Synthesis and antiprotozoal activity of n -alkoxy analogues of the trypanocidal lead compound 4,4′-bis(imidazolinylamino)diphenylamine with improved human blood-brain barrier permeability

Article abstract of DOI:10.1021/jm101335q

To improve the blood-brain barrier permeability of the trypanocidal lead compound 4,4′-bis(imidazolinylamino)diphenylamine (1), five N-alkoxy analogues were synthesized from bis(4-isothiocyanatophenyl)amine and N-alkoxy-N-(2-aminoethyl)-2-nitrobenzenesulfonamides following successive chemical reactions in just one reactor (one-pot procedure). This involved: (a) formation of a thiourea intermediate, (b) removal of the amine protecting groups, and (c) intramolecular cyclization. The blood-brain barrier permeability of the compounds determined in vitro by transport assays through the hCMEC/D3 human cell line, a well-known and characterized human cellular blood-brain barrier model, showed that the N-hydroxy analogue 16 had enhanced blood-brain barrier permeability compared with the unsubstituted lead compound. Moreover, this compound displayed low micromolar IC₅₀ against Trypanosoma brucei rhodesiense and Plasmodium falciparum and moderate activity by intraperitoneal administration in the STIB900 murine model of acute sleeping sickness.

Full text of DOI:10.1021/jm101335q

J. Med. Chem. 2011, 54, 485–494 485
DOI: 10.1021/jm101335q
Synthesis and Antiprotozoal Activity of N-Alkoxy Analogues of the Trypanocidal Lead Compound
4,40-Bis(imidazolinylamino)diphenylamine with Improved Human Blood-Brain Barrier Permeability
,^
Lidia Nieto,†,# Ainhoa Mascaraque,#,† Florence Miller,‡,§ Fabienne Glacial,‡,§ Carlos Rı nez,† Marcel Kaiser,
os Martı
´ ´
Reto Brun, ,^ and Christophe Dardonville*,†
†
‡
´
ꢀ
ꢀ
Instituto de Quımica Medica, CSIC, Juan de la Cierva 3, E-28006 Madrid, Spain, Institut Cochin, Universite Paris Descartes,
CNRS (UMR 8104), Paris, France, §Inserm, U1016, Paris, France, Swiss Tropical and Public Health Institute, Socinstrasse 57,
CH-4002 Basel, Switzerland, and ^University of Basel, Basel, Switzerland. #Both authors contributed equally to this work.
Received June 28, 2010
To improve the blood-brain barrier permeability of the trypanocidal lead compound 4,40-bis-
(imidazolinylamino)diphenylamine (1), five N-alkoxy analogues were synthesized from bis(4-isothio-
cyanatophenyl)amine and N-alkoxy-N-(2-aminoethyl)-2-nitrobenzenesulfonamides following succes-
sive chemical reactions in just one reactor (“one-pot procedure”). This involved: (a) formation of a
thiourea intermediate, (b) removal of the amine protecting groups, and (c) intramolecular cyclization.
The blood-brain barrier permeability of the compounds determined in vitro by transport assays
through the hCMEC/D3 human cell line, a well-known and characterized human cellular blood-brain
barrier model, showed that the N-hydroxy analogue 16 had enhanced blood-brain barrier permeability
compared with the unsubstituted lead compound. Moreover, this compound displayed low micromolar
IC50 against Trypanosoma brucei rhodesiense and Plasmodium falciparum and moderate activity by
intraperitoneal administration in the STIB900 murine model of acute sleeping sickness.
Introduction
are able to cure the acute and CNS stages of HAT remains a
priority in tropical medicine.1
Human African trypanosomiasis (HATa or sleeping sickness),
a neglected tropical disease caused by subspecies of the
protozoan parasite Trypanosoma brucei, is endemic in several
regions of sub-Saharian Africa. It also represents a serious
economic and social burden to some of Africa’s poorest
countries.1 According to the last epidemiologic update by
the World Health Organization (WHO),2 50000-70000 peo-
ple were infected in 2006. However, this may remain only a
short-term view as no long-term systematic screening of the
population at risk is currently ongoing.3
Chemotherapy of HAT relies on four old drugs (suramin,
pentamidine, melarsoprol, and eflornithine) which have un-
acceptable toxicity and require parenteral administration.
This, greatly complicates the treatment of patients in disease
endemic countries with no basic health care systems.4,5 The
treatment optionsavailable for the late-stage disease involving
central nervous system (CNS) infection are particularly defi-
cient, as they mostly rely on a highly toxic arsenical derivative,
melarsoprol, which provokes deadly post-treatment reactive
encephalopathy in up to 10% of the cases.4 Even though the
recent inclusion of the combination therapy Nifurtimox-
eflornithine as simplified stage 2 treatment for T. b. gambiense
sleeping sickness improves the therapeutic arsenal against
late-stage HAT, the discovery of new orally active drugs that
In previous reports, we have shown that 4,40-bis(imidazo-
linylamino)diphenylamine dihydrochloride (1, Chart 1) dis-
played excellent antitrypanosomal6 and antiplasmodial7 activity
in vitro. The crystal structure of 1 bound to its preferred DNA
binding site 50-AATT has suggested a basis for understanding
the antitrypanosomal action of this compound.8 Further
studies in vivo have shown that this trypanocidal lead com-
pound was curative by intraperitoneal administration in
murine models of acute T. b. brucei and T. b. rhodesiense
infections but not in the late-stage disease involving CNS
infection.9 A reason for the lack of effectiveness against the
CNS-stage is a poor brain penetration that is most likely due
to the positively charged imidazolinylamino groups (pKa =
9.9)10 at physiological pH, which limit its passage through the
blood-brain barrier (BBB). The same drawback has been
observed with diamidine drugs such as pentamidine or dimin-
azene, which have been used for decades as a first line
treatment for early stage gambiense sleeping sickness and
cattle trypanosomiasis (Nagana), respectively. Previous re-
ports in the literature have shown that derivatization of the
amidine group as an amidoxime prodrug was useful to over-
come this problem and improve the oral bioavailibility and
CNS delivery of diamidines. These amidoxime prodrugs are
metabolized to the amidine active compounds through
sequential oxidative O-demethylation by cytochrome P450
isoforms 1A1, 1A2, and 1B1 and cytochrome b5 catalyzed
reductive N-dehydroxylation reactions.11-21
*To whom correspondence should be addressed. Phone: þ34
912587490. Fax: þ34 915644853. E-mail: dardonville@iqm.csic.es.
a Abbreviations: BBB, blood-brain barrier; CNS, central nervous
system; DMF, N,N-dimethylformamide; DIPEA, N,N-diisopropylethy-
lamine; EtOAc, ethyl acetate; Et2O, diethyl ether; HAT, human African
trypanosomiasis; LY, lucifer yellow; Ns (nosyl), 2-nitrobenzenesulfo-
namide.
More recently, similar prodrug strategies (e.g., N-substituted
bis-C-alkyloxadiazolones,22 N,N0-dihydroxyamidines,23 O-
carboxymethylamidoximes,24 diacetyldiamidoximeester25)
r
2010 American Chemical Society
Published on Web 12/22/2010
pubs.acs.org/jmc

486 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Nieto et al.
for the oral delivery of amidines and guanidines were also
reported.
functional transporters.30,31 By comparing the permeability
coefficients of the new derivatives and the lead compound 1,
some conclusions regarding the influence of N-alkoxy sub-
stitution on the human BBB permeability can be drawn.
