Synthesis and antimalarial activity of hydroxyethylpiperazine derivatives

Article abstract of DOI:10.1016/j.ejmech.2008.04.009

The antimalarial activity of hydroxyethylpiperazine derivatives, synthesized from the reaction of (2S,3S)Boc-phenylalanine epoxide with benzylpiperazines in good yields (76-96%), has been evaluated in vitro against the Plasmodium falciparum W2 clone (chlo

Full text of DOI:10.1016/j.ejmech.2008.04.009

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 1363e1368
http://www.elsevier.com/locate/ejmech
Preliminary communication
Synthesis and antimalarial activity of hydroxyethylpiperazine derivatives
a
a
a
Wilson Cunico a, , Claudia R.B. Gomes , Marcele Moreth , Diogo P. Manhanini ,
Isabela H. Figueiredo b, Carmen Penido b, Maria G.M.O. Henriques b,
Fernando P. Varotti c, Antoniana U. Krettli c
*
a Fundac¸~ao Oswaldo Cruz, Instituto de Tecnologia em Fa´rmacos, Farmanguinhos R. Sizenando Nabuco, 100,
Manguinhos, 21041-250 Rio de Janeiro, RJ, Brazil
b Instituto de Tecnologia em Fa´rmacos, Farmanguinhos, Departamento de Farmacologia Aplicada, Rio de Janeiro, RJ, Brazil
c Centro de Pesquisas Rene´ Rachou, Fiocruz e Departamento de Parasitologia (UFMG), Belo Horizonte, MG, Brazil
Received 16 February 2008; received in revised form 18 April 2008; accepted 18 April 2008
Available online 29 April 2008
Abstract
The antimalarial activity of hydroxyethylpiperazine derivatives, synthesized from the reaction of (2S,3S )Boc-phenylalanine epoxide with
benzylpiperazines in good yields (76e96%), has been evaluated in vitro against the Plasmodium falciparum W2 clone (chloroquine resistant).
The results show that some compounds have moderate activity against this parasite and none of the active compounds showed cytotoxicity at
high concentration (100 mg/ml).
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Hydroxyethylpiperazine; Malaria; Piperazine
1. Introduction
appear to be involved in the initial steps of the degradation
pathway and therefore make for an attractive antimalarial drug
target [5]. There are four aspartyl proteases present in the food
vacuole of P. falciparum, plasmepsins I, II, IV and Histo-As-
partic-Protease (HAP). The most attractive target from this
group of enzymes is the plasmepsin II [6]. Recent studies have
shown that HIV protease inhibitors can inhibit the plasmepsin
II in vitro [7,8] and in vivo [9] in the food vacuole at pharmaco-
logically relevant concentrations.
A secondary alcohol is usually the structural element of
choice to inhibit aspartic protease. This element mimics the
tetrahedral intermediate during peptide bond cleavage by as-
partic proteases [10]. It has been successfully used to develop
potent inhibitors of these enzymes, for example dihydroxy-
ethylene- [11] and hydroxyethylamine-based [12] molecules
(Fig. 1). In this work, we report the synthesis and in vitro ac-
tivity of novel hydroxyethylpiperazine-based compounds
against P. falciparum. The clone W2 of P. falciparum was
used for the test [13], as described in our previous work
[14,15], and activity compared to that of lopinavir and
Malaria accounts for more than a million deaths each year, of
which over 80% occur in tropical Africa, where malaria is the
leading cause of mortality in children under five years of age
[1]. In Brazil it is estimated that 500,000 cases occur annually,
mainly in the Amazon Rain Forest. Plasmodium falciparum is
the most dangerous form of the four malarial parasites that infect
humans. Despite the importance of this disease, there has been
little economic incentive for the development of new drug-based
antimalarial therapies. There is a growing need for effective
drugs with new mechanism of action, due to the high rate of
mutation of the parasite, which leads to the development of
resistance. One of the critical stages of the life cycle of the par-
asite during human infection is the degradation of hemoglobin
which provides nutrients for its growth and maturation [2]. A
family of aspartic proteases, known as the plasmepsins [3,4],
* Corresponding author. Tel.: þ55 21 3977 2463.
E-mail address: wjcunico@far.fiocruz.br (W. Cunico).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.04.009

1364
W. Cunico et al. / European Journal of Medicinal Chemistry 44 (2009) 1363e1368
R
OH
R
HO
H
N
OH
OH
H
N
H
N
N
N
H
OH
O
R
R
O
R
dihydroxyethylamine
tetrahedral intermediate
of amide hydrolysis
hydroxyethylamines
Fig. 1. Molecules developed for the synthesis of aspartyl protease inhibitors.
saquinavir (Fig. 2), used as in parallel in each test. Lopinavir is
the HIV protease inhibitor which has the best activity against
P. falciparum [8].
of 1 equiv of benzyl chloride to the above warm solution
produced 1 equiv of corresponding monoalkylated piperazine
and 1 equiv of precipitated piperazine dihydrochloride [19].
