Pharmacomodulation on the 3-acetylursolic acid skeleton: Design, synthesis, and biological evaluation of novel N-{3-[4-(3-aminopropyl)piperazinyl]propyl}-3-O-acetylursolamide derivatives as antimalarial agents

Article abstract of DOI:10.1016/j.bmc.2007.10.031

A series of new piperazine derivatives of ursolic acid was synthesized and tested against Plasmodium falciparum strains. They were also tested on their cytotoxicity effects upon MRC-5 cells. Seven new piperazinyl analogues showed significant activity in the nanomolar range (IC₅₀ = 78-167 nM) against Plasmodium falciparum CQ-resistant strain FcB1. A possible mechanism of interaction implicating binding of these compounds to β-hematin was supported by in vitro tests. Moreover, the importance of the hydrophilic framework attached at the terminal nitrogen atom of the bis-(3-aminopropyl)piperazine joined to the triterpene ring was also explored through molecular dynamic simulations.

Full text of DOI:10.1016/j.bmc.2007.10.031

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 771–782
Pharmacomodulation on the 3-acetylursolic acid skeleton:
Design, synthesis, and biological evaluation of novel
N-{3-[4-(3-aminopropyl)piperazinyl]propyl}-3-O-acetylursolamide
derivatives as antimalarial agents
Simone C. B. Gnoatto,a,b Sophie Susplugas,c Luciana Dalla Vechia,a Thais B. Ferreira,a,d
Alexandra Dassonville-Klimpt,b Karine R. Zimmer,b Catherine Demailly,b
Sophie Da Nascimento,b Jean Guillon,e Philippe Grellier,c Hugo Verli,a,d
Grace Gosmanna,* and Pascal Sonnetb,*
aFaculdade de Farma´cia, Universidade Federal do Rio Grande do Sul (UFRGS), Av. Ipiranga, 2752,
Porto Alegre 90610-000, RS, Brazil
bEA 3901 DMAG Faculte´s de Me´decine et de Pharmacie, Universite´ de Picardie Jules Verne,
1 rue des Louvels, 80037 Amiens, Cedex 1, France
cLaboratoire de Biologie Fonctionnelle des Protozoaires, USM 0504, Muse´um National d’Histoire Naturelle,
61 rue Buffon, 75005 Paris, France
dCentro de Biotecnologia, Universidade Federal do Rio Grande do Sul (UFRGS), Av. Bento Gonc¸alves, 9600, Porto Alegre, RS, Brazil
eEA 4138– Pharmacochimie, Universite´ Bordeaux 2, 146 rue Le´o Saignat, 33076 Bordeaux Cedex, France
Received 6 August 2007; revised 21 September 2007; accepted 10 October 2007
Available online 14 October 2007
Abstract—A series of new piperazine derivatives of ursolic acid was synthesized and tested against Plasmodium falciparum strains.
They were also tested on their cytotoxicity effects upon MRC-5 cells. Seven new piperazinyl analogues showed significant activity in
the nanomolar range (IC50 = 78–167 nM) against Plasmodium falciparum CQ-resistant strain FcB1. A possible mechanism of inter-
action implicating binding of these compounds to b-hematin was supported by in vitro tests. Moreover, the importance of the
hydrophilic framework attached at the terminal nitrogen atom of the bis-(3-aminopropyl)piperazine joined to the triterpene ring
was also explored through molecular dynamic simulations.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
dangerous of these infections.2 Until now, several of
the drugs used as antimalarial agents were developed
from natural sources, like quinine, artemisinin, and their
derivatives. Nevertheless, due to the emergence of resis-
tant Plasmodium strains, the therapeutic impact of such
compounds has been diminished creating a great urge to
the development of new and effective antimalarial
drugs.3
Malaria is one of the 10 most prevailing and fatal infec-
tious diseases of the world and has been a public health
problem in about 90 countries; whereas, approximately
40% of the world population is under risk of contamina-
tion. It causes between 1.2 and 2.7 million deaths each
year.1 Four species of Plasmodium cause malaria in
human beings; however P. falciparum is the most
An additional group of natural products showing
promising antimalarial activity can be found in sapo-
nins, naturally amphiphilic compounds composed by an
aglycone (triterpenic or steroidal) and by carbohydrate
chains attached by an ether or ester linkages. Saponins,
as well as triterpenes, have been investigated for numer-
ous biological activities such as anti-inflammatory,
Keywords: Piperazinyl ursolic acid derivatives; Pharmacomodulation;
Antimalarial activity; Molecular dynamic simulation.
*
Corresponding authors. Tel.: +55 51 3308 5526; fax: +55 51 3308
5437 (G.G.); tel.: +33 03 22 82 74 94; fax: +33 03 22 82 74 69
(P.S.); e-mail addresses: grace.gosmann@ufrgs.br; pascal.sonnet@
sa.u-picardie.fr
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.031

772
S. C. B. Gnoatto et al. / Bioorg. Med. Chem. 16 (2008) 771–782
submitted to maceration with EtOH 70% to provide
crude saponin residue. This residue was submitted to
acid hydrolysis to obtain the major aglycone (1) that
was purified as previously mentioned.10,22 Ursolic acid
(1) was converted to its acetyl ester 2 in quantitative
yield. The promising antimalarial activity obtained for
this 3-acetylursolic acid (2) (24.93 lM for 2 versus
52.93 lM for 1, Table 1) led us to use this skeleton as
our bioactive pharmacophore for further pharmaco-
modulation. Thus, coupling reaction of 2 with N-Boc-
bisaminopropylpiperazine and triethylamine (TEA)
was performed after activation of the carboxylic acid
at C-17 with oxalyl chloride. N-Boc-amino protecting
group was, then, removed by a treatment with a trifluo-
roacetic acid/dichloromethane (TFA 10%/CH2Cl2) mix-
ture to result into the deprotected analogue 3. Reductive
amination conditions using sodium triacetoxyborohy-
dride (NaHB(OAc)3)as the reducing agent and appro-
priate aldehydes were used to obtain compounds 4–21
(Scheme 1), according to the literature.20,23 Ten deriva-
tives were synthesized using aromatic (4–11, 19, and
20) and six with aliphatic (12–17) aldehydes, while two
compounds were obtained by the reaction with ferro-
cenecarboxaldehyde (18 and 21).
17
COOH
28
3
HO
1
Figure 1. Ursolic acid.
antimalarial, and anti-HIV.4–8 Our group has been
working for decades on saponins from South American
Ilex species in which ursolic acid (1) (Fig. 1) is found free
or as aglycone of these saponins.9–11 In relation to anti-
malarial activity, ursolic acid (1) produced suppression
of parasitemia against P. berghei berghei in mice.12
Additionally, 1 was also reported to be capable to
reduce parasite proliferation against P. falciparum
3D7, W2, and K1 strains in vitro.8,13 Furthermore, the
potential of triterpenoid scaffolds as pharmacophoric
groups for the development of new antimalarial agents
has been evidenced by other groups.14,15
Recently, the advancement of molecular biology tech-
niques has improved the understanding of the biochemis-
try of malaria parasites concurring to the identification of
potential targets for new drugs.16 Considering the amino-
alkyl side chain as an important requirement for a strong
antimalarial activity,17 the 1,4-bis(3-aminopropyl)piper-
azinyl function was identified as a promising pharmaco-
phoric group associated with antimalarial activity
against both chloroquine (CQ) sensitive and resistant
strains of P. falciparum.18–21 This was related to possible
interactions of protonated piperazine nitrogen atoms
with the carboxylate moiety of heme; thus, promoting
the inhibition of hemozoin formation and preventing
heme detoxification in the digestive parasite’s vacuole.21
Table 1. In vitro sensitivity of P. Falciparum FcB1 strain to
compounds 1–21
R2
N
H
N
N
N
R1
O
AcO
Hence, we describe the synthesis of a new series of deriv-
atives showing promising antimalarial activities based
on the condensation of ursolic acid (1) and 1,4-bis(3-ami-
nopropyl)piperazine. While such approach explores the
postulated interaction between polyamines and heme car-
boxylate groups, it evaluates the possible involvement of
triterpenoid scaffold in inhibition of b-hematin forma-
tion. Additionally, since the major role of aromatic rings
in compounds as chloroquine has been associated to p–p
stacking with heme molecules, the replacement of p-inter-
acting by non-p-interacting structural moieties on anti-
malarial agents has a potential to point new directions
in the search of novel compounds, useful in the treatment
of malaria. Moreover, we further explore the structure–
activity relationship (SAR) on this series of compounds
through molecular dynamic (MD) simulations of the
packing between heme and antimalarial agents.
Compound
R1
R2
IC50 FcB1
(lM)
1
52.93 5.71
24.93 7.44
0.13 0.04
2
Chloroquine
3
R1=R2=
R1=R2=
H
H
0.17 0.07
0.32 0.18
21.12 2.90
8.06 0.65
>100
3a
4
R1=R2= Benzyl
R1=R2= Benzylethyl
5
6
R1=R2= 4-Bromobenzyl
R1=R2= 4-Chlorobenzyl
R1=R2= 4-Fluorobenzyl
R1=R2= 4-Nitrobenzyl
R1=R2= 4-Hydroxybenzyl
R1=R2= 4-Methoxybenzyl
R1=R2= Methylcyclohexyl
R1=R2= Methylcyclopropyl
R1=R2= Heptyl
7
46.04 10.76
20.27 5.46
3.57 1.47
0.16 0.02
6.50 1.74
>100
8
9
10
11
12
13
14
15
16
17
18
19
20
21
0.49 0.21
41.81 6.72
1.00 0.12
0.80 0.13
18.77 1.14
1.21 0.46
1.04 0.33
0.08 0.04
0.77 0.09
R1=R2= Butyl
R1=R2= Propyl
2. Results and discussion
2.1. Chemistry
R1=R2= tert-Butyl
R1=R2= Methylferrocenyl
R1=H
R1=H
R1=H
R2=4-nitrobenzyl
R2=4-hydroxybenzyl
R2=methylferrocenyl
The source of ursolic acid (1) was the leaves of Ilex par-
aguariensis, a native plant in South America, that were
3a, compound 3 not acetylated.