In line with these findings, we have developed a similar
approach (i.e., synthesis of N-alkoxy analogues) to reduce the
pKa of the imidazolinylamino moiety in order to improve the
pharmacokinetics of compound 1. We also sought to establish
the influence of replacing the imidazolylamino moiety with a
closely related heterocycle (i.e., 4,5-dihydrothiazolinylamine)
that has lower pKa value so as to avoid problems of being
ionized at physiological pH (i.e., the pKa of thiazolines is ca. 3
units lower than the pKa of imidazolines; e.g., 2-aminoimida-
zole, pKa = 8.46;26 2-aminothiazole, pKa = 5.39).27 Hence,
five different N-alkoxy substituted analogues of 1 (Chart 1)
and the 4,5-dihydrothiazolinylamino derivative 17 (Scheme 2)
were synthesized. The antitrypanosomal activity of the target
compounds was checked in vitro against the STIB900 Trypa-
nosoma brucei rhodesiense strain. As additional data, we also
checked the antiprotozoal activity of the new compounds
against three related parasites (Trypanosoma cruzi, Leishmania
donovani, and Plasmodium falciparum).
Results
Chemistry. We attempted two different synthetic strategies
in order to obtain the N-alkoxy-substituted derivatives of 1.
Both approaches relied on the reaction of two equivalents of
a functionalized ethylenediamine precursor (4-5 or 9) with
the isothiocyanate 3, previously prepared in 80% yield from
4,40-diaminodiphenylamine (Scheme 1).32 The thiourea in-
termediates obtained (6-7 and 10-11, respectively) were
subsequently subjected to intramolecular cyclization after
adequate removal of the amine protecting groups.
Regarding the first synthetic strategy, the N-alkoxyethy-
lenediamine precursors 4, 5a, and 5b were prepared in low
yield following the protocol previously described for com-
pound 4 (eq ).33,34
Further, the activity of the new compounds was assayed in
vivo in the murine model of acute T. brucei infection. Addi-
tionally, the BBB permeability of the compounds was deter-
mined in vitro by permeability assays through the hCMEC/D3
human endothelial cell monolayer.28,29 This cell line holds
most of the specific properties of in vivo BBB, including cell-
cell junction expression of tight junction proteins (claudin-5,
JAM-A, ZO-1) and the polarized expression of a variety of
The reaction of 3 with 2.5 equiv of 4, or 5a, gave 6 and 7,
respectively (Scheme 1). Attempts to optimize the conditions
for the deprotection of the amino groups with hydrazine/
EtOH and the subsequent in situ cyclization to the target
compound 14 were disappointing, leading to several byproducts.
Chart 1. Lead Compound (1) and New N-Alkoxy Analogues
Scheme 2a
a Reagents and conditions. (i) 2-bromoethanamine hydrobromide,
Et3N, CH2Cl2, rt; (ii) HClg/dioxane, MeOH.
Scheme 1. Synthetic Routes for the Preparation of 1-Alkoxy-2-arylaminoimidazolines

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 487
Table 1. In Vitro Antiprotozoal Activity of N-Substituted Analogues 12-17 and Synthetic Intermediates 6, 7, and 10
a T. brucei rhodesiense STIB900 strain. Control: melarsoprol, IC50 = 12.6 nM. b Selectivity index = [IC50 (L6-cells)/IC50 (parasite)]. cAmastigotes of
T. cruzi Tulahuen C4 strain. Control: benznidazole, IC50 = 1.64 μM. d L. donovani strain MHOM/ET/67/L82 axenically grown amastigotes. Control:
miltefosine, IC50 = 0.275 μM. eP. falciparum K1 erythrocytic stages. Control: chloroquine, IC50 = 0.169 μM. f Rat skeletal myoblast L-6 cells. g Data
taken from refs 6, 7. h Not available. See ref 37 for detailed experimental procedures.
Hence, the thiourea 6 was activated via formation of the
methylisothiouronium salt 8, which was obtained almost
quantitatively (excess CH3I/CH2Cl2/ rt/24 h) with a purity
>80%. Partial deprotection of one benzyl group accounted
for the lower purity of the product. Longer reaction times
slightly increased the yield of the deprotection product.
Attempts of purification by chromatography were unsuccessful,
so the compound was used in the cyclization step without
further purification. The deprotection of the amino groups
(MeNH2/ THF-H2O) and the consequent in situ sponta-
neous cyclization afforded 14, which was isolated in 40%
yield after silica chromatography. Our attempts to scale up
the synthesis of 5a and 5b were unsuccessful, so we explored a
different strategy to obtain the corresponding methoxy 12
and ethoxy analogues 13. As an alternative, we considered
the deprotection of the benzyl protecting groups of 14 and
the subsequent alkylation of 16 to the corresponding alkoxy
derivatives. However, all of the attempts to remove the
benzyl groups, either by catalytic hydrogenolysis or by other
classical methods,35 gave poor yields of the desired product.
Hence, an alternative one-pot procedure strategy was devel-
oped to prepare the 1-methoxy- (12), 1-ethoxy-(13), 1-ben-
zyloxy -(14), and 1-tetrahydropyranyloxy- (15) derivatives
(Scheme 1).36 Treatment of 15 with HClg saturated dioxane
solution afforded the 1-hydroxy analogue 16 as its dihy-
drochloride salt. The intermediates 10a and 10b were isolated
in separate experiments and fully characterized.
were screened in vitro against T. b. rhodesiense and three other re-
lated parasites: T. cruzi, L. donovani, andP. falciparum (Table1).
The N-substituted compounds 12-16 showed submicro-
molar IC50 values against T. b. rhodesiense and P. falciparum.
Nevertheless, these values represented a 5-fold (14, 16), 10-fold
(12, 13), and 20-fold (15) loss of activity against T. brucei,
respectively, compared with the lead compound 1. The anti-
plasmodial activity was also decreased, in comparison with 1,
by 20-fold (14), 50-fold (12, 13), 100-fold (15), and 7-fold for
compound 16. The latter notably conserved nanomolar activity
(IC50 =55 nM) and high selectivity (SI = 1673) against this
parasite. Previous reports have underlined the importance
of the positively charged imidazolylamino moieties of 1 for
antitrypanosomal activity and for binding to DNA minor
groove.7-9 Hence, the 5- to 20-fold higher IC50 observed in
vitro for the uncharged 1-alkoxy-substituted derivatives 12-16
was not unexpected. The lower activity of the 4,5-dihydrothia-
zolinylamino analogue 17 with respect to the imidazolylamino
compound 1 (i.e., 160-fold against T. b. rhodesiense and 270-
fold against P. falciparum) confirmed these findings. The higher
pKa of the imidazoline structure (pKa = 9.9 for 110) ensures a
higher proportion of the ionized form of the molecule at
physiological pH, which proved important for high activity in
this series.