The unavailable benzyl chlorides were synthesized from
corresponding arenealdehydes (Scheme 2, route b). The
reaction of aldehyde with sodium borohydride gives the
corresponding alcohol that is reacted with hydrochloric acid
(37%) to afford the benzyl chlorides [20].
2. Chemistry
The hydroxyethylamine transition-state mimicking
fragment was prepared by selective ring-opening of the
(2S,3S )Boc-phenylalanine epoxide 1 (prepared according to
the method of Beaulieu) [16] with secondary amines (pipera-
zine derivatives) [17]. The epoxide stirred with four different
piperazines 2aed in isopropanol at reflux temperature affords
compounds 3aed in good yields (Scheme 1, route a). Table 1
shows the antimalarial activities of compounds 3aed ex-
pressed as 50% inhibitory concentration (IC50) of P. falcipa-
rum growth.
3. Pharmacological evaluation
Parasites were cultured with human erythrocytes (blood
group Oþ) at 5% hematocrit in RPMI 1640 supplemented
with 10% human plasma as previously described [21]. Test
compounds were solubilized in ethanol prior to in vitro tests.
The antiparasitic effects of the molecules were measured by
the [3H]-hypoxanthine incorporation assay [22]. Briefly,
trophozoite stages in sorbitol-synchronized blood [23] were
cultured at 2% parasitaemia and 2.5% hematocrit, in the
presence of the test compounds (at various concentrations),
diluted with culture medium (RPMI 1640) without hypoxan-
thine; a chloroquine control (as a reference antimalarial
drug) was used in each experiment. Inhibition of parasite
growth was evaluated through the levels of [3H]-hypoxanthine
incorporation plotted to generate doseeresponse curves. The
half-maximal inhibitory response (IC50) compared with
parasite growth in the drug-free controls was estimated by
curve fitting using a software program [Microcal, Origin
Software, Inc. (Northampton, MA, USA)].
In this first screening, we observed that only compound 3d
(entry 4) shows moderate activity. This compound has a CH2
space linker between piperazine and the phenyl group. We
next synthesized more compounds with CH2 space linkers.
A mixture of epoxide 1 and 4-benzylpiperazines 2eej in
refluxing isopropanol for 16 h gave the hydroxyethylpipera-
zine derivatives 3eej in good yields (Scheme 1, route b).
Compound 3k was prepared by a reduction reaction from
compound 3h. The nitro group of compound 3h was reduced
with H2 and Pd/C (10%) using ethanol as the solvent, in quan-
titative yield. Both analytical and spectral data (1H and 13C
NMR) of all compounds are in full agreement with the
proposed structures. 2D-NMR techniques (HMBQ, HMQC
and COSY) helped us to assign the correct signals of com-
pounds. As an example, the 1H NMR for compound 3j
exhibits a double doublet for H1a at 2.96 ppm (J ¼ 14.0 and
2J ¼ 4.8 Hz) and multiplet for H1b at 2.87 ppm. Protons H2
and H3 are shown as a broad signal at 3.80 and 3.62 ppm, re-
spectively, and proton H4 appears together with the piperazine
signals at 2.45 ppm. The signal for the methylene protons of
benzylpiperazine showed a singlet at 3.41 ppm. The 13C
NMR spectra exhibit broad signals at 53.2 and 52.9 ppm for
the piperazine carbons (pp). Other general 13C NMR signals
occur about d 36.4 (C1), 54.5 (C2), 68.0 (C3) and 61.0
(C4) ppm.
Ph
OH
O
H
N
N
NH
O
N
H
O
O
O
Ph
lopinavir
O
H
NH2
OH
Ph
Commercially unavailable precursor piperazines required
to prepare compounds 3eej of Scheme 1 were synthesized
according to a previously reported method that is outlined in
Scheme 2 [18]. The mixture of 1 equiv of piperazine with
1 equiv of piperazine dihydrochloride hydrate in absolute
ethanol (65 ꢀC) gave a solution containing 1 equiv of pipera-
zine monohydrochloride (Scheme 2, route a). Slow addition
H
H
N
N
N
N
H
O
O
N
H
saquinavir
Fig. 2. Structure of HIV protease inhibitors lopinavir and saquinavir.

