl)-2,5-diphenyltetrazolium bromide.
Keywords: Bisnaphthalimidopropyl; Anticancer; Antiparasitic activity;
Polyamines.
*
Corresponding author. Tel.: +44 1224 262818; fax: +44 1224
262828; e-mail: p.kong@rgu.ac.uk
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.09.031

542
J. Oliveira et al. / Bioorg. Med. Chem. 15 (2007) 541–545
O
N
O
O
N
O
CH2Cl2 gave BNIPSpd, BNIPDaoct, BNIPDanon,
BNIPDadec, BNIPDpta and BNIPDeta as their corre-
sponding di- or trihydrobromide salts in yield varying
from 50% to 70%.
N
N
H
H
Point of diversity
2.2. Biological activities
H
N
BNIPSpd
N
N
H
The in vitro cytotoxicity of all the bisnaphthalmidopro-
pyl derivatives described above was studied against co-
lon cancer cell lines Caco-2 and parasite L. infantum.
In the cancer cell line the IC50 values of each compound
were determined after 24 and 48 h drug exposure (Table
1). All compounds except for BNIPDeta (IC50 values,
21.7 and 22.3 lM for 24 and 48 h, respectively, exerted
IC50 values between 0.15 and 8.00 lM. BNIPSpd was
the most active compound (IC50, 0.47 and 0.15 lM at
24 and 48 h, respectively).
H
N
H
BNIPDaoct
N
H
BNIPDanon
BNIPDadec
N
N
H
H
H
N
N
H
BNIPDpta
BNIPdeta
N
H
N
N
H
H
H
N
N
H
N
H
Figure 1. Bisnaphthalimidopropyl derivatives (BNIPSpd, BNIPDaoct,
BNIPDanon, BNIPDadec, BNIPDpta and BNIPDeta).
The removal of a nitrogen atom from the linker chain
does not appear to substantially affect the cytotoxic
properties of these compounds. We previously reported
that when the central alkyl group is a butyl chain, the
compound (BNIPPut) is not soluble in most solvents
and the aqueous solubility of bisnaphthalimidopropyl
compounds is enhanced by introducing a heteroatom
like nitrogen in the central chain.3 Here, by increasing
the length of the alkyl central chain such as in BNIP-
Daoct, BNIPDanon and BNIPDadec also helps aque-
ous solubility. We reason that with the longer alkyl
chain, the two naphthalimido rings do not tend to stack
on top of each other by p–p interactions between the
aromatic rings and hence favour aqueous solubility.
Among the latter compounds, BNIPDadec showed the
highest cytotoxicity against Caco-2 cells with IC50 values
of 0.36 lM (48 h) and 0.77 lM (24 h).
compounds fused with p excessive rings such as furan or
thiophene.11 In a short communication, we reported that
HL60 promyelocytic leukaemia cells treated with bis-
naphthalimidopropylspermidine (BNIPSpd) exhibited
DNA fragmentation, elevated caspase-3 activity and
form ‘condensed bodies’, suggesting strongly that the
mechanism of cell death is by apoptosis.9 We also found
for the first time that bisnaphthalimidopropyl derivatives
exert significant antiproliferative effects on the life cycle of
Leishmania infantum, the causative agent of visceral leis-
maniasis. These drugs also induced the death of promas-
tigotes by apoptosis.12
In this paper, we report the synthesis of analogues bis-
naphthalimidopropyl di- and triamines, BNIPDaoct,
BNIPDanon, BNIPDadec, BNIPDpta and BNIPDeta,
basedonourleadcompoundBNIPSpd(spermidinederiv-
ative) with modification of the central chain as shown in
Figure 1. The modification consists of different alkyl
lengths of the central chain with 2 or 3 nitrogen atoms,
thus modulating the number of positive charges in the
molecules. We also discuss the in vitro cytotoxic proper-
ties of these newly synthesised compounds in colon cancer
cells (Caco-2) and parasites (L. infantum, promastigotes).