None of the new compounds showed significant activity
against axenically grown amastigotes of L. donovani. On the
contrary, one compound, 14, presented an IC50 value three
times higher than the reference drug benznidazole (1.64 μM)
on intracellular T. cruzi amastigotes. The synthetic inter-
mediates (urea derivatives) 6, 7, 10a, and 11b were poorly
active on the four parasites with IC50 in the high micromolar
range (Table 1).
The 4,5-dihydrothiazolinylamino derivative 17 was pre-
pared by reaction of 3 with 2-bromoethanamine hydrobro-
mide in the presence of triethylamine (Scheme 2).
In Vitro Antiprotozoal Activity. The target compounds (12-
16, and 17) and the synthetic intermediates (6, 7, 10, and 11)

488 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Nieto et al.
Table 2. In Vivo Antitrypanosomal Activity of N-Substituted Analogues of 1 in the T. b. rhodesiense (STIB900) Mouse Modela
compd
control
R
dosage routeb
dosage (mg/kg)
curedc/infected
mean relapse days
7d
0/4
1
H
ip
po
4 ꢀ 20
4 ꢀ 50
4/4e
0/4
>60
7.75
12, 13, 14, 15
OMe, OEt, OBn, OTHP
OH
ip
po
4 ꢀ 20
4 ꢀ 50
0/4
0/4
7
7
16
ip
po
4 ꢀ 20
4 ꢀ 50
0/4
0/4
25.3
7
a See Experimental Section for details of STIB 900 (T. b. rhodesiense) model. b ip = intraperitoneal, po = per os. c Number of mice that survive and are
parasite free for 60 days. d Control mice were always positive and were euthanized on day 7. e Data taken from ref 9.
In Vivo Antitrypanosomal Activity. In the T. b. rhodesiense
STIB900 mouse model of acute infection, which mimics the
first stage of the disease, the lead compound 1 was curative
when administered intraperitoneally at 4 ꢀ 20 mg/kg.9 How-
ever, it was hardly effective by oral route in this model (i.e.,
10% increase in mean relapse days compared to control, no
cures), and totally inactive in the GVR35 mouse model that
mimics the late stage of the disease involving CNS infection.9
The newly synthesized N-alkoxy analogues of 1 were designed
as possible prodrugs with improved oral bioavailability and
enhanced CNS uptake. To check their in vivo activity, com-
pounds 12-16 were administered by intraperitoneal (4 ꢀ 20
mg/kg) and oral route (4 ꢀ 50 mg/kg) to T. brucei infected mice
(Table 2). Compounds 12-15 did not show any activity in this
model of first stage HAT (i.e., same mean time of relapse as the
untreated mice), possibly due to a poor bioactivation in vivo.
On the contrary, compound 16 was moderately active by ip
administration, increasing the mean time of relapse 3.6-fold
compared to control.
In Vitro Determination of Human Blood-Brain Barrier
Permeability. We have mentioned before that compound 1
did not show in vivo activity in the murine model of CNS-
stage sleeping sickness.9 Because crossing the BBB is a
requirement for drugs targeting the late-stage disease, we
decided to assess the in vitro BBB permeability of the lead
compound 1 and the new N-alkoxy analogues 12, 13, 14,
and 16. The immortalized human brain endothelial cell line
hCMEC/D329 was used for the drug transport studies. This
cell line has proved useful as a human BBB model,28 so the
results obtained in this study are particularly relevant from a
therapeutic point of view.
The clearance principle (i.e., the slope of the volume
cleared versus time) was used to calculate the permeability
coefficients as usually performed (see Experimental Section
for details).38-40 The fluorescent dye lucifer yellow (LY) was
used as a marker of passive diffusion through cell-cell
junctions; a low permeability to LY reflects the integrity of
the endothelial monolayer and the relevance of cell-cell
junction complexes (see Experimental Section). First, we
validated the good quality of the BBB model in the presence
of the compounds by finding the concentration of the
compounds which could be applied to the endothelial cells
without affecting LY permeability. Hence, permeability
assays of LY in the presence of the new compounds were
performed. Compounds 1, 12, 13, and 16 did not affect the
endothelial monolayer integrity (i.e., a low permeability to
LY was conserved) at concentrations of 100 μM, whereas 14
required a concentration of 10 μM (Figure 1). Compound 14,
at this concentration, made the analysis of the results unreliable
(i.e., beyond the sensitivity limits of our HPLC detection
method). This suggests a very low permeability through the
in vitro BBB for this compound.
The measured permeability values for the lead compound
(1), the O-methoxy (12), and O-ethoxy (13) derivatives were
not significantly different from the one measured for the
poorly permeable compound LY (Figure 2). In contrast, the
1-hydroxy analogue (16) had significant higher permeability
than LY and also than compound 1 (nearly 3-fold). These
results show that N-substitution of the imidazolylamino
group with OH enhances the in vitro BBB permeability of
this molecule, whereas O-alkyl substituents do not improve
the in vitro BBB permeability.
Discussion
Dicationicdrugs arevery effective antimicrobialagents that
have been used for the treatment of African trypanosomiasis
and leishmaniasis for decades. Despite their excellent antipar-
asitic activity, this kind of drugs presents a major drawback,
namely a poor absorption through biological barriers such as
the gastrointestinal tract or the BBB (e.g., the diamidine drugs
pentamidine, berenil, or furamidine are not orally active and
do not cure late-stage HAT). Lowering the pKa of the amidine
moiety by attaching OR groups (R = H, alkyl) to the nitrogen
atoms to generate orally active prodrugs has been a successful
strategy in a number of cases.11,13,21,41 Our objective was the
study of N-alkoxy derivatives of a dicationic bisaminoimida-
zolinyl trypanocidal lead compound, 1, showing the same
drawback as the above-mentioned diamidines.
As expected for a prodrug requiring in vivo metabolic
activation, the N-alkoxy derivatives displayed reduced activ-
ity (in the submicromolar range) in the parasite susceptibi-
lity assay in vitro. Furthermore, one of the new derivatives,
16, was active in vivo in the STIB900 mouse model after
intraperitoneal administration. Even though this moderate
effect could be attributed to the fair intrinsic activity observed
in vitro for this compound (IC50 = 0.37 μM), it is likely due to
a partial bioactivation of 16 to the lead compound 1. In fact,
the 2-aminoimidazolinyl moiety can be viewed as a cyclic
guanidine42 and the in vivo reduction of N-hydroxylated
guanidines and amidines by microsomes and mitochondria
of different organs from different species (e.g., kidney, liver,
brain) has been established before.12,43,44 This result is im-
portant because it is compound 16 that showed the highest in
vitro BBB permeability in our assays. Theinvivo activityof16
in the mouse model of CNS-stage infection was not yet tested
because of its limited efficacy in the acute STIB900 model.
However, the synthesis of this class of prodrugs with more
potent lead candidates is warranted.

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 489
Figure 1. Endothelial monolayer integrity measured as a function of LY permeability in the presence of compounds 1, 12, 13, 14, and 16 at
different concentrations. Increase in LY permeability in presence of the tested compound is an indication of loss of endothelial monolayer
integrity. See Experimental Section for details. Data represent means of three independent culture inserts per condition.
Figure 2. Permeability values of compound 1 and its N-OMe (12), N-OEt (13), and N-OH (16) analogues compared to LY. Permeability
values are represented as a percentage of the permeability value of LY (normalized to 100%). Data represent means of three independent
culture inserts per condition.