W. Cunico et al. / European Journal of Medicinal Chemistry 44 (2009) 1363e1368
1365
R
OH
N
O
a
BocHN
Ph
BocHN
Ph
N
R
N
HN
+
76-96%
route a
3a-d
R = H, CH3, Ph, CH2Ph
2a-d
1
R2
R1
O
OH
N
R2
R1
BocHN
Ph
N
a
BocHN
N
2
+
3
route b
77-89%
HN
4
1
Ph
1
2e-j
3e-j
b
3h R1 = NO2
3k R1 = NH2
93%
Scheme 1. Reagents and conditions: (a) IPA, reflux, 16 h; (b) H2, Pd/C 10%, r.t., 16 h.
4. Cytotoxicity assay
moderate antimalarial activity and also were not cytotoxic to
host cells at high concentration (100 mg/ml). The CH2 space
linker was found to be critical for activity, since compounds
3aec had been without antimalarial activity. Further work
with these hydroxyethylamine derivatives is now undertaken
to increase the antimalarial activity, especially, with
compounds that have the piperonyl piperazine moiety.
Cytotoxicity was determined in the murine monocyte/mac-
rophage cell lineage J774, using the method previously
described [24]. The cells were seeded in a flat bottom 96-
well plate (2 ꢁ 106 cells/well) cultured for 1 h (5% CO2 at
37 ꢀC) in RPMI 1640 enriched with 10% bovine fetal serum,
2 mM L-glutamine, 25 mg/ml gentamicin, pH 7.4. Adherent
cells were cultured in the presence of different concentrations
of the compounds (0.1e100 mg/ml) for 20 h, when MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)
solution (5 mg/ml; 22.5 ml/well) was added to the culture for
4 h. Supernatant was discharged and DMSO (150 ml/well)
was added for solubilization of formazan crystals. The absor-
bance was read at 540 nm.
7. Experimental section
Unless otherwise indicated, all common reagents and
solvents were used as obtained from commercial suppliers
without further purification. All melting points were deter-
mined on a Buchi Melting Point B-545 and are uncorrected.
¨
1H and 13C NMR spectra were recorded on a Bruker DRX
400 spectrometer (1H at 400.14 MHz and 13C at
100.61 MHz) and on a Bruker Avance 500 spectrometer (1H
at 500.13 MHz and 13C at 125.75 MHz) in CDCl3 containing
TMS as an internal standard. Microanalyses were performed
on PerkineElmer Model 2400 instrument and all values
were within ꢂ0.4% of the theoretical values. LCeMS analy-
ses were performed on a LCeMS micromass ZMD using
methanol (100%) as mobile phase with flux of 0.3 ml/min.
5. Results and discussion
The most marked effect is observed when a methylene space
linker was incorporated in piperazine residue. As the IC50 values
in Table 1 indicate, the new series of hydroxyethylpiperazine
derivatives has moderate antimalarial activity. It is evident that
compound 3c which did not have the methylene space linker
has no activity at the maximum concentration (50 mg/ml) used
(entry 3, Table 1). The same results were observed for com-
pounds 3a (R ¼ H) and 3b (R ¼ Me). The compound 3d
(R ¼ CH2Ph) shows moderate antimalarial activity (entry 4).
Introduction of a halogen atom into the para position of the
benzylpiperazine group resulted in an increase in potency
(entries 5 and 6). Compound 3j, with the piperazine ring
substituted with a piperonyl group, has the best antimalarial
activity with an IC50 of 5.1 mg/ml, being as active as saquinavir
mesylate used as the standard (entry 10). The hydroxyethylpi-
perazine 3h, that has a strong electron withdrawing group,
showed poor antimalarial activity (IC50 ¼ 23.5 mg/ml).
7.1. General procedure
Benzylpiperazines 2aej (1.5 mmol) and epoxide
1
(1.6 mmol) were dissolved in 2-propanol (10 ml) and refluxed
overnight (100 ꢀC). The solvent was removed by evaporation
and the crude product was purified by crystallization in meth-
anol/water (7:3) to give hydroxyethylpiperazine derivatives
3aej.