Polyamine derivatives have recently shown promise in
the search for more effective chemotherapeutic agents
against parasite L. infantum. For example, N4,N8-
bis(3-phenylpropyl)spermine, N4,N8-bis(3-naphthylm-
ethyl)spermine and N1,N8-bis(3-naphthylmethyl)spermi-
dine were reported to be potent trypanocides in vitro
with IC50 values ranging from 0.19 to 0.83 lM.13 More
recently, using a combinatorial chemistry approach to
produce a number of diamine libraries, Avery et al.
reported a number of diamine derivatives with very
good in vitro activity against L. infantum with the most
active compound exhibiting an IC50 value of 0.88 lM.14
In our current series of bisnaphthalimidopropyl com-
pounds we observed equally promising cytotoxic prop-
erties against parasite L. infantum (Table 2).
2. Results and discussion
2.1. Chemistry
The synthetic strategy (Scheme 1) adopted to synthesise
bisnaphthalimidopropyl derivatives BNIPSpd, BNIP-
Daoct, BNIPDanon, BNIPDadec, BNIPDpta, and
BNIPDeta was based on methods previously developed
in our laboratory.3,10 Protection and activation of all the
di- and triamines were carried out with mesitylene chlo-
ride in pyridine at room temperature to give compounds
1–6 in high yield. N-alkylation of the latter compounds
with O-tosylpropylnaphthalimide 7 with caesium car-
bonate in anhydrous DMF afforded the fully protected
bisnaphthalimidopropyl derivatives which upon depro-
tection with hydrobromic acid/glacial acetic acid in
In conclusion, the new bisnaphthalimidopropyl deriva-
tives exhibit cytotoxicity that may be further developed
as antitumour and/or antiparasitic therapeutic agents.
We are currently investigating the extent of DNA dam-
age and repair in drug treated cells and these observa-
tions will be reported elsewhere.
2.3. Experimental
Caco-2 cells (ECACC, 86010202) were obtained from
the European Collection of Cell Cultures. All reagents

J. Oliveira et al. / Bioorg. Med. Chem. 15 (2007) 541–545
543
H
N
Mts
1
N
H
N
Mts
Mts
Mts
H
Mts
Mts
N
2
3
N
H
O
N
O
O
Mts
N
H
N
S
O
H
+
H
N
O
Mts
Mts
4
5
N
H
Mts
7
Mts
N
N
N
H
H
Mts
Mts
6
Mts
N
Mts
N
H
N
H
O
S
Mts =
O
1. DMF, Cs2CO3 for 12 hr at 80oC
O
2. CH2Cl2, 20% HBr/galcial CH3COOH
overnight at room temperature
Naphth =
NH
O
H
N
Naphth
Naphth
3HBr
2HBr
BNIPSpd
Naphth
N
H
N
H
H
N
Naphth
Naphth
Naphth
N
H
BNIPDaoct
BNIPDanon
N
H
N
Naphth
2HBr
H
H
N
Naphth 2HBr
BNIPDadec
BNIPDpta
N
H
Naphth
N
H
N
H
N
Naphth
Naphth
H
3HBr
3HBr
H
N
Naphth
N
H
N
BNIPDeta
H
Scheme 1. Synthetic strategy for the synthesis of bisnaphthalimidopropyl derivatives.
Table 1. Cytotoxicity of polyamine analogues against Caco-2 cancer
cells
Table 2. Cytotoxicity of Polyamine derivatives against parasite Leish-
mania infantum
Compounda
IC50 (lM)
Compounda
IC50 (lM)
72 h
24 h
48 h
BNIPSpd
0.47 0.01
0.78 0.049
0.78 0.10
ND
BNIPSpd
0.47 0.12
6.20 1.42
3.60 0.50
0.77 0.15
5.14 1.13
21.7 4.49
0.15 0.04
3.20 0.67
0.67 0.11
0.36 0.08
1.54 0.51
22.3 5.62
BNIPDaoct
BNIPDanon
BNIPDadec
BNIPDpta
BNIPDeta
BNIPDaoct
BNIPDanon
BNIPDadec
BNIPDpta
BNIPDeta
1.54 0.21
0.74 0.11
a Cytotoxicity determined by MTT assay. The results were obtained
after treatment of the promastigote form of the parasite with different
analogue concentrations (0.30–100 lM) after 72 h of incubation. The
results are representative of medium SD at least five assays. ND,
not determined.
a Cytotoxicity determined by MTT assay. Data obtained after treating
Caco-2 cells with varying concentrations of analogues (0.01–40 lM)
for 24 and 48 h. Data are means SD of six replicates.