On the one hand, the lack of in vivo activity of the alkoxy
derivatives 12, 13, and 14, despite of similar inherent activities
in vitro (IC50 values of 0.83, 0.73, and 0.32 μM, respectively),
may possibly be attributed to a poor bioactivation. As far as
we know, the bioactivation of N-alkoxy prodrugs has been
described for amidines but not for guanidines. On the other
hand, this might be explained by a high fraction of protein
binding or other unfavorable pharmacokinetics.
The Pe values measured in vitro with the human brain
endothelial cellline hCMEC/D3 for thelead compound1, and
the O-alkyl substituted derivatives 12 and 13, were compar-
abletothe Pe values ofthe controlmoleculeLY thatpresentsa
very low brain uptake (see Experimental Section for more
details). Thus, compound 1 scarcely cross the BBB, which
possibly explains why this molecule is inactive in the murine
model of late-stage HAT.9 The derivatization of the imidazoline
nitrogen with OMe or OEt did not improve the BBB perme-
ability, whereas a free OH group increasedthe permeabilityby
almost 3-fold compared with the lead compound 1. In com-
parison, the benzodiazepine drug diazepam that is used as
anxiolytic and has large brain uptake45 shows 10-fold higher
permeability than LY using the hCMEC/D3 cell line (data not
shown). This means that compound 16 shows an intermediate
in vitro permeability to the brain.

490 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Nieto et al.
Table 3. Calculated Physicochemical Parameters of the Compounds:
Molecular Weight (MW), Polar Surface Area (PSA), Octanol/Water
Distribution Coefficient at pH 7.4 (log D7.4), H-Bond Donors (HBD),
and pKa
Experimental Section
Chemistry. All dry solvents were purchased from Aldrich
(Sigma-Aldrich Quimica SA, Madrid, Spain) in Sure/Seal bot-
tles. All reactions requiring anhydrous conditions or an inert
atmosphere were performed under a positive pressure of N2. All
reactions were monitored by thin layer chromatography (TLC)
compd
1e
MWa
PSAb
log D7.4
pKa (N3; N30) b,c
HBDd
b
408.33
395.46
423.51
547.65
535.64
367.40
369.51
88.09
85.75
85.75
85.75
104.21
107.75
60.81
0.1
8.02; 8.63
5.73; 6.34
5.81; 6.41
5.77; 6.37
5.85; 6.45
5.64; 6.24
6.76; 7.37
5
3
3
3
3
5
3
12
13
14
15
16
17
2.51
3.22
5.95
4.31
1.76
3.92
~
using silica gel 60 F254 plates (Merck-Espana, Madrid, Spain) or
HPLC-MS. Chromatography was performed with Isolute SI
prepacked columns (Biotage). H and 13C NMR spectra were
1
recorded on a Bruker Advance 300 or Varian Inova 400 spec-
trometer. Chemical shifts of the 1H NMR spectra were internally
referenced to the residual proton resonance of the deuterated
solvents: CDCl3 (7.26ppm), D2O(δ4.6 ppm), CD3OD(3.49ppm),
and DMSO-d6 (δ 2.49 ppm). J values are given in Hz. Melting
points were determined in open capillary tubes with a SMP3-
Stuart Scientific apparatus and are uncorrected. All compounds
are >95% pure by HPLC or combustion analysis otherwise
noted. Elemental analysis was performed on a Heraeus CHN-O
Rapid analyzer. Analytical results were within (0.4% of the
theoretical values unless otherwise noted. Analytical HPLC-
MS was run with an Xbridge C18-3.5 μm (2.1 mm ꢀ 100 mm)
column on a Waters 2695 separation module coupled with a
Waters Micromass ZQ spectrometer using electrospray ioniza-
tion (ESþ). The following HPLC conditions were used: column
temperature = 30 ꢀC, gradient time = 15 min, H2O/CH3CN
(5:95 f 95:5) (HCO2H 0.1%), flow rate=0.25 mL/min, UV
detection diode array (λ=190-400 nm).
a Molecular weight (g/mol). b The polar surface area (A2), LogD7.4
and pKa were calculated using ChemAxon software MarvinSketch
v.5.3.8. c Calculated pKa values of the N3-nitrogen of the imidazoline
rings. d Number of H-bond donors. e Dihydrochloride salt.
This result was somewhat unexpected as increasing the
number of hydrogen bond donors and the polar surface area
is generally associated with a reduced BBB permeability.46 To
try to rationalize these results, we calculated some physico-
chemical parameters that are generally accepted as important
for a compound to be readily BBB permeable (Table 3).47 The
molecular hydrophobicity of a compound, measured as the
octanol/water partition coefficient (log P), is an important
physicochemical property associated with biological mem-
brane permeation. For ionizable molecules to be BBB perme-
able, values of octanol/water distribution coefficients, log
D7.4, in the range between 1 and 3 are recommended.48 As
shown in Table 3, two compounds only, 12 and 16, have log
D7.4 values within this range. Thus, adequate hydrophobicity
and low pKa values may explain the enhanced in vitro BBB
permeability observed for 16. However, these data do not
explain the low permeability of 12 measured in vitro. Taken
together, these findings show that subtle modification of the
nitrogen substituent (e.g., OH T OMe) greatly affects the
BBB permeability of the bis-2-imidazolinylamino compounds.
1. First Synthetic Route for the Synthesis of 1-Alkoxy-2-
Arylaminoimidazolines 12 and 14. Bis(4-isothiocyanatophenyl)-
amine (3). The free diamine was obtained from technical grade
4,40-diaminodiphenylamine sulfate as described earlier.49 Thio-
phosgene (1.67 mL, 21.9 mmol) [CAUTION: wear adequate
protection and work in a well ventilated fumehood] was added with
a syringe to a mechanically stirred mixture of 4,40-diaminodi-
phenylamine (1.985 g, 10 mmol) in 80 mL of Et2O/H2O (3/1 v/v).
The reaction was almost instantaneous. The mixture was stirred
vigorously overnight, and the precipitate was collected by filtration
to give the isothiocyanate 3 as a greenish solid (1.58 g, 56%).
Another crop was recovered from the filtrate (0.85 g, 30%); mp
182-183 ꢀC. 1H NMR (CDCl3, 300 MHz) δ 7.15 (d, J=8.8, 4H),
7.00 (d, J = 8.8, 4H), 5.86 (br, NH, 1H). 13C NMR (CDCl3,
75 MHz) δ 139.2 (C), 132.5 (NCS), 125.3 (CH), 122.6 (C), 117.6
(CH). Anal. (C14H9N3S2) C, H, N.
Conclusions
Several N-alkoxy analogues of compound 1 were designed
to reduce the basicity of the aminoimidazolinyl groups and
potentially improve the oral and CNS absorption of that
trypanocidal lead compound. Four of these derivatives showed
submicromolar IC50 values in vitro against T. brucei and
P. falciparum, but only one compound, the N-hydroxy-2-
imidazolinylamino analogue 16, displayed moderate activity
in vivo in the T. brucei STIB900 murine model at a dosage of
20 mg/kg ip. This modest activity is possibly due to an incom-
plete bioactivation of the prodrug in vivo that does not permit
reaching the curative dose of the active compound. Even though
other pharmacokinetic aspects such as plasma protein binding
cannot be ruled out at this time, the in vivo activity displayed by 16
means that there is a substantial unbound concentration of the
active compound at the therapeutic target in vivo. Further work
to fully understand the pharmacokinetics of these compounds will
be needed, but this shall be the subject of a different study.