7.1.1. Compound 3a
M.p. 145e147 ꢀC. 1H NMR (500 MHz, CDCl3): 7.32e7.21
(m, 5H, Ph); 4.58 (d, 1H, NH, J ¼ 9.0 Hz); 3.82 (br, 1H, H2);
3.65 (br, 1H, H3); 2.97 (dd, 1H, H1a, J ¼ 13.5 Hz,
2J ¼ 4.5 Hz); 2.86 (m, 1H, H1b); 2.73 (br, 2H, pp); 2.49 (br,
8H, H4 and pp); 1.35 (s, 9H, Boc). 13C NMR (125 MHz,
CDCl3): 155.6 (CO); 137.7; 129.6; 129.5; 128.5; 128.4;
6. Conclusion
In summary, the hydroxyethylpiperazines 3aek could be
synthesized easily with good yields. Some compounds showed

1366
W. Cunico et al. / European Journal of Medicinal Chemistry 44 (2009) 1363e1368
Table 1
Yields, in vitro antimalarial activity against P. falciparum W2 clone and cytotoxicity of compounds 3aek
Entry
Compound
R1
R2
Yielda (%)
IC50 mg/mlb (mM)
Cytotoxicityc (% of cell viability)
0.1 mg/ml
1 mg/ml
10 mg/ml
100 mg/ml
1
3a [25]
3b
e
e
89
76
80
96
77
85
89
85
68
86
93
>50
>50
100
94
100
100
95
100
100
94
65
100
58
2
e
e
3
3c [25]
3d
3e
e
H
e
H
>50
100
95
4
14.9 (35.0)
7.5 (16.9)
7.7 (16.7)
6.2 (13.2)
23.5 (50.0)
7.0 (14.1)
5.1 (10.8)
15.8 (34.8)
0.14
100
100
100
100
100
100
98
100
99
100
90
5
F
Cl
H
100
100
100
100
100
100
100
6
3f
3g
H
100
100
98
88
85
7
OMe
NO2
OMe
H
8
3h
3i
H
OMe
81
83
9
10
100
100
98
3j
3k
eOCH2Oe
H
82
85
11
NH2
98
Chloroquine
Saquinavir
Lopinavir
a
7.0 (9.1)
1.7 (2.7)
Yields of isolated compounds.
b
c
IC50 represents concentration inhibitory dose of the parasite growth in relation to control cultures with no drugs.
Percentage of J774 cell viability 24 h after incubation with the compounds determined by the MTT method.
126.3; 79.4 (Boc); 67.9 (C3); 60.9 (C4); 54.3 (C2); 53.8 (br,
pp); 36.2 (C1); 28.3 (Boc). LCeMS (m/z) (%): 350.14
(Mþ þ 1, 100); 249 (37); 182 (100); 96 (15). Anal. Calcd
for C19H31N3O3: C, 65.30; H, 8.94; N, 12.02. Found: C,
65.64; H, 9.14; N, 12.22.
3.68 (br, 1H, H3); 2.99 (dd, 1H, H1a, J ¼ 14.0 Hz,
2J ¼ 4.5 Hz); 2.90 (m, 1H, H1b); 2.78 (br, 2H, pp); 2.50 (br,
8H, H4 and pp); 1.35 (s, 9H, Boc). 13C NMR (125 MHz,
CDCl3): 155.6 (CO); 137.7; 130.4; 129.6; 128.4; 128.3;
127.2; 126.3; 79.4 (Boc); 68.2 (C3); 61.0 (C4); 54.4 (C2);
52.9 and 46.9 (pp); 36.3 (C1); 28.3 (Boc). LCeMS (m/z)
(%): 364.34 (Mþ þ 1, 100); 264 (2). Anal. Calcd for
C25H35N3O3: C, 70.56; H, 8.29; N, 9.87. Found: C, 70.54;
H, 8.30; N, 9.84.
7.1.2. Compound 3b
M.p. 114e115 ꢀC. 1H NMR (500 MHz, CDCl3): 7.31e7.20
(m, 5H, Ph); 4.58 (d, 1H, NH, J ¼ 9.0 Hz); 3.81 (br, 1H, H2);
3.60 (br, 1H, H3); 2.97 (dd, 1H, H1a, J ¼ 14.0 Hz,
2J ¼ 4.0 Hz); 2.87 (m, 1H, H1b); 2.71 (br, 2H, pp); 2.53 (s,
3H, CH3); 2.48 (br, 8H, H4 and pp); 1.34 (s, 9H, Boc). 13C
NMR (125 MHz, CDCl3): 155.6 (CO); 137.8; 129.6; 129.5;
128.3; 128.2; 126.3; 79.3 (Boc); 67.9 (C3); 60.8 (C4); 55.0
(C2); 54.4 and 52.8 (pp); 45.8 (CH3); 36.3 (C1); 28.2 (Boc).
LCeMS (m/z) (%): 364.34 (Mþ þ 1, 100); 264 (2). Anal.
Calcd for C20H33N3O3: C, 66.08; H, 9.15; N, 11.56. Found:
C, 65.92; H, 9.21; N, 11.60.