S. C. B. Gnoatto et al. / Bioorg. Med. Chem. 16 (2008) 771–782
773
2.2. Pharmacological
R1=R2=
R1=R2=
R1=R2=
R1=R2=
12
13
14
15
16
17
18
4
5
6
2.2.1. Antimalarial activity. All compounds were
evaluated for their in vitro antimalarial activities against
P. falciparum CQ-resistant strain FcB1 (IC50CQ =
130 nM). As shown in Table 1, piperazinyl derivatives
(3–21) from ursolic acid (1) were all more active than
R1=R2=
R1=R2=
R1=R2=
Br
their unsubstituted parent (IC50(FcB1
1 = 52.93
strain)
Cl
F
7
8
lM), corroborating the relevance of the 1,4-bis(3-ami-
nopropyl)piperazinyl group to the antimalarial activity.
Additionally, 3, 10, and 20 (170, 160, and 78 nM, respec-
tively) showed equivalent or superior activities when
compared to chloroquine (130 nM), indicating that
hydrogen bonding donor capabilities may be related to
the SAR of our new compounds 1–20. Moreover, the
role of acetate at position 3 of steroids or triterpenoids
is under investigation as already mentioned.24
R1=R2=
R1=R2=
R1=R2=
R1=R2=
O2N
9
R1=R2=
R1=R2=
R1=R2=
R1=R2=
Fe
10
11
R1=H,
R2=
HO
19
O2N
For compounds lacking such interaction, the activity
appeared to be mainly related to the lipophilicity of
the substituent group, as can be observed for aromatic
derivatives 6 (98 lM), 7 (46.04 lM), 4 (21.12 lM), 8
(20.27 lM), and 9 (3.57 lM). Moreover, not only lipo-
philicity but also the flexibility of the substituent group
appeared to be related to activity, as shown with the 2.5-
fold increase in activity from compound 4 to its superior
homologue 5. A similar profile could be observed
between cyclic (less active) and acyclic (more active) sub-
stituents, indicating that the capability of the group at-
tached to R1 and R2 for adopting specific
conformations may be an important structural attribute
for an antimalarial activity in this set of newly synthe-
sized compounds.
MeO
R1=H,
20
21
R2= HO
R1=H,
R2=
Fe
In the aromatic series, substitution on the phenyl ring at
para position was evaluated. The presence of electron-
donating groups, such as OH (10) or OMe (11); or an
electron-withdrawing group as NO2 (9); and halogens
as F (8), Cl (7) or Br (6), was studied. In the aliphatic
series, two cyclic derivatives 12 and 13 were synthesized
bearing a cyclohexyl and a cyclopropyl group, respec-
tively. The effect of the elongation of the chain was stud-
ied in compounds 14–16 together with one compound 17
with branched chain. Finally, two ferrocenyl derivatives
18 and 21 were also synthesized.
The effect of an antimalarial activity related to the
introduction of ferrocenyl moiety to chloroquine and
artemisinin derivatives was previously studied, includ-
ing
a
structure–activity relationship.25–29 Unfortu-
As far as we know, the syntheses of the new N-{3-[4-(3-
aminopropyl)piperazinyl]propyl}-3-O-acetylursolamide
derivatives, using a triterpene skeleton as pharmaco-
phore, were accomplished for the first time.
nately, in our series, the antimalarial activity of
compounds bearing the ferrocene moiety (18, 21) was
not noticeable in comparison to the 4-hydroxybenzyl
derivative 20.
i
ii,iii
H
NH2
N
N
1
COOH
N
O
AcO
AcO
2
3
R2
N
c'
c
H
N
N
N
iv
R1
3
d'
d
b
b'
O
AcO
(compounds 4-21)
Scheme 1. Synthesis of ursolic acid derivatives (2–21). Reagents: (i) pyridine, acetic anhydride; (ii) oxalyl chloride, N-Boc-bisaminopropylpiperazine,
TEA, CH2Cl2; (iii) TFA 10%/CH2Cl2; (iv) RCHO, NaHB(OAc)3.

774
S. C. B. Gnoatto et al. / Bioorg. Med. Chem. 16 (2008) 771–782
Table 3. Inhibition of b-hematin formation
2.2.2. Assays using P. falciparum chloroquine-sensitive
strain Thai and MRC-5 cells’ cytotoxicity. Based on the
results obtained for compounds tested in P. falciparum
FcB1 strain (Table 1), the most active compounds were,
then, assayed against the P. falciparum chloroquine-sen-
sitive strain Thai (Table 2). In addition, they were also
tested against a human diploid embryonic lung cell line
(MRC-5 cell) in order to evaluate their cytotoxicity,
allowing to obtain the resistance index FcB1/Thai and
the selectivity index FcB1/MRC-5 (Table 2).
Compound
Concentration Inhibitiona IC50 (mM) CQ
(mM)
(%)
indexb
Chloroquine
phosphate
5
36.9 8.6
5.9 1.15
1
10
5
97.4 0.2
17.9 8.6
0
1
2
3
ꢁ20
ꢁ3.4
>3.4
1–20
5
12.9 3.1
24.6 3.1
>20
10
9
5.7
11.4
4.2
8.4
3.6 4.86 ꢁ20
ꢁ3.4
18.6 5.84
39.6 25.5 5.25 2.82 0.89
82.2 14.2
The most active ursolic acid derivatives against P. falcipa-
rum CQ-resistant strain FcB1 were also the most active
against sensitive Thai strain, suggesting low levels of
cross-resistance to CQ. The resistance index calculated
from the ratio of the IC50 values of the sensitive and resis-
tant strains of P. falciparum was in the range of 0.37–1.17,
lower than that observed for chloroquine (RI = 11).
10
a Mean standard deviation from three independent experiments.
b IC50 compound/IC50 chloroquine.
lent or superior to chloroquine. This strategy used
hybridization of triterpenic skeleton of ursolic acid (1)
and 1,4-bis(3-aminopropyl)piperazine- both potential
antimalarial pharmacophores- followed by a reductive
amination in order to obtain compounds with different
groups at the terminal nitrogen atom of the second
pharmacophore. After the biological evaluation, we pro-
ceeded to study the complexes formed by heme and the
obtained ursolic acid derivatives through molecular
modeling techniques in order to elucidate their struc-
ture–activity relationship (SAR) at the atomic level.
All tested compounds showed cytotoxicity upon MRC-5
cells in the lM range and, IC50 values varied from 1.57
to 18.28 lM. The selectivity index was defined as the ra-
tio of the IC50 value on the MRC-5 cells to the IC50 va-
lue on the CQ resistant P. falciparum strain FcB1. The
compound 20 presented the highest selectivity among
the compounds tested with a selectivity index of 27
and could be further developed as a new lead for phar-
macological investigations.
2.2.3. Inhibition of b-hematin formation test. Moreover,
compounds 1,2, 3, 9, and 10 were also submitted to an
in vitro evaluation of their capabilities to inhibit b-
hematin formation in order to further explore the mech-
anism of action of these newly synthesized ursolic acid
derivatives. Then, the obtained results were compared
to chloroquine. Considering the results obtained for
the most active compounds, 3 and 10 (Table 3), it can
be postulated that most of the antimalarial activity ob-
served for compounds 1–21 is due to the inhibition of
heme assembly in b-hematin. Such mechanism is in
agreement with previous works suggesting the role of
piperazine nitrogen atoms in complexation to heme car-
boxylate groups.21
So, considering the previous evidences for polar interac-
tions between basic nitrogen atoms from piperazine con-
taining derivatives,21 as well as the influence of
substituents R1 and R2 on the activity of compounds
3–21, molecular dynamic simulations (MD) in explicit
aqueous solutions were employed in order to study the
complexation of compounds with heme. Although such
methodology does not include p–p interactions, it is, in-
deed, capable to describe the effect of solvent over the
solvated compound conformations, diffusion, and com-
plexation at a high level of accuracy.
In fact, MD has been recently employed to study the
interaction of two Fe(III)PPIX molecules in both vac-
uum and aqueous solutions.30,31 So, in order to comple-
ment such observations we decided for the use of
randomly placed heme molecules as the initial configu-
ration for the simulated system. As doing so, a sponta-
neous complexation between two heme molecules in
water was observed (Fig. 2a). As far as we know, such
observation is one of the first descriptions, at the atomic
level, of a non-enzymatic and spontaneous formation of
an intermolecular precursor of the b-hematin dimer in
solution. Additionally, the so obtained conformational
ensemble confirmed previous descriptions of an ꢀ180ꢁ
orientation between carboxylate groups, strongly sup-
porting the proposal that aqueous Fe(III)PPIX dimers
consist of coplanar back-to-back complexes between
two porphyrins.31 Besides, the distance between two
heme molecules is in agreement with previous data.32
2.3. Structure–activity of ursolic acid derivatives
The strategy employed in the design of derivatives 3–21
resulted in compounds with biological activities equiva-
Table 2. In vitro sensitivity of P. falciparum Thai strain and MRC-5
Compound
IC50 (lM)
Resistance Selectivity
index FcB1 index
Thai
MRC-5
3
0.46 0.04 1.61 0.14
3.05 0.27 18.28 0.10
0.37
1.17
0.46
0.51
0.79
0.81
1.06
0.43
0.68
9.6
5.1
9.7
5.1
2.5
3.5
5.2
27
9
10
13
16
18
19
20
21
0.35 0.03
0.95 0.01
1.00 0.55
1.57 0.67
2.53 0.51
1.96 0.78
4.08 0.09
5.39 0.10
2.10 0.20
2.00 0.20
NA
1.48
0
0.98 0.21
0.18 0.03
1.13 0.12
2.6
NA
Since the nanosecond time scale simulations of heme
molecules were capable to, adequately, describe the for-
mation of b-hematin dimer we used the same approach
Chloroquine 0.01 0.01
11
NA, not available.

S. C. B. Gnoatto et al. / Bioorg. Med. Chem. 16 (2008) 771–782
775
Figure 2. (a) Spontaneous association of heme molecules in aqueous solution during a MD simulation. (b and c) Complexes between heme and
chloroquine (CQ), 3, 6, and 16, obtained after spontaneous diffusion in solvent shown in space filing and tube representations, respectively.
to evaluate the interaction of heme with the synthesized
compounds. Based on such approach, compounds 16, 6,
3 and chloroquine were observed to spontaneously
interact with heme molecules by diffusion, forming sta-
ble complexes during the 10.0 ns trajectories (Fig. 2b
and c). Furthermore, it is important to note that the
geometry observed for the complex between chloroquine
and heme is in agreement with the interatomic distances
previously described to occur in such complex by NMR
methods.33
perform strong electrostatic interactions with carbox-
ylate groups of heme molecules, that is, more hydro-
phobic and steric imposing groups would abolish or
reduce such interaction and so decrease the activity
of the compounds;
2. The molecular basis for the N-[1,4-bis(3-aminopro-
pyl)piperazinyl pharmacophoric group can be ratio-
nalized in terms of its interaction with the
carboxylate groups of heme: in systems composed
only by heme molecules the carboxylate groups are
in opposite orientations, when the heme molecules
are packed with antimalarial agents, all the negatively
charged groups show a re-orientation and point to
the side where the positively charged amino groups
are placed. As a result, several salt bridges can be per-
formed simultaneously in solution between ligands
and hematin. A similar scheme of interactions was
recently proposed to occur with diethyl-amino-alk-
oxyxanthones,34 supporting these observations;
3. One of the main interactions expected to take place in
most of antimalarial agents evolves a p–p packing
between the flat and aromatic rings from compounds
Related to the ursolic acid derivative complexations to
heme molecules, important details could be obtained
from the performed simulations, confirming and
expanding, at the atomic level, a SAR for this class of
compounds, as follows (Chart 1):
1. The inverse relation between the hydrophobicity of
the substituent attached to the terminal nitrogen
atom of the N-[1,4-bis(3-aminopropyl)piperazinyl
pharmacophoric group and the antimalarial activity
could be explained on the basis of its capability to
hydrophobic
interactions with
hemozoin
R2
H
N
N
N
N
R1
O
AcO
pharmacophore
Interaction with hemozoin
carboxylate
pharmacophore
increase of lipophilicity
•
•
tertiary amines were less active than their
monosubstituted counterparts → exposure of the amine
More bulky substituents → less active compounds
Chart 1. Structure–activity relationships of piperazinyl ursolic acid derivatives.