More importantly, this compound also had the highest
BBB permeability of the series (i.e., almost 3-fold increase with
respect to lucifer yellow) in the in vitro transport assay
involving the human brain endothelial cell line hCMEC/D3.
These results suggest that N-hydroxy-2-imidazolinylamino
analogues may possibly be used as prodrugs of the imidazoli-
nylamino group with enhanced brain penetration. Efforts are
ongoing toward the discovery of more potent drugs able to
cross the BBB for the treatment of late-stage HAT.
2-(2-(Benzyloxyamino)ethyl)isoindoline-1,3-dione (4).33,34
A
mixture of O-benzylhydroxylamine hydrochloride (12.71 mmol,
2.03 g, 1 equiv), sodium hydroxide 5% solution (25 mL), and
diethyl ether (Et2O) (75 mL) was stirred for 15 min. The organic
layer was extracted, dried over MgSO4, and evaporated under
vacuum to give the free base of O-benzylhydroxylamine (BnONH2)
as colorless oil (1.59 g, 100%). A mixture of BnONH2 (1.59 g,
12.68 mmol, 2 equiv) and 2-bromoethylphthalimide (1.61 g,
6.34 mmol, 1 equiv) was heated at 80 ꢀC for 4 days. Ethyl acetate
(EtOAc) was added to the cold reaction, and the precipitate was
filtered off. The filtrate was concentrated under vacuum to give
a yellow oil. Recrystallization from MeOH yielded white crys-
tals (433 mg, 23%): Rf=0.38 (hexane/EtOAc 2:1); mp (MeOH)
83-85 ꢀC (with previous softening) [lit.33 92-94 ꢀC]. 1H NMR
(CDCl3, 300 MHz) δ 7.84-7.68 (m, 4H, ArHPht), 7.36-7.25 (m,
5H, ArH), 5.63 (s, 1H, NH), 4.69 (s, 2H, CH2O), 3.89 (t, J=5.7,
2H, NCH2), 3.20 (t, J=6.0, 2H, CH2NH). 13C NMR (CDCl3,
75 MHz) δ 168.9 (CO), 138.1 (C-1, Ar), 134.3 (CH, Pht), 132.6
(C, Pht), 129.4 (CH-4, Ar), 129.3 (CH-3, Ar), 128.2 (CH-2, Ar),
123.5 (CHCO, Pht), 76.3 (OCH2), 50.4 (CH2NH), 36.6 (NCH2).
LRMS (ESþ) m/z=297.0 [(M þ H), 100%]. Anal. (C17H16N2O3)
C, H, N.
2-(2-(Methoxyamino)ethyl)isoindoline-1,3-dione (5a). N,N-Diiso-
propylethylamine (DIPEA) (3.7 mmol, 0.6 mL, 2 equiv) was added
to O-methylhydroxylamine hydrochloride (3.7 mmol, 307 mg, 2
equiv) in acetonitrile under N2 atmosphere. The mixture was stirred

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 491
for few minutes, and 2-bromoethylphthalimide (1.8 mmol, 467 mg,
1 equiv) was added. The reaction was heated at 80 ꢀC for 43 h. The
cold reaction mixture was diluted with EtOAc and the precipitate
was filtered off. The filtrate was concentrated under vacuum to give
a yellow oil. Purification by flash chromatography with hexane/
EtOAc (9:1) yielded the product as a white solid (80 mg, 20%): Rf =
3.54 (s, 8H, CH2CH2). 13C NMR (75 MHz, CD3OD) δ 162.3
(CdN), 144.6 (C; 4,40-Ph), 136.6 (C, Bn), 130.9 (CHm, Bn), 130.2
(CHp, Bn), 129.8 (CHo, Bn), 128.2 (C; 1,10-Ph), 127.5 (CHPh),
119.2 (CHPh), 79.7 (OCH2), 52.7 (BnONCH2), 42.1 (CH2N).
LRMS (ESþ) m/z 548.39 (M þ H). Anal. (C32H35I2N7O2 2H2O)
3
Calcd: C, 45.78; H, 4.68; N, 11.68. Found: C, 45.65; H, 2.87; N,
12.09%. HPLC > 92% pure.
1
0.26 (hexane/EtOAc 2:1); mp 78-80 ꢀC. H NMR (CDCl3, 300
MHz) δ 7.85-7.68 (m, 4H, ArHPht), 5.63 (t, 1H, NH), 3.88 (t, J =
5.7, 2H, NCH2), 3.51 (s, 3H, OCH3), 3.19 (q, J=5.7, 2H, CH2NH).
13C NMR (CDCl3, 75 MHz) δ 168.8 (CO), 134.4 (CH, Pht), 132.5
(C, Pht), 123.7 (CH, Pht), 62.4 (OCH3), 49.9 (CH2NH), 35.9
(NCH2). LRMS (ESþ) m/z = 221.0 [(M þ H), 100%]. Anal.
2. Second Synthetic Route for the Synthesis of 1-Alkoxy-2-
arylaminoimidazolines 12-16. The 1-methoxy- (12), 1-ethoxy-
(13), 1-benzyloxy- (14), and 1-tetrahydropyranyloxy- (15) deri-
vatives were synthesized following the one-pot procedure
previously reported by us. The products showed satisfactory
spectroscopic and analytical data as reported previously.36 In
separate experiments, the thiourea intermediates (10a and 10b)
were also isolated and fully characterized. The spectroscopic
data are given below.
(C11H16N2O5 2H2O) Calcd: C, 51.56; H, 6.29; N, 10.93. Found:
3
C, 51.74; H, 4.90; N, 11.39.
1,10-(4,40-Azanediylbis(4,1-phenylene))bis(3-(benzyloxy)-3-(2-(1,
3-dioxoisoindolin-2-yl)ethyl)thiourea) (6). Compound 4 (860 mg,
2.9 mmol, 2.5 equiv) was added to the isothiocyanate 3 (329 mg,
1.7 mmol, 1 equiv) in dry N,N-dimethylformamide (DMF) (5 mL).
The reaction mixture was stirred 4 days at room temperature.