7.1.4. Compound 3d
1
M.p. 96e98 ꢀC. H NMR (500 MHz, CDCl3): 7.34e7.19
(m, 10H, Ph); 4.57 (d, 1H, NH, J ¼ 8.5 Hz); 3.86 (br, 1H,
H2); 3.64 (br, 1H, H3); 3.52 (s, 2H, CH2); 2.96 (dd, 1H,
2
H1a, J ¼ 14.0 Hz, J ¼ 4.0 Hz); 2.87 (m, 1H, H1b); 2.70 (br,
2H, pp); 2.48 (br, 8H, H4 and pp); 1.34 (s, 9H, Boc). 13C
NMR (125 MHz, CDCl3): 155.6 (CO); 137.8; 129.6; 129.2;
128.4; 128.3; 127.2; 126.3; 79.3 (Boc); 67.9 (C3); 62.9
(CH2); 61.0 (C4); 54.3 (C2); 52.9 and 52.8 (pp); 36.2 (C1);
28.2 (Boc). LCeMS (m/z) (%): 440.27 (Mþ þ 1, 100); 384
(6); 267 (4); 182 (98); 177 (47); 91 (97). Anal. Calcd for
7.1.3. Compound 3c
M.p. 183e185 ꢀC. 1H NMR (500 MHz, CDCl3): 7.32e7.22
(m, 10H, Ph); 4.64 (d, 1H, NH, J ¼ 9.0 Hz); 3.85 (br, 1H, H2);
a
R2
R1
HN
2x
NH.HCl
HN
NH + ClH.HN
NH.HCl
N
HN
e
route a
2e-j
O
OH
50-75%
+
R2
R1
R2
R1
R2
R1
d
Cl
H
b, c
H
ClH.HN
NH.HCl
route b
Scheme 2. Reagents and conditions: (a) EtOH, 78 ꢀC, 1 h; (b) NaBH4 (0.5 equiv), EtOH, 0 ꢀC, r.t., 18 h; (c) HCl 15%; (d) SOCl2, py, r.t., 4 h; (e) EtOH, 78 ꢀC, 2 h.

W. Cunico et al. / European Journal of Medicinal Chemistry 44 (2009) 1363e1368
1367
C26H37N3O3: C, 71.04; H, 8.48; N, 9.56. Found: C, 71.44; H,
8.59; N, 9.86.
364 (100); 301 (31); 264 (15). Anal. Calcd for C26H36N4O5:
C, 64.44; H, 7.49; N, 11.56. Found: C, 64.32; H, 7.51; N,
11.68.
7.1.5. Compound 3e
M.p. 115e117 ꢀC. 1H NMR (400 MHz, CDCl3): 7.30e7.20
(m, 7H, Ph); 7.00 (d, 2H, Ph, J ¼ 8.5 Hz); 4.57 (d, 1H, NH,
J ¼ 7.6 Hz); 3.80 (br, 1H, H2); 3.72 (br, 1H, H3); 3.50 (s,
2H, CH2); 2.98 (dd, 1H, H1a, J ¼ 14.0 Hz, 2J ¼ 4.4 Hz);
2.87 (m, 1H, H1b); 2.78 (br, 2H, pp); 2.55 (br, 8H, H4 and
pp); 1.34 (s, 9H, Boc). 13C NMR (100 MHz, CDCl3): 162.2
(d, JCF ¼ 245.1 Hz); 155.6 (CO); 137.7; 130.7 (d,
2JCF ¼ 20.9 Hz); 79.5 (Boc); 68.0 (C3); 61.8 (CH2); 61.4
(C4); 54.4 (C2); 53.3 and 52.1 (pp); 36.3 (C1); 28.3 (Boc).
LCeMS (m/z) (%): 458.58 (Mþ þ 1, 100); 364 (22); 303
(15). Anal. Calcd for C26H36FN3O3: C, 68.25; H, 7.93; N,
9.18. Found: C, 68.41; H, 8.20; N, 9.32.
7.1.9. Compound 3i
M.p. 125e126 ꢀC. 1H NMR (500 MHz, CDCl3): 7.30e7.20
(m, 7H, Ph); 6.81 (d, 2H, Ph, J ¼ 8.5 Hz); 4.57 (d, 1H, NH,
J ¼ 10.0 Hz); 3.89 (s, 3H, OCH3); 3.87 (s, 3H, OCH3); 3.81
(br, 1H, 2H); 3.69 (br, 1H, H3); 3.55 (s, 2H, CH2); 2.97 (dd,
2
1H, H1a, J ¼ 13.5 Hz, J ¼ 4.0 Hz); 2.87 (m, 1H, H1b); 2.73
(br, 2H, pp); 2.51 (br, 8H, H4 and pp); 1.34 (s, 9H, Boc).