776
S. C. B. Gnoatto et al. / Bioorg. Med. Chem. 16 (2008) 771–782
and heme. However, in molecules as artemisinin and
these results could provide suitable information for the
development of new antimalarial compounds.
the ursolic acid derivatives such interaction is not
possible. At least for this last class of molecules, we
observed that the substitution of an aromatic hetero-
cyclic group by a triterpene skeleton is accompanied
by the creation of a wide hydrophobic surface inter-
acting with heme molecules (Fig. 2b). Besides, such
group shows a perpendicular orientation related to
the aromatic ring in compounds as chloroquine
(Fig. 2c). That orientation can still be related to sim-
ilar hydrophobic nucleus in complex with heme pro-
teins.35 If confirmed, such new orientation may
represent a completely novel lead for future optimiza-
tions, exploring original interactions with heme and,
mainly, the iron atom.
4. Experimental
4.1. Chemicals
All commercially available reagents were used without
further purification unless otherwise stated. The solvents
used were all of AR grade and were distilled under po-
sitive pressure of dry nitrogen atmosphere where neces-
sary. All reactions were performed in pre-dried
apparatus under an atmosphere of nitrogen unless
otherwise stated. The progress of the reactions was mon-
itored by analytical thin-layer chromatography (TLC)
performed on Merck silica gel 60 F254 plates. Visualiza-
tion was performed using phosphomolybdic acid in
methanol. Column chromatography was carried out
using matrix silica 60 (35–70 lm). The melting points
were determined on a Kofler bench melting point appa-
ratus and are not corrected. High-resolution spectra
were obtained on a Micromass-Waters Q-TOF Ultima
spectrometer. The IR spectra through the range from
4000 to 600 cmꢂ1 were run on a Jasco FT/IR-4200 spec-
3. Conclusion
A series of new piperazine derivatives from ursolic acid
were successfully synthesized through a rational design,
based on the inhibition of b-hematin formation as one
molecular mechanism of the malaria disease. These
compounds were tested for their antimalarial in vitro
activity upon the P. falciparum chloroquine-resistant
strain FcB1 and, the most active compounds were tested
against the P. falciparum chloroquine-sensitive strain
Thai. Seven new piperazinyl analogues of ursolic
acid (1) showed significant activity in the nanomolar
range including compounds 20 (IC50 = 78 nM), 10
(IC50 = 161 nM), and 3 (IC50 = 167 nM), while chloro-
quine presented an IC50 of 130 nM. A comparison of
the IC50 values related to inhibition of growth of the
resistant and the sensitive strains of P. falciparum sug-
gested low levels of cross-resistance to chloroquine. All
tested compounds showed cytotoxicity upon the human
diploid embryonic lung cell line (MRC-5 cells) in the
micromolar range and, IC50 values varied from 1.57 to
18.28 lM. The most active compounds showed an
in vitro inhibition of b-hematin formation, suggesting
an effect on this target. These results indicate that they
may share some similarities in their mechanism of action
with chloroquine; but, this should be more deeply
investigated.
1
trometer. H and 13C NMR spectra were recorded on a
Bruker AC 500 spectrometer operating at 500 and
125 MHz, respectively, with tetramethylsilane as inter-
nal standard, using deuterated chloroform. The chemi-
cal shifts are expressed as d values in parts per million
(ppm) and the coupling constants (J) are given in hertz
(Hz). Yields were of purified compounds and were not
optimized.
Dried-powdered leaves of Ilex paraguariensis A. St Hil.
were submitted to maceration using 70% EtOH and,
after evaporation of ethanol under vacuum, the
saponins present in the resulting aqueous solution were
submitted to acid hydrolysis to afford 1.10 After
crystallization using AcOEt:MeOH (70:30), ursolic acid
(1) was obtained with 10% yield and its purity (>95%)
was determined by HPLC (Rt = 15 min).22 This com-
pound was identified by comparison of its NMR data
with the literature.36,37
Furthermore, the importance of the triterpene skeleton
bearing an acetyl group at C-3 for antimalarial activity
was demostrated and, the piperazine moiety was also
identified as a required pharmacophore in this series.
The importance of the hydrophilic framework attached
at the terminal nitrogen atom of the bis-(3-aminopro-
pyl)piperazine joined to the triterpene ring was also
characterized through MD simulations, supporting a
mechanism of action for these new ursolic acid deriva-
tives on the binding of triterpene to heme. Based on
the obtained results, the most active compounds could
constitute suitable candidates for supplementary physi-
cochemical and biological studies in order to use these
molecules to improve our knowledge about the molecu-
lar mechanism involved in malaria. This means to
understand their mechanism of action as antimalarial
compounds and/or their capacity to interfere in the par-
asite mechanism of resistance. Finally, we believe that
4.2. 3-O-acetylursolic acid (2)
This compound was prepared from 1 (91 mg, 0.2 mmol)
20
D
as previously described10 (100% yield): mp 170 ꢁC; ½aꢃ
+99.3ꢁ (CHCl3, c 0.1); IR (ATR): 3562 (OH), 2947
(OH), 2866, 1732 (C@O acetyl), 1697 (C@O acid),
1460 (C–O), 1385, 1306 (C–O), 1248 (C–O–C), 1030
(C–OH). HRMS: C32H49O4 497.3635 [M + H]+(100%).
4.3. N-{3-[4-(3-aminopropyl)piperazinyl]propyl}-3-O-
acetylursolamide (3) and 3a
To a solution of 2 (1.48 g, 3 mmol) in dry CH2Cl2
(30 mL) at 0 ꢁC was added dropwise oxalyl chloride
(7.8 mL, 9 mmol) and stirred for 3 h at room tempera-
ture. The reaction mixture was allowed to cool at
0 ꢁC, and TEA (25 mL, 18 mmol) and N-tert-butoxycar-
bonyl-1,4-bis(3-aminopropyl)piperazine (2.7 g, 9 mmol)