The solvent was evaporated, and the resulting yellow oil was
purified by flash chromatography (10 g SI cartridge) with
hexane/EtOAc: 4/1f1/1. Further rerystallization from EtOAc/
hexane yielded 6as a yellow solid (427 mg, 42%): Rf=0.26 (EtOAc/
N,N0-(2,20-(4,40-Azanediylbis(4,1-phenylene)bis(azanediyl))bis-
(thioxomethylene)bis (azanediyl)bis(ethane-2,1-diyl))bis(N-methoxy-
2-nitrobenzenesulfonamide) (10a). A solution of isothiocyanate 3
(0.30 mmol, 85 mg, 1 equiv) in dry DMF (1 mL) was added to a
solution of N-(2-aminoethyl)-N-methoxy-2-nitrobenzenesulfo-
namide (9, R1=Me)36 (0.60 mmol, 277 mg, 2 equiv) in dry DMF
(5 mL). The resulting mixture was stirred at room temperature
for 2 days. Water was added to give a precipitate that was
filtered off. Purification by flash chromatography with hexane/
ethyl acetate (1:1) yielded the product as a yellowish solid (67 mg,
27%): Rf=0.65 (EtOAc/hexane 8:2); mp (CH2Cl2) 120-122 ꢀC
(with previous softening). 1H NMR (CDCl3, 300 MHz) δ 8.01
(d, J=7.8, 2H, ArHNs), 7.85-7.67 (m, 4H, ArHNs), 7.75 (s, 2H,
NHCS), 7.58 (d, J=7.8, 2H, ArHNs), 7.13 (m, 8H, ArH), 6.27 (t,
J=4.8, 2H, NHCH2), 6.07 (s, 1H, NH), 3.90 (m, 4H), 3.78 (s,
6H, OCH3), 3.35 (t, J=4.4, 4H). 13C NMR (CDCl3, 75 MHz) δ
181.7 (CS), 150.0 (CNO2), 142.7 [ArC-NH), 135.7 (CH-5, Ns),
132.8 (CH-3, Ns), 131.7 (CH-6, Ns), 128.9 (CSO2, Ns), 127.9
(CH-3, Ar), 126.2 (C-4, Ar), 124.2 (CH-4, Ns), 119.5 (CH-2, Ar),
66.1 (OCH3), 52.4, 42.9. LRMS (ESþ) m/z=833.7 [(M þ H),
1
hexane 1:1); mp 118-120 ꢀC (EtOAc/hexane). H NMR (300
MHz, CDCl3) δ 8.32 (s, 2H, PhNHCS), 7.79 (dd, J=3.0 and 5.4,
2H, ArHPht), 7.65 (dd, J=3.0 and 5.4, 2H, ArHPht), 7.35-7.31
(m, 10H, ArHBn), 7.01 (d, J=8.7, 4H, ArH), 6.90 (d, J=8.7, 4H,
ArH), 5.85 (s, 1H, PhNHPh), 4.87 (s, 4H, OCH2Ph), 4.52 (t, J =
4.6, 4H), 4.09 (t, J=4.6, 4H). 13C NMR (75 MHz, CDCl3) δ
181.2 (CS), 168.2 (CO), 141.2 (NHC), 133.9 (CCH2O þ CHPht),
132.1 (CPht), 130.8 (CNHCS), 129.3 (CHm, Bn), 129.2 (CHp, Bn),
128.8 (CHo, Bn), 126.8 (CH, aniline), 123.1 (CCHCH, Pht), 117.4
(CH, aniline), 60.3 (OCH2), 48.8 (CH2N), 34.8 (CH2N). LRMS
(ESþ) m/z 877.0 (M þ H). Anal. (C48H41N7O6S2) C, H, N.
1,10-(4,40-Azanediylbis(4,1-phenylene))bis(3-(2-(1,3-dioxoisoindo-
lin-2-yl)ethyl)-3-methoxythiourea) (7). Compound 7was obtained
as an orange solid (55 mg, 83%) by the same procedure used for
6: Rf=0.74 (CH2Cl2/MeOH 95:5); mp 194-196 ꢀC (decomp).
1H NMR (300 MHz, DMSO-d6) δ 9.82 (s, 2H, NHCS), 8.10 (s,
1H, PhNHPh), 7.86-7.78 (m, 8H, Pht), 7.05 (d, J=8.7, 4H, Ph),
6.92 (d, J=8.7, 4H, Ph), 4.38 (t, J=4.7, 4H, CH2CH2), 3.90
(t, J=4.7, 4H, CH2CH2), 3.68 (s, 6H, OCH3). 13C NMR (75 MHz,
DMSO-d6) δ 179.5 (CS), 168.1 (CO), 141.5 (NHC), 134.6 (CHPht),
132.3 (CPht), 131.6 (CNHCS), 128.0 (CH, aniline), 123.3 (CHPht),
116.3 (CH, aniline), 61.6 (OCH3), 47.7 (CH2N), 35.0 (CH2N).
LRMS (ESþ) m/z 724.10 [(M þ H), 100%]. Anal. (C36H33N7-
O6S2) C, H, N.
100%]. Anal. (C32H35N9O10S4 3H2O) Calcd: C, 43.28; H, 4.65;
3
N, 14.20; S, 14.44. Found: C, 43.86; H, 4.78; N, 14.05; S, 13.01.
HPLC > 90% pure.
N,N0-(2,20-(4,40-Azanediylbis(4,1-phenylene)bis(azanediyl))bis-
(thioxomethylene)bis(azanediyl)bis(ethane-2,1-diyl))bis(N-ethoxy-
2-nitrobenzenesulfonamide) (10b). A solution of isothiocyanate 3
(0.708 mmol, 200.4 mg, 1 equiv) was added to a solution of N-(2-
aminoethyl)-N-ethoxy-2-nitrobenzenesulfonamide36 (9, R1=
Et) (1.56 mmol, 450 mg, 2.2 equiv) in dry DMF (15 mL). The
reaction mixture was stirred overnight at room temperature.
Water was added to give a purple precipitate that was collected
by filtration and purified by flash chromatography (10g SI
cartridge) with hexane/EtOAc (1:1) to yield 10b as yellow oil
(291 mg, 47%): Rf = 0.59 (EtOAc/hexane 5:1). 1H NMR
(CDCl3, 300 MHz) δ 8.03 (dd, J=7.7 and 1.35, 2H, ArHNs),
7.83-7.71 (m, 4H, ArHNs), 7.59 (dd, J = 7.78 and 1.0, 2H,
ArHNs), 7.58 (s, 2H, NHCS), 7.14 (m, 8H, ArH), 6.24 (t, 2H,
NHCH2), 5.94 (s, 1H, NH), 4.06 (q, J=7.0, 4H, OCH2), 3.92 (m,
4H, NHCH2), 3.39 (t, J=4.8, 4H, NCH2), 1.19 (t, J=7.1, 6H,
CH3). 13C NMR (CDCl3, 75 MHz) δ 181.2 (CS), 149.5 (CNO2),
142.2 (ArC-NH), 135.2 (CH-5, Ns), 132.4 (CH-3, Ns), 131.2
(CH-6, Ns), 128.4 (CSO2, Ns), 127.4 (CH-3, Ar), 125.9 (C-4,
Ar), 123.8 (CH-4, Ns), 119.1 (CH-2, Ar), 73.9 (OCH2), 52.1
(CH2), 42.5 (CH2), 13.5 (CH3). LRMS (ESþ) m/z = 862.47
(M þ H). HPLC > 90% pure.