13C NMR (125 MHz, CDCl3): 155.6 (CO); 148.8; 148.2;
137.7; 129.6; 129.1; 128.4; 126.3; 121.4; 110.7; 79.4 (Boc);
67.9 (C3); 62.9 (CH2); 61.0 (C4); 55.9 (OCH3); 55.8
(OCH3); 54.4 (C2); 53.3 and 52.7 (pp); 36.3 (C1); 28.3
(Boc). LCeMS (m/z) (%): 500.43 (Mþ þ 1, 100); 400 (3);
151 (3). Anal. Calcd for C28H41N3O5: C, 67.31; H, 8.27; N,
8.41. Found: C, 66.98; H, 8.48; N, 8.23.
3JCF ¼ 7.6 Hz);
129.6;
128.4;
126.4;
115.2
(d,
7.1.6. Compound 3f
M.p. 128e129 ꢀC. 1H NMR (500 MHz, CDCl3): 7.30e7.19
(m, 9H, Ph); 4.57 (d, 1H, NH, J ¼ 9.0 Hz); 3.81 (br, 1H, H2);
3.64 (br, 1H, H3); 3.47 (s, 2H, CH2); 2.96 (dd, 1H, H1a,
7.1.10. Compound 3j
M.p. 103e105 ꢀC. 1H NMR (400 MHz, CDCl3): 7.30e7.18
(m, 5H, Ph); 6.84 (s, 1H, Ph); 6.74 (s, 2H, Ph); 5.94 (s, 2H,
OCH2O); 4.57 (d, 1H, NH, J ¼ 8.4 Hz); 3.80 (br, 1H, H2);
3.62 (br, 1H, H3); 3.41 (s, 2H, CH2); 2.96 (dd, 1H, H1a,
2
J ¼ 14.0 Hz, J ¼ 4.0 Hz); 2.88 (m, 1H, H1b); 2.69 (br, 2H,
pp); 2.48 (br, 8H, H4 and pp); 1.33 (s, 9H, Boc). 13C NMR
(125 MHz, CDCl3): 155.6 (CO); 137.8; 136.5; 132.8; 130.4;
129.6; 128.4; 128.3; 126.3; 79.3 (Boc); 67.9 (C3); 62.1
(CH2); 61.0 (C4); 54.3 (C2); 52.9 and 52.8 (pp); 36.3 (C1);
28.3 (Boc). LCeMS (m/z) (%): 474.58 (Mþ þ 1, 100); 364
(63); 264 (14); 214 (10). Anal. Calcd for C26H36ClN3O3: C,
65.88; H, 7.65; N, 8.86. Found: C, 65.90; H, 7.66; N, 8.86.
2
J ¼ 14.0 Hz, J ¼ 4.8 Hz); 2.87 (m, 1H, H1b); 2.67 (m, 2H,
pp); 2.45 (br, 8H, H4 and pp); 1.34 (s, 9H, Boc). 13C NMR
(100 MHz, CDCl3): 155.6 (CO); 147.7; 146.7; 137.9; 131.9;
129.6; 128.3; 126.3; 122.2; 109.5; 107.9; 100.9 (OCH2O);
79.3 (Boc); 68.0 (C3); 62.7 (CH2); 61.0 (C4); 54.5 (C2);
53.2 and 52.9 (pp); 36.4 (C1); 28.3 (Boc). LCeMS (m/z)
(%): 484.49 (Mþ þ 1, 100); 384 (4). Anal. Calcd for
C27H37N3O5: C, 67.06; H, 7.71; N, 8.69. Found: C, 67.06;
H, 7.72; N, 8.69.
7.1.7. Compound 3g
1
M.p. 110e112 ꢀC. H NMR (500 MHz, CDCl3): 7.28 (d,
2H, Ph, J ¼ 7.5 Hz); 7.25e7.21 (m, 5H, Ph); 6.87 (d, 2H,
Ph, J ¼ 8.5 Hz); 4.57 (d, 1H, NH, J ¼ 9.5 Hz); 3.82 (br, 1H,
H2); 3.81 (s, 3H, OCH3); 3.66 (br, 1H, H3); 3.49 (s, 2H,
CH2); 2.97 (dd, 1H, H1a, J ¼ 13.9 Hz, 2J ¼ 4.5 Hz); 2.88
(m, 1H, H1b); 2.74 (br, 2H, pp); 2.51 (br, 8H, H4 and pp);
1.34 (s, 9H, Boc). 13C NMR (100 MHz, CDCl3): 159.0;
155.7 (CO); 138.1; 130.6; 129.8; 128.5; 126.5; 113.8; 79.5
(Boc); 68.2 (C3); 62.5 (CH2); 61.2 (C4); 55.5 (OCH3); 54.6
(C2); 53.4 and 53.0 (pp); 36.5 (C1); 28.5 (Boc). LCeMS
(m/z) (%): 470.67 (Mþ þ 1, 100); 364 (15); 264 (15). Anal.
Calcd for C27H39N3O4: C, 69.05; H, 8.37; N, 8.95. Found:
C, 69.39; H, 8.23; N, 8.88.