were purchased from Aldrich, Fluka and Lancaster and
were used without purification. TLC was performed on
Kieselgel plates (Merck) 60 F254 in chloroform: metha-
nol (97:3 or 99:1). Column chromatography was done
with silica gel 60, 230–400 meshes using chloroform
and methanol as eluent. FAB-mass spectra were ob-
tained on a VG Analytical AutoSpec (25 kV) spectro-
meter, EC/CI spectra were performed on a Micromass
Quattro II (low resolution) or on a VG Analytical

544
J. Oliveira et al. / Bioorg. Med. Chem. 15 (2007) 541–545
1
ZAB-E instrument (accurate mass). H and 13C NMR
spectra were recorded on a JEOL JNM-EX90 FT
NMR spectrometer.
161.70 (C@O), 135.70–122.90 (aromatic carbons), 74.90,
59.90, 30.90 (3· CH2).
Naphthalimidopropanol (5.10 g, 20 mmol) was dis-
solved in anhydrous pyridine (80 ml). The solution was
stirred for 15 min at 0 ꢁC. Tosyl chloride (5.72 g,
30 mmol) was added, in small portions, over 30 min.
The solution was left overnight at 4 ꢁC and was poured
into ice water (200 ml) to form a solid on standing. The
solid was filtered off and washed thoroughly with water.
The crude product was recrystallised from either ethanol
or ethyl acetate to give O-tosylpropylnaphthalimide 6
(53%). 1H NMR (CDCl3): d 8.65–7.80 (m, 6H, aromatic
protons), 4.45 (t, 2H, CH2), 4.35 (t, 2H, CH2), 2.50 (s,
3H, CH3), 2.25 (p, 2H, CH2). 13C NMR (CDCl3): d
161.30 (C@O), 145.10–123.10 (aromatic carbons),
73.10, 67.90, 28.70 (3· CH2), 22.10 (CH3). LRMS
(FAB): Calcd for C12H19NO6S 425.09; found: 426
[MH]+.
BNIPSpd was synthesised according to our methods
previously reported.3,10
2.4. General method for the synthesis of mesitylated
di- or triamine (1–6)
Corresponding diamine or triamine was dissolved in
anhydrous pyridine followed by the addition of mesityl-
ene chloride (2.1 M excess for diamine and 3.1 M excess
for triamine). The resulting solution was stirred at room
temperature for 4 h. Removal of the pyridine followed
by the addition of cold water resulted in the formation
of a precipitate. The latter was filtered off and washed
thoroughly with water. The crude product was recrystal-
lised from absolute ethanol.
2.4.1. N1,N8-Dimesityloctane 2. (70%), 13C NMR
(CDCl3) d 20.82 (CH3, Mts), 22.85 (CH3, Mts), 26.34
(CH2), 28.70 (CH2), 29.41 (CH2), 41.05 (N–CH2),
47.58 (CH2), 133 (aromatic carbons, Mts).
2.6. General N-alkylation reaction (step 1 in Scheme 1)
Mesitylated polyamines (1–6) (0.651 mmol) were dis-
solved in anhydrous DMF (13.5 ml) followed by the
addition of 7 (0.13 mmol) and caesium carbonate
(1.06 g). The solution was left at 80 ꢁC. Completion of
the reaction was monitored by thin-layer chromatogra-
phy. DMF was removed under vacuo, and the residue
was poured into cold water and the resulted precipitate
filtered and washed thoroughly with water. After drying,
the crude product was recrystallised from ethanol to
give the fully protected pure product in high yield (75–
85%).
2.4.2. N1,N9-Dimesitylnonane 3. (36%), 13C NMR
(CDCl3) d 20.82 (CH3, Mts), 22.85 (CH3, Mts), 26.34
(CH2), 28.70 (CH2), 29.41 (CH2), 41.05 (N–CH2),
47.58 (CH2), 133 (aromatic carbons, Mts).
2.4.3. N1,N10-Dimesityldecane 4. (48%), 13C NMR
(CDCl3) d 20.82 (CH3, Mts), 22.85 (CH3, Mts), 26.34
(CH2), 28.70 (CH2), 29.41 (CH2), 41.05 (N–CH2),
47.58 (CH2), 133 (aromatic carbons, Mts).