(N,N0Z,N,N0Z)-Dimethyl-N,N0-4,40-azanediylbis(4,1-phenylene)-
bis(N-benzyloxy-N-(2-(1,3-dioxoisoindolin-2-yl)ethyl)carbamimido-
thioate) (8). An excess of methyl iodide (2 mL) was added to a
stirred solution of 6 (408 mg, 0.5 mmol) in CH2Cl2 (9 mL). The
mixture was stirred at room temperature for 6 days. Vacuum
evaporation of the solvents gave the product as a yellow solid
(470 mg, quantitative). The product was pure enough to be used
1
in the next reaction: Rf =0.72 (EtOAc/hexane 1:1). H NMR
(300 MHz, CDCl3) δ 7.85-7.73 (m, 8H, Pht), 7.39-7.30 (m,
10H, CH2Ph), 7.07 (m, 8H, Ph), 5.08 (s, 4H, OCH2Ph), 4.31 (t,
4H, CH2N), 4.04 (t, 4H, CH2N), 2.12 (s, 6H, SCH3). LRMS
(ESþ) m/z 904.5 (M þ H). HPLC=80% pure.
N1-(1-(Benzyloxy)-4,5-dihydro-1H-imidazol-2-yl)-N4-(4-(1-(ben-
zyloxy)-4,5-dihydro-1H-imidazol-2-ylamino)phenyl)benzene-1,4-
diamine (14). An excess of methylamine 2.0 M solution in THF
(15 mL) was added to a solution of 8 (1.37 g, 1.5 mmol) in H2O
(10 mL). The reaction mixture was stirred for 24 h and evapo-
rated to dryness. The oily residue was washed with H2O, filtered
off, and purified by flash chromatography with CH2Cl2/MeOH
(97:3) to yield the title compound as a brown solid (250 mg,
30%). Dihydroiodide salt of 14: mp 152-154 ꢀC (with previous
softening). 1H NMR (300 MHz, CD3OD) δ 7.56-7.45 (m, 10H,
CH2Ph), 7.23 (m, 8H, Ph), 5.49 (s, 1H, NH), 5.10 (s, 4H, OCH2),
4,40-Bis(1-hydroxyimidazolinylamino)diphenylamine (16). The
THP-protected product 1536 (80 mg, 0.15 mmol) was dissolved
in MeOH (2 mL) and treated with HClg saturated dioxane
solution (5 mL). The reaction was stirred at room temperature
for 5 h, and the solvent was removed under vacuum. The crude
product was dissolved in MeOH, and Et2O was added. The flask
was allowed to stand in the fridge overnight, whereupon a
precipitate settled at the bottom of the flask. The supernatant
was discarded, and Et2O was added. The precipitate was

492 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Nieto et al.
triturated with a spatula, collected, and dried under vacuum to
give the dihydrochloride salt of 16 as brown solid (56 mg, 85%);
mp >91.4 ꢀC (decomp). 1H NMR (DMSO-d6, 300 MHz) δ
10.94 (brs, 2H, OH), 10.62 (s, 2H, NH), 8.87 (s, 2H, NH), 7.21 (d,
J=9.0, 4H, ArH-3), 7.16 (d, J=9.0, 4H, ArH-2), 3.82-3.46 (m,
8H). 13C NMR (DMSO-d6, 75 MHz) δ 160.8 (CdN), 142.7 (C),
126.9 (C), 126.4 (C), 117.6 (CH), 53.3 (CH2), 40.6 (CH2). LRMS
(ESþ) m/z 368.15 [(M þ H)]. HPLC g 95% pure.
So, briefly, after 10-12 days of culture, coated culture inserts
with and without endothelial cell were transferred to 6-well
plates containing 2 mL of transport buffer (Hanks buffer saline
solution with CaCl2 and MgCl2, 10 mM HEPES, and 1 mM
sodium pyruvate) in the abluminal chamber. At time 0, trans-
port buffer, containing tested compound with or without LY
(50 μM dissolved in cell culture tested water), was placed in each
luminal chamber. Transport incubations occurred at 37 ꢀC,
95% humidity and 5% CO2. At different times (10, 25, and
45 min) each culture insert (with and without cell) was trans-
ferred to a new lower compartment containing fresh transport
buffer. The amount of each compound in the lower compart-
ments at different time points, in the upper one at the end of the
experiment, and in the working solution, was quantified either
by fluorimetry (for LY) or by HPLC (for compounds 1, 12, 13,
14, and 16).
Endothelial permeability was calculated from the clearance
rate and the surface area, as previously described.38,39 In this
way, a concentration-independent permeability value was ob-
tained. The increment in cleared volume between successive
sampling events is calculated by dividing the amount of solute
during the interval time by the donor chamber concentration,
which is the luminal one in this study, as described below:
The total cleared volume at each time point is calculated by
summing the incremental cleared volumes up to the given time
point:
4,40-Bis(thiazolinylamino)diphenylamine (17). Triethylamine
(3.18 mmol, 0.44 mL, 3 equiv) was added dropwise to a solution
of isothiocyanate 3 (1.06 mmol, 300 mg, 1 equiv) and 2-bro-
moethanamine hydrobromide (2.65 mmol, 543 mg, 2.5 equiv) in
CH2Cl2 (20 mL). The reaction mixture was stirred at room
temperature overnight. The precipitate was collected by filtra-
tion and rinsed with dichloromethane and water, successively.
Recrystallization from boiling methanol/acetonitrile yielded a
green solid (92 mg, 15%): Rf=0.22 (CH2Cl2/MeOH 2/1); mp
82-84 ꢀC (with previous softening). A methanolic solution of 17
(2 mL) was stirred with HClg saturated dioxane solution (3 mL).
The solvents were removed under vacuum affording the dihy-
drochloride salt of 17: 1H NMR (DMSO-d6, 300 MHz) δ 8.64
(brs, 2H, NH), 7.67 (s, 1H, NH), 7.25 (d, J=7.9, 4H, ArH-2),
6.88 (d, J=8.7, 4H, ArH-3), 3.85 (t, J=7.1, 4H, NCH2), 3.24
(t, J=7.1, 4H, SCH2). 13C NMR (DMSO-d6, 75 MHz) δ 157.2
(CdN), 138.9 (Ar, C-2), 137.1 (Ar, C-4), 120.2 (Ar, 2-CH), 117.3
(Ar, 3-CH), 57.33 (CH2N), 32.9 (CH2S). LRMS (ESþ) m/z =
369.9 [(M þ H), 100%]. Anal. (C18H19N5S2 2HCl 2H2O) C, H,
3
3
clearance ðmLÞ ¼ X=Cd
N. HPLC > 95% pure.
Biology. In Vitro BBB Permeability Studies. Cell Line Culture.
Immortalized human brain endothelial cells (hCMEC/D3
cell line) were seeded at the density of 50000 cells per cm2 in the
upper compartment of six-well tissue culture plate inserts
(Millicell CM inserts, 0.4 μm porosity, Millipore, Bellerica,
MA, USA), precoated with rat tail collagen type I (Cultrex rat
Collagen, R&D Systems, Trevigen). They were then maintained
for 10-12 days in EBM-2 basal medium (Lonza, Basel,
Switzerland) containing 5% fetal bovine serum Gold (PAA,
The Cell Culture Company, Pasching, Austria), 1% penicillin-
streptomycin (Invitrogen, Carlsbad, CA, USA), 1.4 μM hydro-
cortisone (Sigma, St. Louis, MO, USA), 10 mM HEPES (PAA
Laboratories, Pasching, Austria), 5 μg mL-1 ascorbic acid
(Sigma, St Louis, MO, USA), and 1 ng mL-1 bFGF (Sigma,
St Louis, MO, USA), at 37 ꢀC in 5% CO2 atmosphere. The
medium was changed every 3-4 days. Twenty-four hours prior
to permeability assays, fresh medium was supplemented with
Simvastatin 1 nM (Calbiochem, La Jolla, CA, USA). In sum-
mary, endothelial cells were cultured in Boyden chamber-like
system allowing them to separate into two compartments, the
upper or luminal compartment and the lower or abluminal one,
which represent the blood and the cerebral one, respectively.