7.1.11. Compound 3k
To a stirred solution of compound 3h (280 mg, 0.57 mmol)
in absolute ethanol (15 ml) under a blanket of nitrogen was
added 10% palladium on activated charcoal (5 mg). The reac-
tion was placed under a hydrogen atmosphere and stirred over-
night. The reaction mixture was filtered through Celite, and
the solvent was removed under reduced pressure to yield 3k
as a brown oil. The residue was purified by chromatography
on silica, eluting with 3:1 hexane/EtOAc. 3k: m.p. 139e
142 ꢀC. 1H NMR (500 MHz, CDCl3): 7.30e7.20 (m, 5H,
Ph); 7.08 (d, 2H, Ph, J ¼ 8.0 Hz); 6.96 (d, 2H, Ph,
J ¼ 8.0 Hz); 4.62 (d, 1H, NH, J ¼ 9.5 Hz); 3.81 (m, 1H,
H2); 3.61 (br, 1H, H3); 3.42 (s, 2H, CH2); 2.96 (dd, 1H,
7.1.8. Compound 3h
1
M.p. 148e150 ꢀC. H NMR (500 MHz, CDCl3): 8.17 (d,
2
2H, Ph, J ¼ 9.0 Hz); 7.50 (d, 2H, Ph, J ¼ 8.5 Hz); 7.30e7.19
(m, 5H, Ph); 4.56 (d, 1H, NH, J ¼ 9.0 Hz); 3.81 (br, 1H,
H2); 3.60 (br, 1H, H3); 3.59 (s, 2H, CH2); 2.95 (dd, 1H,
H1a, J ¼ 15.0 Hz, J ¼ 4.0 Hz); 2.86 (m, 1H, H1b); 2.69 (br,
2H, pp); 2.45 (br, 8H, H4 and pp); 1.34 (s, 9H, Boc). 13C
NMR (125 MHz, CDCl3): 155.5 (CO); 145.5; 137.7; 130.5;
129.7; 129.5; 128.3; 128.2; 126.3; 126.2; 115.2; 79.2 (Boc);
67.9 (C3); 62.3 (CH2); 61.7 (C4); 54.3 (C2); 52.6 and 52.3
(pp); 36.2 (C1); 28.3 (Boc). IR (KBr): 3450 (NH2); 3354
(NH); 2970 and 2924 (CH2 and CH3); 1706 (CO). Anal. Calcd
for C26H38N4O3: C, 68.69; H, 8.43; N, 12.32. Found: C, 68.42;
H, 8.63; N, 12.15.
2
H1a, J ¼ 14.0 Hz, J ¼ 4.5 Hz); 2.87 (m, 1H, H1b); 2.67 (br,
2H, pp); 2.45 (br, 8H, H4 and pp); 1.34 (s, 9H, Boc). 13C
NMR (125 MHz, CDCl3): 155.6 (CO); 147.1; 146.2; 137.8;
129.6; 129.5; 128.3; 126.3; 123.5; 79.3 (Boc); 67.9 (C3);
62.0 (CH2); 60.8 (C4); 54.3 (C2); 53.3 and 53.1 (pp); 36.3
(C1); 28.3 (Boc). LCeMS (m/z) (%): 485.33 (Mþ þ 1, 96);

1368
W. Cunico et al. / European Journal of Medicinal Chemistry 44 (2009) 1363e1368
´
[12] (a) D. Noteberg, E. Hamelink, J. Hulten, M. Wahlgren, L. Vrang,
B. Samuelsson, A. Hallberg, J. Med. Chem. 46 (2003) 734;
(b) D. Muthas, D. Noteberg, Y.A. Sabnis, E. Hamelink, L. Vrang,
Acknowledgements
The authors thank Farmanguinhos, FAPERJ and CNPq for
the financial support of the research.
´
B. Samuelsson, A. Karlen, A. Hallberg, Bioorg. Med. Chem. 13 (2005)
5371;
(c) T.S. Haque, A.G. Skillman, C.E. Lee, H. Habashita, I.Y. Gluzman,
T.J. Ewing, D.E. Goldberg, I.D. Kuntz, J.A. Ellman, J. Med. Chem. 42
(1999) 1428.
References
[1] (a) W. Cunico, S.A. Carvalho, C.R.B. Gomes, G.H. Marques, Rev. Bras.
Farm. 89 (2008) 49;
[13] M.G. Zalis, L. Pang, M.S. Silveira, W.K. Milhous, D.F. Wirth, Am. J.
Trop. Med. Hyg. 58 (1998) 630.
(b) B.M. Greenwood, K. Bojang, C.J. Whitty, G.A. Targett, Lancet 365
(2005) 1487.