S. C. B. Gnoatto et al. / Bioorg. Med. Chem. 16 (2008) 771–782
777
were added; then, it was allowed to warm at room tem-
perature and stirred overnight. The reaction was
quenched with H2O (10 mL), and the organic phase
was separated. The aqueous phase was extracted with
CH2Cl2 (3 · 30 mL). The combined organic phases were
dried over Na2SO4, filtered, and evaporated under vac-
uum. The crude residue was dissolved in CH2Cl2
(20 mL), acidified with TFA 10% (50 mL) for deprotec-
tion reaction, and stirred for 5 h. The reaction was
quenched using saturated aqueous potassium carbonate
solution (60 mL). The aqueous phase was extracted with
CH2Cl2 (3 · 30 mL). The organic phases were washed
with H2O (3 · 30 mL), dried over Na2SO4, filtered,
and evaporated under vacuum to afford 3 as a white so-
lid (80%): mp 138 ꢁC; IR (ATR): 3325 (NH2), 2873,
2807, 1731 (C@O acetyl), 1683 (C@O amide), 1637
(C@C), 1462 (C–O), 1367, 1308 (C–O–C), 1245 (C–N).
1H NMR (CDCl3): 0.81 (d, 3 H,3J = 6.9, CH3-30), 0.87
(m, 1H, CH-5), 0.91 (d, 3H, 3J = 6.6, CH3-29), 0.93
(m, 1H, CH2(Ha)-1), 0.94 (s, 3H, CH3-25), 0.95 (m, 1H,
CH-11), 0.96 (m, 1H, CH2-20), 0.99 (s, 3H, CH3-24),
1.00 (s, 3H, CH3-23), 1.01 (s, 3H, CH3-26), 1.14 (s,
3H, CH3-27), 1.21 (m, 1H, CH2(Ha)-7), 1.33 (m; 1H,
CH-19), 1.41 (m, 2H, CH2-2), 1.55 (m, 4H, CH2(Hb)-6,
CH2(Hb)-7, CH-9, CH2(Ha)-21), 1.62 (m, 4H, CH2(Hb)-1,
CH2(Ha)-15, CH2(Hb)-16, CH2(Hb)-21), 1.70 (m, 5H,
with CH2Cl2 (3 · 1 mL). The combined organic phases
were washed with H2O (2 · 1 mL); then, it was dried
over Na2SO4, filtered, and evaporated under vacuum.
The resultant crude products were purified by flash
chromatography on silica gel using 0–10% MeOH in
CH2Cl2 as eluent.
4.5. N-{3-[4-(3-(Bisbenzylamino)propyl)piperazinyl]pro-
pyl}-3-O-acetyl-ursolamide (4)
This compound was prepared from benzaldehyde
(15.2 lL, 0.15 mmol, 3 eq) to afford 4 as yellow oil
(55%): IR (ATR): 3351, 2926, 1732, 1643, 1519, 1452,
1
3
1367, 1244. H NMR (CDCl3): 0.81 (d, 3H, J = 6.9,
CH3), 0.87 (1H, CH), 0.91 (d, 3H,3J = 5.2, CH3), 0.93
(1H, CH2), 0.96 (s, 3H, CH3), 0.95 (1H, CH2), 0.99 (sl,
4H, CH3, CH-20), 1.00 (s, 3H, CH3), 1.01 (s, 3H,
CH3), 1.14 (s, 3H, CH3), 1.21 (m, 1H, CH2), 1.33 (m,
3H, CH, CH2-2), 1.48 (m, 4H, CH2, CH), 1.60 (m,
3
4H, CH2), 1.71 (m, 5H, CH2), 1.77 (td, 1H, J = 14.0
and 4.0, CH2), 1.84 (2H, CH2), 1.99 (m, 1H, CH2),
2.10 (s, 3H, CH3COO), 2.49 (m, 13H, CH2-piperazine,
1H, CH), 3.09 (m, 2H, CH2-d0), 3.47 (m, 2H, CH2-d),
3
3.60 (s, 4H, –N(CH2)2), 4.54 (dd, 1H, J = 9.5 and 5.9,
CH), 5.35 (tl, 1H, CH), 6.48 (sl, 1H, NH), 7.27 (dd, 2·
3
3
1H, J = 7.1, CH-para), 7.34 (dd, 2· 2H, J = 7.3, CH-
3
3
CH2-c, CH2-c0, CH2(Ha)-6), 1.77 (td, 1H, J = 14.0 and
meta), 7.39 (d, 2· 2H, J = 7.5, CH-ortho). 13C NMR
4.0, CH2(Ha)-16), 1.83 (m, 2H, CH2-22), 2.00 (m, 1H,
CH2(Hb)-15), 2.10 (s, 3H, CH3COO), 2.51 (m, 13H,
CH2-piperazine, CH2-b, CH2-b0, CH-18), 3.03 (m, 2H,
(CDCl3): 15.8, 17.1, 17.4, 17.7, 18.6, 21.6 (C-30), 21.7
(CH3COO), 23.8, 23.9, 24.1, 25.1, 26.2, 28.2, 28.5,
31.3, 32.8 (C-c0), 33.4, 37.3, 37.8, 38.1, 38.5, 38.7
(C-d), 39.5, 39.8, 39.9, 42.5, 47.9 (C-9 and C-17), 51.7
(C-b0), 52.9 (piperazine), 53.9 (C-d0), 54.0, 55.6, 56.6
(C-b), 58.9 (C-e), 81.3, 123.9, 127.3 (C-para), 128.6
(C-meta), 129.2 (C-ortho), 140.1, 145.3 (C-ipso), 171.4,
178.1 (C-28). HRMS [M+H]+ C56H85N4O3 requires
861.6622, found 861.6649.
3
CH2-d0), 3.49 (m, 2H, CH2-d), 4.54 (dd, 1H, J = 13.8
and 7.4, CH-3), 5.34 (tl, 1H, CH-12), 6.46 (sl, 1H,
NH). 13C NMR: 15.9 (C-25), 17.1 (C-26), 17.3 (C-24),
17.6 (C-29), 18.6 (C-6), 21.7 (C-30 and CH3COO),
23.8 (C-11), 23.9 (C-27), 24.1 (C-2), 25.1 (C-16), 26.2
(C-c), 28.2 (C-23), 28.5 (C-15), 31.2 (C-21), 31.8 (C-c0),
33.1 (C-7), 37.2 (C-10), 37.8 (C-22), 38.1 (C-4), 38.5
(C-1), 39.2 (C-d), 39.4 (C-19), 39.9 (C-20), 40.0 (C-8),
40.2 (C-d0), 42.6 (C-14), 47.8 (C-9), 47.9 (C-17), 53.4
(C-18), 54.7 (piperazine), 55.6 (C-b and C-b0), 55.7 (C-5),
81.2 (C-3), 123.1 (C-12), 139.5 (C-13), 171.4 (CH3COO),
179.5 (C-28). HRMS [M+H]+ C42H73N4O3 requires
681.5683, found 681.5667.
4.6. N-{3-[4-(3-(Bis(3-phenylpropyl)amino)propyl)piper-
azinyl]propyl}-3-O-acetylursolamide (5)
This compound was prepared from phenylpropionalde-
hyde (26.5 lL, 0.15 mmol, 3 eq) to afford 5 as yellow oil
(30%): IR (ATR): 3318, 2922, 2854, 2811, 1731, 1648,
1455, 1370, 1245. 1H NMR (CDCl3): 0.81 (d, 3H,
3
Compound 3 was submitted to deacetylation reaction
using potassium carbonate according to the literature38
3J = 7.0), 0.87 (1H, CH), 0.91 (d, 3H, J = 5.0, CH3),
0.93 (1H, CH2), 0.94 (1H, CH2), 0.98 (s, 3H, CH3),
0.99 (sl, 7H, CH3, CH), 1.00 (s, 3H, CH3), 1.14 (s, 3H,
CH3), 1.21 (1H, CH2), 1.33 (m, 1H, CH), 1.42 (2H,
CH2), 1.53 (m, 4H, CH2, CH), 1.63 (m, 4H, CH2),
1.74 (m, 6H, CH2), 1.97 (2H, CH2-22), 2.02 (m, 5H,
CH2(Hb)-15, 2· –CH2CH2Ar), 2.10 (s, 3H, CH3), 2.54
(m, 21H, CH2-piperazine, CH2-b, CH2-b0, CH-18,
–N(CH2)2, 2· –CH2Ar), 3.10 (m, 2H, CH2), 3.48 (m,
1
to obtain compound 3a. Its H and 13C NMR spectral
data were similar to those of 3 except the absence of
the acetyl signals.
4.4. General procedure for the synthesis of 4–21 through
reductive amination
3
To a solution of 3 (34 mg, 0.05 mmol) in dry THF
(1 mL) were added the appropriate aldehyde (0.15 mmol,
3 eq) and NaHB(OAc)3 (32.8 mg, 0.15 mmol, 3 eq) as
the reducing agent to obtain compounds 4–18 according
to the literature.20,23 Similar reaction conditions using
1.5 equivalents of the appropriate aldehyde and reduc-
ing agent were used to obtain the compounds 19–21.
The mixture was stirred at room temperature for 5 h;
then, it was quenched with an aqueous solution of
1 M NaOH (0.5 mL). The aqueous phase was extracted
2H, CH2), 4.27 (dd, 1H, J = 8.2 and 6.2, CH), 5.34
(tl, 1H, CH), 6.51 (sl, 1H, NH), 7.21 (d, 2· 2H,
3
3J = 7.1, CH-para), 7.24 (d, 2· 2H, J = 7.3, CH-meta),
7.32 (d, 2· 2H, 3J = 6.2, CH-ortho). 13C NMR (CDCl3):
15.9; 17.1, 17.4, 17.7, 18.6, 21.6, 21.7, 23.8, 23.9, 24.2,
25.1, 26.1, 28.2, 28.5, 30.1 (2· –CH2CH2Ar), 31.3,
33.2, 33.9, 37.3, 37.8, 38.1, 38.7, 39.2, 39.5, 39.9, 40.1,
42.7, 47.9, 52.0, 53.3, 53.5, 54.1, 55.7, 56.6, 57.4, 68.6
(2· –CH2Ar), 81.3, 125.8, 126.3 (C-para), 128.7 (C-
meta), 129.2 (C-ortho), 134.3 (C-ipso), 140.1, 171.4,

778
S. C. B. Gnoatto et al. / Bioorg. Med. Chem. 16 (2008) 771–782
178.2. HRMS [M+H]+ C60H93N4O3 requires 917.7248,
found 917.7242.
3H, 3J = 7.0, CH3), 0.87 (1H, CH), 0.91 (d, 3H,
3J = 5.8, CH3), 0.94 (1H, CH2), 0.95 (s, 3H, CH3),
0.94 (1H, CH2), 0.98 (sl, 4H, CH3,CH), 0.99 (s, 3H,
CH3), 1.00 (s, 3H, CH3), 1.14 (s, 3H, CH3), 1.20 (m,
1H, CH2), 1.33 (m, 1H, CH), 1.41 (m, 2H, CH2), 1.54
(m, 4H, CH2, CH), 1.62 (m, 4H, CH2), 1.70 (m, 5H,
4.7. N-{3-[4-(3-(Bis(4-bromobenzyl)amino)propyl)piper-
azinyl]propyl}-3-O-acetylursolamide (6)
3
This compound was prepared from 4-bromobenzalde-
hyde (27.5 mg, 0.15 mmol, 3 eq) to afford 6 as yellow
oil (56%): IR (ATR): 3325, 2927, 2872, 2810, 1730,
CH2), 1.77 (td, 1H, J = 13.6 and 3.9, CH2), 1.84 (2H,
CH2), 1.97 (m, 1H, CH2), 2.10 (s, 3H, CH3), 2.45 (m,
13H, CH2, CH-18), 3.10 (m, 2H, CH2), 3.47 (m, 2H,
CH2), 3.54 (s, 4H, CH2), 4.54 (dd, 1H, 3J = 9.3 and
5.5, CH), 5.33 (tl, 1H, CH), 6.49 (sl, 1H, NH), 7.03
1
1644, 1519, 1368, 1245, 1069. H NMR (CDCl3): 0.79
(d, 3H, 3J = 7.8, CH3), 0.86 (1H, CH), 0.90 (d, 3H,
3J = 6.2, CH3), 0.93 (1H, CH2), 0.94 (1H, CH2), 0.98
(s, 7H, CH3, CH), 0.99 (sl, 6H, CH3), 1.13 (s, 3H,
CH3), 1.19 (1H, CH2), 1.31 (m, 1H, CH), 1.41 (2H,
CH2), 1.51 (m, 4H, CH2, CH), 1.59 (m, 4H, CH2),
1.72 (m, 6H, CH2), 1.84 (2H, CH2-22), 2.11 (s, 3H,
CH3), 2.41 (m, 13H, CH2, CH), 3.07 (m, 2H, CH2),
3.48 (m, 2H, CH2), 3.50 (s, 4H, CH2), 4.53 (dd, 1H,
3J = 9.7 and 5.8, CH), 5.33 (tl, 1H, CH), 6.56 (sl, 1H,
3
(dd, 2· 2H, JHH = 8.5,3JHF = 8.6, CH-meta), 7.32 (dd,
3
3
2· 2H, JHH = 8.2, JHF = 5.7, CH-ortho).13C NMR
(CDCl3): 15.9; 17.1, 17.4, 17.7, 18.6, 21.6, 21.7, 23.8,
23.9, 24.1, 25.1, 26.1, 28.2, 28.5, 31.3, 33.1, 33.4, 37.3,
37.8, 38.1, 38.7, 39.2, 39.5, 39.8, 40.1, 42.8, 47.9, 51.7,
53.5, 53.6, 53.8, 55.6, 56.8, 57.9, 81.3, 115.5 (C-meta),
122.9 125.6 (C-ipso), 130.6 (C-ortho), 140.2, 161.3 (C-
para), 171.4, 178.2. HRMS [M+H]+ C56H83N4O3F2
requires 897.6433, found 897.6407.
3
NH), 7.25 (d, 2· 2H, J = 8.1, CH-ortho), 7.47 (d, 2·
2H, 3J = 8.2, CH-meta). 13C NMR (CDCl3): 15.9;
17.1, 17.4, 17.7, 18.6, 21.6, 21.7, 23.8, 23.9, 24.1, 25.1,
26.2, 28.2, 28.5, 31.3, 33.1, 37.3, 37.8, 38.1, 38.7, 39.2,
39.5, 39.9, 40.1, 42.8, 47.9, 51.8, 53.4, 53.6, 54.1, 55.7,
56.6, 58.2, 81.3, 121.1 (C-ortho) 125.8, 130.8 (C-meta),
131.7 (C-ipso), 139.0 (C-para), 140.2, 171.4, 178.2.
HRMS [M+H]+ C56H83N4O3Br2 requires 1017.4832,
found 1017.4812.
4.10. N-{3-[4-(3-(Bis(4-nitrobenzyl)amino)propyl)piper-
azinyl]propyl}-3-O-acetylursolamide (9)
This compound was prepared from 4-nitrobenzaldehyde
(22.6 mg, 0.15 mmol, 3 eq) to afford 9 as green oil (40%):
IR (ATR): 3325, 2924, 2854, 1729, 1644, 1597, 1518,
1454, 1368, 1245. 1H NMR (CDCl3): 0.80 (d, 3H,
3
3J = 6.9, CH3), 0.89 (1H, CH), 0.90 (d, 3H, J = 5.7,
4.8. N-{3-[4-(3-(Bis(4-chlorobenzyl)amino)propyl)piper-
azinyl]propyl}-3-O-acetylursolamide (7)
CH3), 0.93 (1H, CH2), 0.96 (s, 3H, CH3), 0.94 (1H,
CH2), 0.97 (sl, 4H, CH3, CH-20), 0.99 (s, 6H, CH3),
1.12 (s, 3H, CH3), 1.19 (1H, CH2), 1.31 (m, 1H, CH),
1.40 (2H, CH2), 1.57 (m, 8H, CH2, CH), 1.72 (m, 6H,
CH2), 1.97 (2H, CH2), 2.02 (m, 1H, CH2), 2.11 (s, 3H,
CH3), 2.48 (m, 13H, CH2, CH), 3.09 (m, 2H, CH2),
3.51 (m, 2H, CH2), 3.70 (s, 4H, CH2), 4.53 (sl, 1H,
CH), 5.34 (tl, 1H, CH), 6.46 (sl, 1H, NH), 7.58 (d, 2·
2H, J = 8.2, CH-ortho), 8.24 (d, 2· 2H, J = 8.3, CH-
meta).13C NMR (CDCl3): 15.9, 17.1, 17.4, 17.7, 18.6,
21.6, 21.7, 23.8, 23.9, 24.1, 25.1, 26.1, 28.2, 28.5, 31.3,
33.1, 33.4, 37.2, 37.8, 38.1, 38.7, 39.2, 39.5, 39.9, 40.1,
42.8, 47.9, 51.8, 53.3, 53.5, 54.1, 55.4, 56.4, 58.4, 81.3,
124.1 (C-ortho), 125.8, 129.6 (C-meta), 139.1, 143.0 (C-
ipso), 148.1 (C-para), 171.4, 178.1. HRMS [M+H]+
C56H83N6O7 requires 951.6323, found 951.6326.
This compound was prepared from 4-chlorobenzalde-
hyde (21.9 mg, 0.15 mmol, 3 eq) to afford 7 as green
oil (56%): IR (ATR): 3374, 2944, 2874, 1731, 1645,
1490, 1367, 1246. 1H NMR (CDCl3): 0.82 (d, 3H,
3J = 6.9, CH3), 0.88 (1H, CH), 0.91 (d, 3H, 3J = 5.6,
CH3), 0.93 (1H, CH2), 0.94 (1H, CH2), 0.96 (s, 3H,
CH3), 0.99 (sl, 4H, CH3, CH), 1.00 (s, 3H, CH3), 1.01
(s, 3H, CH3), 1.14 (s, 3H, CH3), 1.21 (1H, CH2), 1.34
(m, 1H, CH), 1.42 (2H, CH2), 1.53 (m, 4H, CH2, CH),
1.63 (m, 4H, CH2), 1.72 (m, 6H, CH2), 1.97 (2H, CH2-
22), 2.02 (m, 1H, CH2), 2.10 (s, 3H, CH3), 2.49 (m,
13H, CH2, CH), 3.09 (m, 2H, CH2), 3.47 (m, 2H,
CH2), 3.54 (s, 4H, CH2), 4.55 (dd, 1H, 3J = 9.3 and
5.3, CH), 5.34 (tl, 1H, CH), 6.46 (sl, 1H, NH), 7.30 (d,
3
3
3
3
2· 2H, J = 8.8, CH-ortho), 7.32 (d, 2· 2H, J = 8.5,
CH-meta). 13C NMR (CDCl3): 15.9; 17.1, 17.4, 17.7,
18.6, 21.6, 21.8, 23.8, 23.9, 24.1, 25.1, 26.2, 28.3, 28.5,
31.3, 33.2, 33.4, 37.3, 37.8, 38.1, 38.7, 39.1, 39.5,
39.8, 40.0, 42.5, 47.9, 51.8, 53.3, 53.5, 54.1, 55.7, 56.6,
58.1, 81.3, 122.9 128.8 (C-meta), 130.4 (C-ipso), 133.0
(C-para), 138.5(C-ortho), 140.2, 171.3, 178.2. HRMS
[M+H]+ C56H83N4O3Cl2 requires 929.5842, found
929.5825.
4.11. N-{3-[4-(3-(Bis(4-hydroxybenzyl)amino)propyl)pip-
erazinyl]propyl}-3-O-acetylursolamide (10)
This compound was prepared from 4-hydroxybenzalde-
hyde (18.3 mg, 0.15 mmol, 3 eq) to afford 10 as green oil
(30%): IR (ATR): 3309, 2925, 2872, 1728, 1629, 1514,
1453, 1367, 1246. 1H NMR (CDCl3): 0.79 (d, 3H,
3
3J = 7.0, CH3), 0.87 (1H, CH), 0.91 (d, 3H, J = 5.8,
CH3), 0.93 (1H, CH2), 0.96 (s, 3H, CH3), 0.94 (1H,
CH2), 0.96 (sl, 4H, CH3, CH), 0.97 (s, 3H, CH3), 1.00
(s, 3H, CH3), 1.13 (s, 3H, CH3), 1.21 (1H, CH2), 1.38
(m, 3H, CH, CH2), 1.53 (m, 4H, CH2, CH), 1.62 (m,
4H, CH2), 1.69 (m, 6H, CH2), 1.95 (2H, CH2), 1.98
(m, 1H, CH2), 2.11 (s, 3H, CH3), 2.48 (m, 13H, CH2,
CH), 3.06 (m, 2H, CH2), 3.45 (m, 2H, CH2), 3.54 (s,
4H, CH2), 4.54 (dd, 1H, 3J = 9.3 and 5.5, CH), 5.33
(tl, 1H, CH), 6.39 (sl, 1H, NH), 6.77 (d, 2· 2H,
4.9. N-{3-[4-(3-(Bis(4-fluorobenzyl)amino)propyl)piperaz-
inyl]propyl}-3-O-acetylursolamide (8)
This compound was prepared from 4-fluorobenzalde-
hyde (16.2 lL, 0.15 mmol, 3 eq) to afford 8 as green
oil (70%): IR (ATR): 3361, 2927, 2874, 1731, 1644,
1507, 1454, 1368, 1245. 1H NMR (CDCl3): 0.81 (d,