2.7. General deprotection reaction (step 2 in Scheme 1)
2.4.4. N1,N5,N9-Trimesityldipropyltriamine 5. (67%), 13
C
NMR (CDCl3) d 20.85 (CH3, Mts), 22.79 (CH3, Mts),
27.69 (CH2), 39.50 (N–CH2), 43.11 (N–CH2), 132.17
(aromatic carbons, Mts), 139.98 (aromatic carbons,
Mts).
The fully protected polyamine derivatives (0.222 mmol)
were dissolved in anhydrous dichloromethane (10 ml)
followed by the addition of hydrobromic acid/glacial
acetic acid (1 ml). The solution was left stirring at room
temperature for 24 h. The yellow precipitate formed was
filtered off and washed with dichloromethane, ethyl ace-
tate and ether.
2.4.5. N1,N3,N6-Trimesityldiethyltriamine 6. (59%), 13C
NMR (CDCl3) d 21.35 (CH3, Mts), 23.06 (CH3, Mts),
41.05 (N–CH2), 47.58 (N–CH2), 133 (aromatic carbons,
Mts).
2.7.1. BNIPSpd. (75%) DMSO-d6, d 22.20 (CH2), 24.70
(CH2), 44.10 (N–CH2), 44.20 (N–CH2), 45.00 (N–CH2),
130 (aromatic carbons) 164.87 (C@O). LRMS (FAB):
Calcd for C37H44N5O4Br3 862.1 ([MÀ3HBr]+ 619.3);
found: 620.4 [MÀ2H–3Br]+.
2.5. Synthesis of O-tosylpropylnaphthalimide 7
Naphthalic anhydride (6.34 g, 0.032 mol) was dissolved
in DMF (50 ml) followed by the addition of aminopro-
panol
3
(2.45 g, 0.032 mol) and DBU (4.87 g,
2.7.2. BNIPDaoct. (85%), DMSO-d6, d 24.43 (CH2),
25.30 (CH2), 25.66 CH2), 28.07 (CH2), 44.72 (N–CH2),
46.60 (N–CH2), 121.99, 127.13, 130.62, 131.21, 134.31
(aromatic carbons), 163.61 (C@O). HRMS (FAB):
Calcd for C38H44N4O4 Br2 778.1729, ([MÀ2HBr]+
618.3206); found: 619.3282 [MÀH–2Br]+.
0.032 mol). The solution was left stirring at 85 ꢁC for
4 h. The DMF was removed under reduced pressure
and the resulting residue was poured into cold water
with stirring (200 ml) to form a precipitate. The latter
was filtered using a Buchner funnel and washed thor-
oughly with (i) water and (ii) saturated bicarbonate solu-
tion. The yield of the reaction was found to be 95%. This
compound, naphthalimidopropanol, was pure enough
and was used in the next step with no further purifica-
tion. NMR (CDCl3): d 8.65–7.80 (m, 6H, aromatic pro-
tons), 4.39 (t, 2H, –N–CH2), 3.69 (t, 2H, CH2–O–), 3.20
(br s, 1H, OH), 2.06 (p, 2H, CH2). 13C NMR (CDCl3): d
2.7.3. BNIPDanon. (85%), DMSO-d6, d 24.88 (CH2),
25.84 (CH2), 26.16 CH2), 28.76 (CH2), 45.29 (N–CH2),
47.29 (N–CH2), 121.94, 127.51, 131.12, 131.42, 134.76
(naphthalimido aromatic carbons), 164.00 (C@O).
HRMS (FAB): Calcd for C39H46N4O4 Br2 792.1886,
([MÀ2HBr]+ 632.3363), found: 633.3440 [MÀH–2Br]+.