[14] W. Cunico, C.A. Cechinel, H.G. Bonacorso, M.A.P. Martins, N. Zanatta,
M.V.N. de Souza, I.O. Freitas, R.P.P. Soares, A.U. Krettli, Bioorg. Med.
Chem. Lett. 16 (2006) 649.
[2] G.H. Coombs, D.E. Goldberg, M. Klemba, C. Berry, J. Kay,
J.C. Mottram, Trends Parasitol. 17 (2001) 532.
[15] W. Cunico, M.L.G. Ferreira, T.G. Ferreira, C. Penido,
M.G.M.O. Henriques, L.G. Krettli, F.P. Varotti, A.U. Krettli, Lett.
Drug Des. Discov. 5 (2008) 178.
[3] C. Boss, S. Richard-Bildstein, T. Weller, W. Fischli, S. Meyer,
C. Binkert, Curr. Med. Chem. 10 (2003) 883.
´
´
[4] H. Gutierrez-de-Teran, M. Nervall, K. Ersmark, P. Liu, L.K. Janka,
B. Dunn, A. Hallberg, J. Aqvist, Biochemistry 45 (2006) 10529.
[5] C. Dash, A. Kulkarni, B. Dunn, M. Rao, Crit. Rev. Biochem. Mol. Biol.
38 (2003) 89.
[16] P.L. Beaulieu, D. Wernic, J.-S. Duceppe, Y. Guindon, Tetrahedron Lett.
36 (1995) 3317.
[17] (a) H. Tamamura, Y. Koh, S. Ueda, Y. Sasaki, T. Yamasaki, M. Aoki,
K. Maeda, Y. Watai, H. Arikuni, A. Otaka, H. Mitsuya, N. Fujii, J.
Med. Chem. 46 (2003) 1764;
[6] K. Ersmark, B. Samuelsson, A. Hallberg, Med. Res. Rev. 26 (2006) 626.
[7] T.S. Skinner-Adams, J.S. McCarthy, D.L. Gardiner, P.M. Hilton,
K.T. Andrews, J. Infect. Dis. 190 (2004) 1998.
(b) P.L. Beaulieu, P.C. Anderson, D.R. Cameron, G. Croteau, V. Gorys,
C. Grand-Maitre, D. Lamarre, F. Liard, W. Paris, L. Plamondon,
F. Soucy, D. Thibeault, D. Wernic, C. Yoakin, J. Med. Chem. 43
(2000) 1094.
[8] S. Parikh, J. Gut, E. Istvan, D.E. Goldberg, D.V. Havlir, P.J. Rosenthal,
Antimicrob. Agents Chemother. 49 (2005) 2983.
[9] K.T. Andrews, D.P. Fairlie, P.K. Madala, J. Ray, D.M. Wyatt,
P.M. Hilton, L.A. Melville, L. Beattie, D.L. Gardioner, R.C. Reid,
M.J. Stoermer, T. Skinner-Adams, C. Berry, J.S. McCarthy, Antimicrob.
Agents Chemother. 50 (2006) 639.
[18] A.S. Mehanna, J.Y. Kim, Bioorg. Med. Chem. 13 (2005) 4323.
[19] J.C. Craig, R.J. Young, Org. Syntheses Coll. 5 (1973) 88.
[20] S.J. Howell, N. Spencer, D. Philp, Tetrahedron 57 (2001) 4945.
[21] W. Trager, J.B. Jensen, Science 193 (1976) 673.
[22] R.E. Desjardins, C.J. Canfield, J.D. Haynes, J.D. Chulay, Antimicrob.
Agents Chemother. 16 (1979) 710.
[10] R.C. Ogden, C.W. Flexner (Eds.), Protease Inhibitors in AIDS Therapy,
Marcel Dekker Inc., New York, 2001.
[11] (a) K. Ersmark, I. Feierberg, S. Bjelic, E. Hamelink, F. Hackett,
[23] C. Lambros, J.P. Vanderberg, J. Parasitol. 65 (1979) 418.
[24] M. Ferrari, M.C. Fornasiero, A.M. Isetta, J. Immunol. Methods 131
(1990) 165.
´
M.J. Blackman, J. Hulten, B. Samuelsson, J. Aqvist, A. Hallberg, J.
Med. Chem. 47 (2004) 110;
´
´
(b) K. Ersmark, M. Nervall, H. Gutierrez-de-Teran, E. Hamelink,
L.K. Janka, J.C. Clemente, B.M. Dunn, A. Gogoll, B. Samuelsson,
J. Aqvist, A. Hallberg, Bioorg. Med. Chem. 14 (2006) 2197.
[25] R.M. McConnell, A.W. Green, C.J. Trana, M.S. McConnell,
J.F. Lindley, K. Sayyar, W.E. Godwin, S.E. Hatfield, Med. Chem. 2
(2006) 27.