S. C. B. Gnoatto et al. / Bioorg. Med. Chem. 16 (2008) 771–782
779
3J = 8.1, CH-ortho), 7.16 (d, 2· 2H, 3J = 8.2, CH-meta).
4.14. N-{3-[4-(3-(Bis(cyclopropylmethyl)amino)pro-
pyl)piperazinyl]propyl}-3-O-acetylursolamide (13)
13C NMR (CDCl3): 15.9, 17.1, 17.3, 17.7, 18.5, 21.6,
21.7, 23.8, 23.9, 24.1, 25.2, 26.2, 28.2, 28.5, 31.2, 33.0,
33.4, 37.2, 37.7, 38.1, 38.7, 39.1, 39.5, 39.9, 40.1, 42.6,
47.8, 48.1, 51.3, 52.7, 53.8, 53.8, 54.2, 55.6, 56.4,
58.3, 81.3, 115.8 (C-meta), 125.9, 130.6 (C-ortho),
130.9 (C-ipso), 140.2, 156.0 (C-para), 171.6, 179.6.
HRMS [M+H]+ C56H83N4O5 requires 893.6493, found
893.6498.
This compound was prepared from cyclopropanecar-
boxaldehyde (11.4 lL, 0.15 mmol, 3 eq) to afford 13 as
yellow oil (30%): IR (ATR): 3322, 2922, 2852, 1733,
1
1646, 1524, 1457, 1369, 1244. H NMR (CDCl3): 0.82
(d, 3H, 3J = 6.7, CH3), 0.87 (1H, CH), 0.92 (d, 3H,
3J = 5.5, CH3), 0.94 (1H, CH2), 0.95 (s, 3H, CH3),
0.93 (1H, CH2), 1.00 (sl, 4H, CH3, CH), 1.01 (s, 6H,
CH3), 1.14 (s, 3H, CH3), 1.21 (1H, CH2), 1.32 (m,
11H, CH-19, CH2-2, 4· CH2 cyclopropyl), 1.55 (m,
6H, CH2, 2· CH cyclopropyl), 1.72 (m, 10H, CH2),
4.12. N-{3-[4-(3-(Bis(4-methoxybenzyl)amino)propyl)pip-
erazinyl]propyl}-3-O-acetylursolamide (11)
3
This compound was prepared from 4-methoxybenzalde-
hyde (18.2 lL, 0.15 mmol, 3 eq) to afford 11 as yellow oil
(30%): IR (ATR): 3363, 2927, 2873, 1731, 1645, 1510,
1456, 1367, 1244. 1H NMR (CDCl3): 0.81 (d, 3H,
3J = 6.9, CH3), 0.87 (1H, CH), 0.91 (d, 3H, 3J = 5.3,
CH3), 0.93 (1H, CH2), 0.96 (s, 3H, CH3), 0.94 (1H,
CH2), 0.99 (sl, 4H, CH3, CH), 1.00 (s, 3H, CH3), 1.01
(s, 3H, CH3), 1.14 (s, 3H, CH3), 1.23 (m, 1H, CH2),
1.33 (1H, CH), 1.41 (m, 2H, CH2), 1.52 (m, 4H, CH2,
CH), 1.62 (m, 4H, CH2), 1.73 (m, 6H, CH2), 1.87 (2H,
CH2), 2.00 (m, 1H, CH2), 2.10 (s, 3H, CH3), 2.50 (m,
13H, CH2, CH-18), 3.10 (m, 2H, CH2), 3.47 (m, 2H,
CH2), 3.52 (s, 4H, CH2), 3.85 (s, 6H, CH3O-), 4.54
2.01 (m, 3H, CH2), 2.09 (d, 4H, J = 6.0, –N(CH2)2),
2.10 (s, 3H, CH3), 2.55 (m, 13H, CH2, CH), 3.10 (m,
3
2H, CH2), 3.51 (m, 2H, CH2), 4.55 (dd, 1H, J = 9.2
and 5.5, CH), 5.35 (tl, 1H, CH), 6.55 (sl, 1H, NH).13C
NMR (CDCl3): 15.9, 17.1, 17.4, 17.7, 18.6, 21.6, 23.8,
23.9, 24.1, 25.1, 26.1, 28.3, 28.5, 29.7 (4· CH2 cyclopro-
pyl), 30.1 (2· CH cyclopropyl), 31.9, 32.3, 33.2, 37.3,
37.8, 38.1, 38.7, 39.3, 39.5, 39.9, 40.1, 42.8, 47.9, 51.9,
53.6, 53.8, 54.1, 55.7, 57.7, 59.2, 81.3, 125.8, 140.1,
171.4, 178.1. HRMS [M+H]+ C50H85N4O3 requires
789.6622, found 789.6609.
4.15. N-{3-[4-(3-(Bisheptylamino)propyl)piperazinyl]pro-
pyl}-3-O-acetyl-ursolamide (14)
3
(dd, 1H, J = 9.5 and 5.9, CH), 5.35 (tl, 1H, CH), 6.50
3
(sl, 1H, NH), 6.89 (d, 2· 2H, J = 8.4, CH-ortho), 7.29
(d, 2· 2H, 3J = 8.4, CH-meta).13C NMR (CDCl3):
15.9, 17.1, 17.4, 17.7, 18.6, 21.6, 21.7, 23.8, 23.9, 24.1,
25.1, 26.2, 28.2, 28.5, 31.3, 33.1, 37.2, 37.8, 38.1, 38.7,
39.1, 39.5, 39.8, 40.1, 42.5, 47.9, 51.7, 52.9, 53.1, 54.0,
55.6, 56.6 (CH3O-), 57.9, 81.3, 113.9 (C-meta), 123.9,
125.8 (C-ipso), 130.3 (C-ortho), 140.1, 158.9 (C-para),
171.4, 178.1. HRMS [M+H]+ C58H89N4O5 requires
921.6833, found 921.6792.
This compound was prepared from heptaldehyde
(22 lL, 0.15 mmol, 3 eq) to afford 14 as yellow oil
(30%): IR (ATR): 3318, 2926, 2854, 1735, 1640, 1519,
1456, 1366, 1244. 1H NMR (CDCl3): 0.82 (d, 3H,
3
3J = 6.8, CH3), 0.87 (1H, CH), 0.91 (d, 3H, J = 6.7,
CH3), 0.93 (1H, CH2), 0.94 (8H, CH2, 2· CH3 heptyl),
0.95 (s, 3H, CH3), 0.97 (sl, 4H, CH3, CH), 0,.99 (s,
3H, CH3), 1.01 (s, 3H, CH3), 1.14 (s, 3H, CH3), 1.21
(1H, CH2), 1.32 (m, 23H, CH-19, CH2-2, 10· CH2 hep-
tyl), 1.53 (m, 4H, CH2, CH), 1.74 (m, 10H, CH2), 1.99
(m, 3H, CH2), 2.10 (s, 3H, CH3), 2.46 (m, 17H, CH2,
CH, –N(CH2)2), 3.09 (m, 2H, CH2), 3.48 (m, 2H,
CH2), 4.56 (dd, 1H, 3J = 9.3 and 5.5, CH), 5.35 (tl,
1H, CH), 6.53 (sl, 1H, NH).13C NMR (CDCl3): 14.4
(2· CH3 heptyl), 15.9, 17.1, 17.4, 17.7, 18.6, 21.6, 23.0
(2· –CH2CH3 heptyl), 23.8, 23.9, 24.1, 25.1, 26.1, 27.9
(2· –CH2(CH2)3CH3 heptyl), 28.3, 28.5, 28.7 (2·
–CH2(CH2)2CH3 heptyl), 29.7 (2· –CH2(CH2)4CH3
heptyl), 30.1 (2· –CH2CH2CH3 heptyl), 31.3, 32.3, 33.2,
37.3, 37.8, 38.1, 38.7, 39.3, 39.5, 39.9, 40.1, 42.8, 47.9,
48.0, 52.3, 53.6, 54.1, 54.4, 55.7, 57.1, 57.8, 81.3, 125.8,
140.1, 171.4, 178.1. HRMS [M+H]+ C56H101N4O3
requires 877.7874, found 877.7901.
4.13. N-{3-[4-(3-(Bis(cyclohexylmethyl)amino)pro-
pyl)piperazinyl]propyl}-3-O-acetylursolamide (12)
This compound was prepared from cyclohexanecarbox-
aldehyde (18.14 lL, 0.15 mmol, 3 eq) to afford 12 as
yellow oil (50%): IR (ATR): 3310, 2920, 2849, 1733,
1
1643, 1520, 1448, 1368, 1242. H NMR (CDCl3): 0.83
(d, 3H, 3J = 6.7, CH3), 0.87 (1H, CH), 0.92 (d, 3H,
3J = 6.4, CH3), 0.93 (1H, CH2), 0.94 (1H, CH2), 0.96
(s, 3H, CH3), 0.97 (sl, 4H, CH3, CH), 0.99 (s, 3H,
CH3), 1.01 (s, 3H, CH3), 1.15 (s, 3H, CH3), 1.20
(1H, CH2), 1.25 (m, 12H, 6· CH2 cyclohexyl), 1.33
(m, 13H, CH-19, CH2-2, 4· CH2 cyclohexyl), 1.54
(m, 6H, CH2, 2· CH cyclohexyl), 1.74 (m, 10H,
CH2), 1.99 (m, 3H, CH2), 2.10 (s, 3H, CH3), 2.13 (d,
4H, 3J = 6.3, –N(CH2)2), 2.48 (m, 13H, CH2, CH),
3.10 (m, 2H, CH2), 3.49 (m, 2H, CH2), 4.55 (dd, 1H,
3J = 9.3 and 5.5, CH), 5.35 (tl, 1H, CH), 6.53 (sl, 1H,
NH).13C NMR (CDCl3): 15.9, 17.1, 17.4, 17.7, 18.6,
21.6, 23.8, 23.9, 24.1, 25.1, 26.1, 26.6 (6· CH2 cyclo-
hexyl), 27.4 (4· CH2 cyclohexyl) 28.3, 28.5, 31.3,
32.3, 33.2, 36.7 (2· CH cyclohexyl), 37.3, 37.9,
38.1, 38.7, 39.3, 39.5, 40.0, 40.1, 42.9, 47.9, 53.0,
53.5, 53.6, 54.1, 55.7, 57.0, 57.7, 81.3, 125.8, 140.1,
171.3, 178.1. HRMS [M+H]+ C56H97N4O3 requires
873.7561, found 873.7553.
4.16. N-{3-[4-(3-(Bisbutylamino)propyl)piperazinyl]pro-
pyl}-3-O-acetyl-ursolamide (15)
This compound was prepared from butyraldehyde
(13.2 lL, 0.15 mmol, 3 eq) to afford 15 as yellow oil
(30%): IR (ATR): 3320, 2926, 2870, 1733, 1640, 1520,
1461, 1368, 1242. 1H NMR (CDCl3): 0.82 (d, 3H,
3
3J = 6.9, CH3), 0.88 (1H, CH), 0.91 (d, 3H, J = 6.7,
CH3), 0.94 (2H, CH2), 0.93 (m, 6H, 2· CH3 butyl),
0.95 (s, 3H, CH3), 0.97 (sl, 4H, CH3, CH), 0.98 (s, 3H,
CH3), 1.00 (s, 3H, CH3), 1.14 (s, 3H, CH3), 1.21 (1H,