J. Oliveira et al. / Bioorg. Med. Chem. 15 (2007) 541–545
545
2.7.4. BNIPDadec. (75%), DMSO-d6, d 24.97 (CH2),
25.90 (CH2), 26.22 CH2), 28.79 (CH2), 29.00 (CH2),
45.35 (N–CH2), 47.38 (N–CH2), 121.05, 127.66,
131.30, 131.51, 134.94 (naphthalimido aromatic car-
bons), 164.21 (C@O). LRMS (FAB): Calcd for
C40H48N4O4 Br2 806.2, ([MÀ2HBr]+ 646.4); found:
647.4 [MÀH–2Br]+.
of culture) were incubated with a serial range of concen-
trations of each drug for 3 days at 27 ꢁC and the growth
of parasites was determined by the MTT assay. Briefly,
the MTT solution was added from a 5 mg/ml stock solu-
tion to have 0.25 mg/ml in the wells. The plates were
incubated at 27 ꢁC for 4 h. At the end of the incubation
period 50 ll of a solution containing 20% of SDS, 50%
of DMF and pH of 4.8 was added. After 1 h at 37 ꢁC
the absorbance was read at 550 nm on a micro plate
reader. The IC50 is the concentration of the drug re-
quired to inhibit the growth by 50% was determined
by linear regression analysis.
2.7.5. BNIPDpta. (85%), DMSO-d6, d 22.20 (CH2),
24.70 (CH2), 44.10 (N–CH2), 44.20 (N–CH2), 45.00
(N–CH2), 130 (aromatic carbons) 164.87 (C@O). LRMS
(FAB): Calcd for C36H42N5O4 Br3 850.7, ([MÀ3HBr]+
605.3); found: 606.4 [MÀ2H–3Br]+.
2.7.6. BNIPDeta. (67%), DMSO-d6, d 22.20 (CH2),
24.70 (CH2), 44.10 (N–CH2), 44.20 (N–CH2), 45.00
(N–CH2), 130 (aromatic carbons). HRMS (FAB): Calcd
for C34H38N5O4 Br3 817.0474, ([MÀ3HBr]+ 577.2689);
found: 578.2760 [MÀ2H–3Br]+.
Acknowledgments
The authors thank The Robert Gordon University, The
Scottish Executive Environment and Rural Affairs
Department (SEERAD) and the Royal Society of
Chemistry for financial support, The Centre of Mass
Spectrometry at the University of Wales, Swansea, for
mass spectroscopic analyses and FCT BD/SFRH/
18137/2004 (fellowship to J.T., Project No: POCI/
SAU-FCF/59837/2004).
2.8. Cytotoxic studies
Cytotoxicity was evaluated for Caco-2 colon carcinoma
and L. infantum using the MTT assay with protocols
appropriate for the individual test system.10,12 Caco-2
cells were maintained in Earle’s Minimum Essential
Medium (Sigma), supplemented with 10% foetal calf ser-
um (Biosera), 2 M L-glutamine (Sigma), 1% non-essen-
tial amino acids (Sigma), 100 IU/ml penicillin and
100 lg/ml streptomycin (Sigma). Exponentially growing
cells were plated at 2 · 104 cells cmÀ2 into 96-well plates
and incubated for 24 h before the addition of drugs.
Stock solutions of compounds were initially dissolved
in 20% DMSO and further diluted with fresh complete
medium.
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After 24 and 48 h of incubation at 37 ꢁC, the medium
was removed and 200 ll of MTT reagent (1 mg/ml) in
serum-free medium was added to each well. The plates
were incubated at 37 ꢁC for 4 h. At the end of the incu-
bation period, the medium was removed and pure
DMSO (200 ll) was added to each well. The metabo-
lized MTT product dissolved in DMSO was quantified
by reading the absorbance at 560 nm on a micro plate
reader (Dynex Technologies, USA). IC50 values are de-
fined, as the drug concentrations required to reduce the
absorbance by 50% of the control values. The IC50 val-
ues were calculated from the equation of the logarith-
mic line determined by fitting the best line (Microsoft
Excel) to the curve formed from the data. The IC50 val-
ue was obtained from the equation for y = 50 (50%
value).
Leishmania infantum (clone MHOM/MA671TMA-
P263) promastigotes were grown at 27 ꢁC in RPMI
medium (Gibco) supplemented with 10% of heat-inacti-
vated foetal bovine serum (FBS-Gibco), 2 mM L-gluta-
mine (Gibco), 20 mM Hepes (Gibco), 100 U/ml
penicillin (Gibco) and 100 lg/ml streptomycin (Gibco).
The parasites (106/ml) in the logarithmic phase (2 days
14. Labadie, G. R.; Choi, S-R.; Avery, M. A. Bioorg. Med.
Chem. Lett. 2004, 14, 615.