Permeability Assays. Prior to compound permeability study,
it is essential to find a working concentration of the compounds
that will not disturb the cellular complex between endothelial
cells. For this purpose, endothelial monolayer quality was checked
by following the passage of the small hydrophilic fluorescent
molecule lucifer yellow (LY, Sigma, St Louis, MO, USA) when a
fixed concentration of the studied compound (i.e., 1, 12, 13, 14,
or 16) was put on the cells in the luminal compartment. Note
that LY is widely used and accepted as a marker of tight junction
integrity.40 If LY permeability was affected by the presence of
the compound, the concentration of the compound was de-
creased until a nonaffecting dose was reached.
where X is the amount of drug in the receptor chamber (the
abluminal one) and Cd is the donor chamber concentration at
each time-point. The average volume cleared is plotted versus
time and the slope estimated by linear regression analysis, which
allows calculating total endothelial monolayer permeability
noted PSe:
1=PSe ¼ 1=PSt - 1=PSf and Pe ¼ PSe=A
where PSe is the permeablity-surface area product value for the
endothelial cell monolayer, A is the surface area of the filter (in
cm2), and PSt and PSf are the slopes of the clearance curves for
the cell monolayer on coated culture insert filter and for the
coated culture insert filter alone (meaning without cell mono-
layer), respectively. The PSe values are divided by the surface
area (A, in cm2) of the culture insert to generate the endothelial
permeability coefficient (Pe, in cm per min). These Pe values
allow classifying the molecules into three categories: low-,
intermediate- or high-brain uptake.
Compound permeability values were expressed as an increase
or decrease compared with a model of low brain uptake, LY
permeability (Pe=3.04 ꢀ 10-3 cm/min), which is given the value
of 100%. LY was chosen as paracellular diffusion marker
because of its properties: it is a small (457.3 Da) and hydrophilic
molecule that has no transporter on endothelial cells (data not
shown).
HPLC Analysis of the Results. One mL samples for each time
point were taken directly from the basal compartment and
transferred to 1.5 mL Waters HPLC vials that were stored in
the freezer. The samples were defrosted and shaken with an
orbital shaker for 20 min just before running the HPLC ana-
lysis. Analytical HPLC was run with a Waters Sunfire C18-
3.5 μm (4.6 mm ꢀ 50 mm) column on a Waters 2695 separation
module coupled with a Waters Micromass ZQ spectrometer
using electrospray ionization (ESþ). The following HPLC con-
ditions were used: column temperature=30 ꢀC, flow rate=
1 mL/min, gradient time=5 min. The solvent mixture was H2O:
CH3CN (HCO2H 0.1%%) with the following proportions:
compound 1 (100:0 f 90:10), compound 12 (98:2 f 50:50),
compounds 13 and 14 (95:5 f 50:50), and compound 16 (98:2 f
70:30). The compounds were detected by UV at the following
wavelengths: λ=296 nm (compound 1), 300 nm (compounds 12
and 13), 303 nm (compound 14), and 298 nm (compound 16).
Compound 1 was directly dissolved in transport buffer,
whereas the N-alkoxy derivatives (12, 13, 14, and 16) were
dissolved in DMSO (Sigma) and then diluted with transport
buffer to reach a concentration of 100 μM. The maximum
quantity of DMSO in the stock solutions was 0.1%. The
endothelial monolayer integrity in presence of 0.1% DMSO
was checked, showing no significant difference compared with
the buffer alone (see Figure 1).

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 493
Each sample was injected three times, and the mean value of the
compound UV peak area was used to determine the concentra-
tion of the sample according to the calibration curves. Calibra-
tion curves were obtained for each compound using six different
concentrations (1, 10, 30, 75, 150, 375 μM). Each concentration
was tested in triplicate, and the mean value of the compound UV
peak area vs concentration was plotted. The equation for the
calibration curve was obtained by linear regression analysis
using the Microsoft Excel program.
In Vitro Antiprotozoal Activity. IC50 values against blood-
stream forms of T. b. rhodesiense strain STIB900, amastigotes of
L. donovani strain MHOM/ET/67/L82, and cytotoxicity on rat
myoblasts L-6 cells were determined using the Alamar blue
assay.50,51 IC50 values against erythrocytic stages of P. falciparum
was determined by a [3H]hypoxanthine incorporation assay using
the chloroquine- and pyrimethamine-resistant strain K1.52 IC50
values against trypomastigote forms of T. cruzi Tulahuen strain
C2C4 containing the β-D-galactosidase (LacZ) gene were deter-
mined using a colorimetric assay with the substrate chlorophe-
nyl red β-D-galactopyranoside (CPRG)-Nonidet.53 Detailed
experimental protocols for all of these assays have been reported
before.37
In Vivo Antitrypanosomal Activity. The STIB900 acute mouse
model mimics the first stage of the disease. Experiments were
performed as previously reported,16 with minor modifications.
Briefly, female NMRI mice were infected intraperitoneally (ip)
with 2 ꢀ 104 bloodstream forms of T. b. rhodesiense (strain
STIB900). Experimental groups of four mice were treated ip or
orally (per os (po)) with compounds on four consecutive days
from days 3 to 6 postinfection. A control group was infected but
remained untreated. The tail blood of all mice was checked for
parasitemia until 60 days after infection. Surviving and aparasi-
temic mice at day 60 were considered cured and then euthanized.
The day of death of the animals was recorded (including the
cured mice, as >60) to calculate the mean survival time in days
(MSD). Mean relapse days were determined as day of relapse
postinfection of mice. All protocols and procedures used in the
current study were reviewed and approved by the local veterinary
authorities of the Canton Basel-Stadt.
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Acknowledgment. This work was supported by grants
ꢀ
from the Spanish Ministerio de Ciencia e Innovacion (grants
SAF2006-04698, SAF2009-10399) and the CSIC (grant PIE
200680I121, bilateral projects grant 2008GB0021 and PA100-
2103) to C.D., and the UNDP/World Bank/WHO Special
Program for Research and Training in Tropical Diseases (R.B.).
We gratefully acknowledge the IQM Mass Spectrometry
Service Centre for HPLC-MS analysis and Dr. Angel de la
Puerta for helpful discussions regarding HPLC analytical
methods. C.R. was recipient of a Ph.D. fellowship for the
government of Panama (SENACYT grant BIDP-2008-030),
Florence Miller was supported by EUSTROKE seventh
framework programme theme health-2007-2.4.2-3, Combat-
ing Stroke. Thanks are given to Dr. Pierre-Olivier Couraud
for logistic collaboration and proofreading of the manuscript.
We thank Dr. Raj Chari for kindly revising the English of this
manuscript.
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