780
S. C. B. Gnoatto et al. / Bioorg. Med. Chem. 16 (2008) 771–782
CH2), 1.35 (m, 11H, CH-19, CH2-2, 4· CH2butyl), 1.53
(m, 4H, CH2, CH), 1.73 (m, 10H, CH2), 1.99 (m, 3H,
CH2), 2.10 (s, 3H, CH3), 2.51 (m, 17H, CH2, CH,
–N(CH2)2), 3.10 (m, 2H, CH2), 3.47 (m, 2H, CH2),
4.19. N-{3-[4-(3-(Bisferrocenylamino)propyl)piperazi-
nyl]propyl}-3-O-acetyl-ursolamide (18)
This compound was prepared from ferrocenecarboxal-
dehyde (65.5 mg, 0.15 mmol, 3 eq) to afford 18 as yellow
oil (60%): IR (ATR): 3328, 2924, 2854, 1733, 1646, 1518,
1455, 1369, 1244. 1H NMR (CDCl3): 0.81 (d, 3H,
3
4.54 (dd, 1H, J = 9.3 and 5.5, CH), 5.35 (tl, 1H, CH),
6.51 (sl, 1H, NH).13C NMR (CDCl3): 14.0 (2· CH3
butyl), 15.8, 17.1, 17.4, 17.7, 18.6, 20.6 (2· –CH2CH3
butyl), 21.6, 21.7, 23.7, 23.9, 24.1, 25.1, 25.4 (2·
–CH2CH2CH3 butyl), 26.2, 28.3, 28.5, 30.1, 31.3, 33.1,
37.3, 37.9, 38.1, 38.5, 39.3, 39.5, 39.9, 40.0, 42.7, 47.9,
48.1, 51.2, 52.8, 53.4, 53.7, 54.7, 54.8, 55.6, 81.3, 125.8,
140.2, 171.4, 178.1. HRMS [M+H]+ C50H89N4O3
requires 793.6935, found 793.6981.
3
3J = 6.0, CH3), 0.86 (1H, CH), 0.90 (d, 3H, J = 6.3,
CH3), 0.91 (1H, CH2), 0.94 (1H, CH2), 0.98 (s, 3H,
CH3), 0.99 (sl, 4H, CH3, CH), 1.00 (s, 6H, CH3), 1.13
(s, 3H, CH3), 1.20 (1H, CH2), 1.32 (m, 3H, CH, CH2),
1.53 (m, 4H, CH2,CH), 1.62 (m, 4H, CH2), 1.72 (m,
6H, CH2), 1.84 (2H, CH2), 2.02 (m, 1H, CH2), 2.11 (s,
3H, CH3), 2.34 (m, 13H, CH2, CH), 3.08 (m, 2H,
CH2), 3.48 (m, 2H, CH2), 3.53 (s, 4H, –N(CH2)2), 4.17
(s, 10H, ferrocene), 4.26 (m, 8H, ferrocene), 4.54 (dd,
4.17. N-{3-[4-(3-(Bispropylamino)propyl)piperazinyl]pro-
pyl}-3-O-acetyl-ursolamide (16)
3
1H, J = 9.0 and 5.3, CH), 5.33 (tl, 1H, CH), 6.54 (sl,
This compound was prepared from propionaldehyde
(11.2 lL, 0.15 mmol, 3 eq) to afford 16 as yellow oil
(45%): IR (ATR): 3325, 2923, 2870, 1734, 1639, 1523,
1457, 1369, 1243. 1H NMR (CDCl3): 0.80 (d, 3H,
3J = 7.0, CH3), 0.88 (1H, CH), 0.91 (d, 3H, 3J = 6.5,
CH3), 0.93 (1H, CH2), 0.93 (m, 6H, 2· CH3 propyl),
0.94 (1H, CH2), 0.96 (s, 3H, CH3), 0.99 (s, 3H, CH3,
1H, CH), 1.00 (s, 3H, CH3), 1.01 (s, 3H, CH3), 1.14
(s, 3H, CH3), 1.21 (m, 1H, CH2), 1.32 (m, 7H, CH,
2· CH2 propyl), 1.53 (m, 4H, CH2, CH), 1.62 (m,
4H, CH2), 1.74 (m, 6H, CH2), 1.99 (m, 3H, CH2),
2.10 (s, 3H, CH3), 2.56 (m, 17H, CH2, CH,
–N(CH2)2), 3.08 (m, 2H, CH2), 3.49 (m, 2H, CH2),
4.54 (dd, 1H, 3J = 9.3 and 5.5, CH), 5.35 (tl, 1H,
CH), 6.43 (sl, 1H, NH).13C NMR (CDCl3): 12.3 (2·
CH3 propyl), 15.9, 17.1, 17.4, 17.7, 18.6, 20.5 (CH2
propyl), 21.6, 23.8, 23.9, 24.1, 25.1, 26.1, 28.2,
28.5, 31.3, 32.3, 33.2, 37.3, 37.8, 38.1, 38.7, 39.3,
39.5, 39.9, 40.1, 42.8, 47.9, 48.1, 52.5, 53.7, 53.9,
54.1, 55.7, 56.6, 57.8, 81.3, 125.8, 140.2, 171.6, 179.0.
HRMS [M+H]+ C48H85N4O3 requires 765.6622, found
765.6600.
1H, NH).13C NMR (CDCl3): 15.9, 17.1, 17.4, 17.7,
18.6, 21.6, 21.7, 23.8, 23.9, 24.1, 25.1, 26.1, 28.3, 28.5,
29.7, 31.3, 33.1, 37.3, 37.8, 38.1, 38.7, 39.0, 39.5, 39.9,
40.1, 42.8, 47.9, 53.1, 53.4, 54.0, 54.1, 55.7, 69.1 (ferro-
cene), 69.7 (ferrocene), 70.0 (ferrocene), 70.9, 81.3, 125.8,
140.1, 171.4, 179.2. HRMS [M+H]+ C64H93N4O3Fe2
requires 1077.5946, found 1077.5945.
4.20. N-{3-[(4-(3-(4-Nitrobenzyl)amino)propyl)piperazi-
nyl]propyl}-3-O-acetyl-ursolamide (19)
This compound was prepared from 4-nitrobenzalde-
hyde (13.7 mg, 0.075 mmol) to afford 19 as green oil
20
D
(20%): ½aꢃ +29.1ꢁ (CHCl3, c 0.1); IR (ATR): 3332
(NH); 2854; 1729 (C@O acetyl); 1644 (C@O amide);
1597 (C@C); 1518 (aromatic-NO2); 1454 (C–O);
1368; 1245 (C–N); 854 (aromatic p-substituted). 1H
NMR (CDCl3): 0.81 (d, 3H, 3J = 7.0, CH3-30), 0.89
(1H, CH-5), 0.90 (d, 3H, 3J = 5.8, CH3-29), 0.96 (s,
3H, CH3-25), 0.95 (2H, CH2-1, CH2-11), 0.97 (sl,
4H, CH3-24, CH-20), 1.00 (s, 6H, CH3-23, CH3-26),
1.13 (s, 3H, CH3-27), 1.19 (1H, CH2(Ha)-7), 1.36 (m,
3H, CH2-2, CH-19), 1.57 (m, 8H, CH2(Hb)-1,
CH2(Hb)-6, CH2(Hb)-7, CH-9, CH2(Ha)-15, CH2(Hb)-16,
CH2-21), 1.74 (m, 8H, CH2-c, CH2H-c0, CH2(Ha)-6,
CH2( Ha)-16, CH2-22), 2.04 (m, 1H, CH2(Hb )-15),
2.11 (s, 3H, H3CCOO), 2.47 (m, 13H, CH2-a1, CH2-
a10, CH2-a2, CH2-a20, CH2-b, CH2-b0, CH-18), 3.04
(m, 2H, CH2-d0), 3.50 (m, 2H, CH2-d), 3.75 (s, 2H,
–NCH2), 4.53 (dd, 1H, 3J = 11.7 and 4.6, CH-3),
5.34 (tl, 1H, CH-12), 6.57 (sl, 1H, NH), 7.58 (d,
4.18. N-{3-[4-(3-(Bisisobutylamino)propyl)piperazi-
nyl]propyl}-3-O-acetyl-ursolamide (17)
This compound was prepared from isobutyraldehyde
(26.4 lL, 0.15 mmol, 3 eq) to afford 17 as yellow oil
(39%): IR (ATR): 3335, 2926, 2868, 1735, 1637, 1521,
1458, 1368, 1244. 1H NMR (CDCl3): 0.82 (3H, d,
3J = 6.5, CH3), 0.87 (1H, CH), 0.91 (m, 15H, CH3-29,
2· CH3 isobutyl), 0.93 (sl, 6H, CH3, CH2), 0.94 (sl,
4H, CH3, CH), 0.97 (s, 6H, CH3), 1.12 (s, 3H, CH3),
1.20 (1H, CH2), 1.31 (m, 5H, CH, CH2, 2· CH isobu-
tyl), 1.54 (m, 4H, CH2, CH), 1.73 (m, 10H, CH2), 1.99
(m, 3H, CH2), 2.10 (s, 3H, CH3), 2.41 (m, 17H, CH2,
CH, –N(CH2)2), 3.09 (m, 2H, CH2), 3.50 (m, 2H,
CH2), 4.54 (dd, 1H, 3J = 9.0 and 5.7, CH), 5.35 (tl,
1H, CH), 6.62 (sl, 1H, NH).13C NMR (CDCl3): 15.9,
17.1, 17.4, 17.7, 18.6, 21.3 (4· CH3 isobutyl), 21.6,
21.7, 23.8, 23.9, 24.1, 25.1, 26.0, 27.0 (2· CH isobutyl),
28.3, 28.5, 30.1, 31.3, 33.2, 37.3, 37.8, 38.1, 38.7, 39.3,
39.5, 39.9, 40.1, 42.8, 47.8, 47.9, 53.0, 53.6, 54.1,
54.5, 55.7, 57.2, 57.7, 81.3, 125.8, 140.1, 171.4, 178.2.
HRMS [M+H]+ C50H89N4O3 requires 792.6935, found
793.6942.
3
3
2H, J = 8.2, H-ortho), 8.22 (d, 2H, J = 8.3, H-meta).
13C NMR (CDCl3): 15.9 (C-25), 17.1 (C-26), 17.4 (C-
24), 17.7 (C-29), 18.6 (C-6), 21.6 (C-30), 21.7
(H3CCOO), 23.8 (C-11), 23.9 (C-27), 24.1 (C-2),
25.1 (C-16), 26.0 (C-c), 28.2 (C-23), 28.5 (C-15),
30.2 (C-c0), 31.3 (C-21), 33.1 (C-7), 37.2 (C-10),
37.8 (C-22), 38.1 (C-4), 38.7 (C-1), 39.2 (C-d), 39.5
(C-19), 39.9 (C-20), 40.1 (C-8), 42.8 (C-14), 47.9 (C-
17), 48.0 (C-9), 48.8 (C-b), 53.5 (C-a1, C-a10, C-a2,
C-a20), 53.5 (C-d0), 54.1 (C-18), 55.6 (C-5), 57.1 (C-
b0), 57.5 (C-e), 81.3 (C-3), 124.1 (C-ortho), 125.8
(C-12); 129.6 (C-meta); 139.2 (C-13); 143.0 (C-ipso);
148.0 (C-para); 171.5 (H3CCOO); 178.1 (C-28).
HRMS [M+H]+ C49H78N5O5 requires 816.6003,
found 816.5983.

S. C. B. Gnoatto et al. / Bioorg. Med. Chem. 16 (2008) 771–782
781
4.21. N-{3-[(4-(3-(4-Hydroxybenzyl)amino)propyl)piper-
azinyl]propyl}-3-O-acetylursolamide (20)
using explicit water molecules and counter-ions to neu-
tralize the system.
This compound was prepared from 4-hydroxybenzalde-
hyde (9.15 mg, 0.075 mmol) to afford 20 as green oil
4.24. In vitro P. falciparum culture and drug assays
20
D
(62%): ½aꢃ +58.3 (CHCl3, c 0.15). IR (ATR): 3309;
Plasmodium falciparum strains were maintained
continuously in culture on human erythrocytes as
described by Trager and Jensen.45 In vitro antiplasmo-
dial activity was determined using a modification of
the semi-automated microdilution technique of Desjar-
dins et al.46 P. falciparum CQ-sensitive (Thai) and
CQ-resistant (FcB1) strains were used in sensitivity
testing. Stock solutions of chloroquine diphosphate
and test compounds were prepared in sterile distilled
water and DMSO, respectively. Drug solutions were
serially diluted with culture medium and added to
asynchronous parasite cultures (1% parasitemia and
1% final hematocrite) in 96-well plates for 24 h, at
37 ꢁC, prior to the addition of 0.5 lCi of [3H]hypo-
xanthine (1–5 Ci/mmol; Amersham, Les Ulis, France)
per well, for 24 h. The growth inhibition for each drug
concentration was determined comparing the radioac-
tivity incorporated into the treated culture with that in
the controlled culture (without drug) maintained on
the same plate. The concentration causing 50% inhibi-
tion (IC50) was obtained from the drug concentration-
response curve and the results were expressed as
means standard deviations determined from several
independent experiments. The DMSO concentration
never exceeded 0.1% and did not inhibit the parasite
growth.
2853; 1732; 1637; 1518; 1457; 1369; 1246. 1H NMR
3
(CDCl3): 0.81 (d, 3H, J = 6.9, CH3), 0.87 (1H, CH),
0.91 (d, 3H, 3J = 5.9, CH3), 0.96 (s, 3H, CH3), 0.95
(2H, CH2), 0.96 (sl, 4H, CH3 and CH), 0.99 (s, 3H,
CH3), 1.01 (s, 3H, CH3), 1.14 (s, 3H, CH3), 1.21
(1H, CH2), 1.37 (m, 3H, CH and CH2), 1.53 (m, 4H,
CH2, CH), 1.62 (m, 4H, CH2), 1.75 (m, 8H, CH2),
2.01 (m, 1H, CH2), 2.11 (s, 3H, CH3), 2.44 (m, 13H,
CH2, CH), 3.04 (m, 2H, CH2), 3.45 (m, 2H, CH2),
3.75 (s, 2H), –N(CH2), 4.55 (dd, 1H, 3J = 11.3 and
5.5, CH), 5.34 (tl, 1H, CH), 6.47 (sl, 1H, NH), 6.78
(d, 2H, 3J = 8.1, CH-ortho), 7.20 (d, 2H, 3J = 8.2,
CH-meta). 13C NMR (CDCl3): 15.9, 17.1, 17.3, 17.7,
18.6, 21.6, 21.7, 23.8, 23.9, 24.1, 25.1, 26.0, 28.2,
28.5, 30.1, 31.2, 33.1, 37.2, 37.8, 38.1, 38.7, 39.2,
39.5, 39.9, 40.1, 42.8, 47.9, 48.0, 48.9, 53.5, 54.1,
54.2, 55.6, 57.3, 57.7, 81.3, 115.7 (C-meta), 125.9
(C-12), 130.3 (C-orthoet C-ipso), 140.1, 156.0 (C-para),
171.5 (H3CCOO), 178.6.
4.22. N-{3-[(4-(3-Ferrocenylamino)propyl)piperazi-
nyl]propyl}-3-O-acetyl-ursolamide (21)
This compound was prepared from ferrocenecarboxal-
dehyde (16.5 mg, 0.075 mmol) to afford 21 as yellow
oil (52%): IR (ATR): 3328, 2813, 1731, 1639, 1524,
1
1457, 1370, 1246, 1105, 1031. H NMR (CDCl3): 0.81
4.25. Inhibition of b-hematin formation test
(3H, d, 3J = 6.0, CH3), 0.87 (1H, CH), 0.91 (d,
3J = 6.3, 3H, CH3), 0.93 (2H, CH2), 0.96 (m, 7H, CH3,
CH), 1.00 (s, 6H, CH3), 1.14 (s, 3H, CH3), 1.21 (1H,
CH2), 1.34 (m, 3H, CH, CH2), 1.52 (m, 4H, CH2,
CH), 1.61 (m, 4H, CH2), 1.73 (m, 6H, CH2), 1.86 (2H,
CH2), 2.01 (m, 1H, CH2), 2.10 (s, 3H, CH3), 2.36 (m,
13H, CH2, CH), 3.06 (m, 2H, CH2), 3.47 (m, 2H,
CH2), 3.78 (s, 2H, –NCH2), 4.21 (s, 5H, CH), 4.28 (m,
The methodology was the same as Baelmans and
co-workers.47 Deferoxamine mesylate presented 0%
inhibition from 1 to 20 mM.
Acknowledgments
3
4H, CH), 4.54 (dd, J = 11.3, 1H, 5.5, CH ferrocene),
5.35 (tl, 1H, CH ferrocene), 6.41 (sl, 1H, NH). 13C
NMR (CDCl3): 15.9, 17.1, 17.3, 17.7, 18.6, 21.6, 23.8,
23.9, 25.1, 26.2, 28.2, 28.5, 30.1, 31.3, 33.1, 37.3, 37.8,
38.1, 38.7, 39.0, 39.5, 39.9, 40.1, 42.8, 47.9, 48.2, 48.6,
53.4, 53.6, 54.1, 55.6, 57.3, 58.1, 68.5 (ferrocene), 69.2
(ferrocene), 69.6 (ferrocene), 81.3, 125.8, 140.2, 171.4,
178.5.
This work was supported by Accord CAPES/COFE-
CUB (Brazil/France) 418/03 and Conselho Nacional
´
´
de Desenvolvimento Cientıfico e Tecnologico (CNPq,
Brazil). The authors are also grateful to the Programa
´
ˆ ˆ
de Pos-Graduac¸a˜o em Ciencias Farmaceuticas/UFRGS
(Brazil).
4.23. Molecular modeling
References and notes
1. WHO, 2007. http://www.who.int/tdr/diseases/malaria/
direction.htm.
2. (a) Breman, J. G. Am. J. Trop. Med. Hyg. 2001, 64, 1; (b)
Klan, S. M.; Waters, A. P. Trends Parasitol. 2004, 20, 575;
(c) WHO 2007 http://www.who.int/csr/drugresist/malaria/
en/.
3. Biot, C.; Chibale, K. Infect. Disorders-Drug Targets 2006,
6, 173.
4. Hostettmann, K.; Marston, A. Saponins; Cambridge
University: Cambridge, 1995.
The calculations employed methodology previously de-
scribed by the group.39–41 Briefly, the compounds were
constructed and geometry optimized at the HF/3-21G
level using GAMESS.42 The so-obtained geometries
were submitted to a single-point calculation at the HF/
6-31G** level in order to obtain atomic charges suitable
for MD simulations. These charges were included in
PRODRG derived topologies43 for each compound,
allowing the description of the molecules through MD.
All simulations were performed under GROMACS sim-
ulation suite44 and GROMOS96 force field for 10.0 ns
5. Liu, J. J. Ethnopharmacol. 1995, 49, 57.
6. Liu, J. J. Ethnopharmacol. 2005, 100, 92.

782
S. C. B. Gnoatto et al. / Bioorg. Med. Chem. 16 (2008) 771–782
7. Mahato, S. B.; Garai, S. In Progress in the chemistry of
organic natural products; Herz, W., Ed.; Springer: Wien,
1998; pp 1–196.
27. Delhaes, L.; Abessolo, H.; Biot, C.; Berry, L.; Delcourt,
P.; Maciejewski, L.; Brocard, J.; Camus, D.; Dive, D.
Parasitol. Res. 2001, 87, 239.
8. Steele, J. C. P.; Warhhurst, D. C.; Kirby, G. C.;
Simmonds, M. S. J. Phytother. Res. 1999, 13, 115.
9. Gosmann, G.; Schenkel, E. P.; Seligmann, O. J. Nat. Prod.
1989, 52, 1367.
10. Gosmann, G.; Guillaume, D.; Taketa, A. T. C.; Schenkel,
E. P. J. Nat. Prod. 1995, 58, 438.
11. Schenkel, E. P.; Gosmann, G.; Montanha, J. A.; Heinz-
mann, B. M.; Athayde, M. L.; Taketa, A. T. C.; Pires, V.
S.; Guillaume, D. Cien. Cult. (Sa˜o Paulo) 1997, 49, 359.
12. Amusan, O. O. G.; Adesogan, E. K.; Makinde, J. M.
Phytother. Res. 1996, 10, 692.
28. Biot, C.; Dessolin, J.; Ricard, I.; Dive, D. J. Organomet.
Chem. 2004, 689, 4678.
29. Biot, C.; Taramelli, D.; Frofar-Bares, I.; Maciejewski, L.;
Boyce, M.; Nowogrocki, G.; Brocard, J.; Basilico, N.;
Olliaro, P.; Egan, T. J. Mol. Pharm. 2005, 2, 185.
30. Egan, T. J.; Chen, J. Y.-J.; de Villiers, K. A.; Mabotha, T.
E.; Naidoo, K. J.; Ncokazi, K. K.; Langford, S. J.;
McNaughton, D.; Pandiancherri, S.; Wood, B. R. FEBS
Lett. 2006, 580, 5105.
31. de Villiers, K. A.; Kaschula, C. H.; Egan, T. J.; Marques,
H. M. J. Biol. Inorg. Chem. 2007, 12, 101.
13. Traore-Keita, F.; Gasquet, M.; Di Giorgio, C.; Ollivier,
E.; Delmas, F.; Keita, A.; Duombo, O.; Balansard, G.;
Timon-David, P. Phytother. Res. 2000, 14, 45.
32. Dorn, A.; Vippagunta, S. R.; Matile, H.; Jaquet, C.;
Vennerstrom, J. L.; Ridley, R. G. Biochem. Pharmacol.
1998, 55, 727.
14. Pathak, A.; Singh, S. K.; Biabani, M. A. F.; Kulshreshtha,
D. K.; Puri, S. K.; Srivastava, S.; Kundu, B. Comb. Chem.
High Throughput Screen 2002, 5, 241.
15. Suksamrarn, A.; Tanachatchairatana, T.; Kan-
okmedhakul, S. J. Ethnopharmacol. 2003, 88, 275.
16. Fidock, D. A.; Rosenthal, P. J.; Croft, S. L.; Brun, R.;
Nwaka, S. Nat. Rev. Drug Discovery 2004, 3, 509.
17. Egan, T. J.; Hunter, R.; Kaschula, C. H.; Marques, H. M.;
Misplon, A.; Walden, J. J. Med. Chem. 2000, 43, 283.
33. Leed, A.; Dubay, K.; Ursos, K. M. B.; Sears, D.; Dios, A.
C.; Roepe, P. D. Biochemistry 2002, 41, 10245.
34. Solomonov, I.; Osipova, M.; Feldman, Y.; Baehtz, C.;
Kjaer, K.; Robinson, I. K.; Webster, G. T.; Mcnaughton,
D.; Wood, B. R.; Weissbuch, I.; Leiserowitz, L. J. Am.
Chem. Soc. 2007, 129, 2615.
35. Podust, L. M.; Yermalitskaya, L. V.; Lepesheva, G. I.;
Podust, V. N.; Dalmasso, E. A.; Waterman, M. R.
Structure 2004, 12, 1937.
`
18. Girault, S.; Grellier, P.; Berecibar, A.; Maes, L.; Lemiere,
36. Mahato, S.; Kundu, A. P. Phytochemistry 1994, 37, 1517.
37. Tkachev, A. V.; Denisov, A. Y.; Gatilov, Y. V.; Bagr-
yanskaya, I. Y.; Shevtsov, S. A.; Rybalova, T. V.
Tetrahedron 1994, 50, 11459.
38. Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic
Synthesis, 3rd ed.; John Wiley: Hoboken, 1999, p. 154.
39. Verli, H.; Guimara˜es, J. A. Carbohydr. Res. 2004, 339,
281.
40. Verli, H.; Guimara˜es, J. A. J. Mol. Graph. Model. 2005,
24, 203.
41. Becker, C. A.; Guimara˜es, J. A.; Verli, H. Carbohydr. Res.
2005, 340, 1499.
42. Schmidt, M. W.; Baldridge, K. K.; Boatz, J. A.; Elbert, S.
T.; Gordon, M. S.; Jensen, J. H.; Koseki, S.; Matsunaga,
N.; Nguyen, K. A.; Su, S.; Windus, T. L.; Dupuis, M.;
Montgomer, J. A., Jr. J. Comput. Chem. 1993, 14, 1347.
43. van Aalten, D. M. F.; Bywater, R.; Findlay, J. B. C.;
Hendlich, M.; Hooft, R. W. W.; Vriend, G. J. Comput.
Aided Mol. Des. 1996, 10, 255.
44. van der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.;
Mark, A. E.; Berendsen, H. J. C. J. Comput. Chem. 2005,
26, 1701.
P.; Mouray, E.; Davioud-Charvet, E.; Sergheraert, C.
J. Med. Chem. 2001, 44, 1658.
19. Ryckebusch, A.; Deprez-Poulain, R.; Debreu-Fontaine,
M. A.; Vandaele, R.; Mouray, E.; Grellier, P.; Sergheraert,
C. Bioorg. Med. Chem. Lett. 2002, 12, 2595.
20. Ryckebusch, A.; Deprez-Poulain, R.; Maes, L.; Debreu-
Fontaine, M. A.; Mouray, E.; Grellier, P.; Sergheraert, C.
J. Med. Chem. 2003, 46, 542.
21. Guillon, J.; Grellier, P.; Labaied, M.; Sonnet, P.; Leger, J.
M.; Deprez-Poulain, R.; Forfar-Bares, I.; Dallemagne, P.;
Lemaitre, N.; Pehourcq, F.; Rochette, J.; Sergheraert, C.;
Jarry, C. J. Med. Chem. 2004, 47, 1997.
22. Gnoatto, S. C. B.; Schenkel, E. P.; Bassani, V. L. J. Braz.
Chem. Soc 2005, 16, 723.
23. Abdel-magid, A. F.; Carson, K. G.; Harris, B. D.;
Maryanoff, C. A.; Shah, R. D. J. Org. Chem. 1996, 61,
3849.
24. Gros, L.; Lorente, S. O.; Jimenez, C. J.; Yardley, V.;
Rattray, L.; Wharton, H.; Little, S.; Croft, S. L.; Ruiz-
Perez, L. M.; Gonzalez-Pacanowska, D.; Gilbert, I. H.
J. Med. Chem. 2006, 49, 6094.
25. Biot, C.; Glorian, G.; Maciejewski, L. A.; Brocard, J. S.
J. Med. Chem. 1997, 40, 3715.
26. Delhaes, L.; Biot, C.; Berry, L.; Maciejewski, L.; Camus,
D.; Brocard, J.; Dive, D. Bioorg. Med. Chem. 2000, 8,
2739.
45. Trager, W.; Jensen, J. B. Science 1976, 193, 673.
46. Dejardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J.
D. Antimicrob. Agents Chemother. 1979, 16, 710.
47. Baelmans, R.; Deharo, E.; Mun˜oz, V.; Sauvain, M.;
Ginsburg, H. Exp. Parasitol. 2000, 96, 243.