Imidazolidin-4-one peptidomimetic derivatives of primaquine: Synthesis and antimalarial activity

Article abstract of DOI:10.1016/j.bmcl.2008.05.076

The synthesis of imidazolidin-4-one derivatives of primaquine containing the five-membered ring at the C-terminus of a dipeptide backbone coupled to the parent drug is described. These peptidomimetic derivatives were active against a chloroquine-resistant Plasmodium falciparum strain and inhibited the development of the sporogonic cycle of Plasmodium berghei, affecting the appearance of oocysts in the midguts of the mosquitoes. The novel imidazolidin-4-ones are extremely stable, both in human plasma and in pH 7.4 buffer, as a result of N¹-acylation. Thus, 'internal' imidazolidin-4-ones derived from dipeptidyl 8-aminoquinolines represent a new entry in antimalarial structure-activity relationships.

Full text of DOI:10.1016/j.bmcl.2008.05.076

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4150–4153
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Imidazolidin-4-one peptidomimetic derivatives of primaquine: Synthesis
and antimalarial activity
a
a
b
c
c
b
´
Nuno Vale , Joana Matos , Jiri Gut , Fátima Nogueira , Virgılio do Rosário , Philip J. Rosenthal ,
Rui Moreira d, Paula Gomes a,
*
a CIQUP, Departamento de Qu´ımica, Faculdade de Ciências, Universidade do Porto, P-4169-007 Porto, Portugal
b Department of Medicine, San Francisco General Hospital, University of California, San Francisco, CA 94143-0811, USA
c CMDT, Instituto de Higiene e Medicina Tropical, P-1349-008 Lisboa, Portugal
d iMed.UL, CECF, Faculdade de Farmácia de Lisboa, P-1600-083 Lisboa, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of imidazolidin-4-one derivatives of primaquine containing the five-membered ring at
the C-terminus of a dipeptide backbone coupled to the parent drug is described. These peptidomimetic
derivatives were active against a chloroquine-resistant Plasmodium falciparum strain and inhibited the
development of the sporogonic cycle of Plasmodium berghei, affecting the appearance of oocysts in the
midguts of the mosquitoes. The novel imidazolidin-4-ones are extremely stable, both in human plasma
and in pH 7.4 buffer, as a result of N1-acylation. Thus, ‘internal’ imidazolidin-4-ones derived from
dipeptidyl 8-aminoquinolines represent a new entry in antimalarial structure–activity relationships.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 5 May 2008
Revised 16 May 2008
Accepted 20 May 2008
Available online 23 May 2008
Keywords:
Antimalarial
Gametocytocidal
Imidazolidin-4-one
Malaria
Peptidomimetic
Primaquine
Primaquine, 1, is the only currently available drug that is active
against both the latent liver forms of the relapsing malaria caused
by Plasmodium vivax and Plasmodium ovale and the gametocytes
from all species of parasite causing human malaria.1 However, pri-
maquine presents a short plasma half-life (ca. 6 h),2 presumably
due to its rapid oxidative deamination to carboxyprimaquine 2.3–5
Blood toxicity, in particular hemolysis secondary to the ability of
primaquine to induce oxidation of oxyhemoglobin to methemoglo-
bin, has also been a source of concern.6 Peptide and amino acid
derivatives of primaquine have been prepared to reduce toxicity
of the parent drug as well as to suppress the metabolic pathway
leading to 2,7–10 but many of these derivatives are rapidly hydro-
lyzed to primaquine by aminopeptidases and endopeptidases.8,10
Imidazolidin-4-one formation is often used to protect the N-ter-
minal amino acid residue of peptides and peptidomimetics.11,12
Recently, we prepared imidazolidin-4-ones, 3, as potential pepti-
dase-stable prodrugs of amino acid derivatives of primaquine.13–15
than 12 h).14,16 We now set out to incorporate the imidazolidin-4-
one moiety into dipeptide derivatives of primaquine, for example,
5 (Scheme 1), both to (i) introduce a terminal basic amino group
reported as relevant for activity,17 and (ii) effectively suppress
hydrolysis of the imidazolidin-4-one due to acylation of the N1
nitrogen atom. We herein report the synthesis and evaluation of
the antiplasmodial activity of imidazolidin-4-ones 5 (Scheme 1).
N
N
R1
NH
NH
MeO
MeO
O
Me
Me
NH
N
R3
X
R2
Compounds
3 displayed gametocytocidal activity against
1, X=CH2NH2
2, X=CO2H
3
Plasmodium berghei comparable to that of primaquine, as well as
activity against P. falciparum asexual stages, while presenting high
stability in pH 7.4 buffer (half-lives for hydrolysis typically higher
Compounds 5 were prepared as depicted in Scheme 1.
a-Ami-
noacyl derivatives of primaquine, 4, were converted into the corre-
sponding imidazolidin-4-ones 3 by refluxing with propanone,
cyclopentanone, or cyclohexanone in MeOH.18 Preliminary con-
densation of 3 with N-Boc-protected amino acids (BocAAOH) using
* Corresponding author. Tel.: +351 220402563; fax: +351 220402659.
E-mail address: pgomes@fc.up.pt (P. Gomes).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.076

N. Vale et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4150–4153
4151
suggest that the effectiveness of the acylation increases along with
the solvent’s dielectric constant. When 3a was reacted with bulkier
BocAAOH (AA = Ala, Val, Leu, Phe, and Met) using DIPCDI/HOBt in
DMF, the resulting compounds 6b–f were obtained only in moder-
ate yield (Scheme 1 and Table 1). In contrast, acylation of alanine-
based imidazolidin-4-one 3b (R1 = R2 = R3 = Me) proceeded
successfully with BocGlyOH to give 6g in moderate yield. Further
increase in the size of substituents at the imidazolidin-4-one
C-5 position failed to give the corresponding derivatives 6, with
exception of 3c (R1 = CH2CH2SMe, R2 = R3 = Me) that gave 6h in
very poor yield (1.6%) by reaction with BocGlyOH. Unfortunately,
attempts to prepare the spiro counterparts of 6, starting from imi-
dazolidin-4-ones 3 derived from cyclopentanone or cyclohexa-
none, were also unsuccessful. Removal of Boc from 6 using 30%
TFA in DCM gave compounds 5 almost quantitatively,21 with the
exception of 6h, which decomposed under these conditions.
The structure of imidazolidin-4-ones 5 and 6 follows from their
spectroscopic data, which reveal the presence of two diastereo-
mers resulting from using racemic primaquine as starting material.
For example, the imidazolidin-4-one C-2 methyl proton signals of
6, 5b–g appear as two sets of two singlets (1:1:1:1 integration).22
Similarly, the two methyl groups at C-2 appear as four signals in
the 13C NMR spectra (cf. Supporting Information). The 1H NMR sig-
nal of the C-5 CH2 of 6, 5a–f indicates the diastereotopic nature of
the two protons, which appear as an AB system at d ca. 4 ppm, with
2J = 14–16 Hz. All attempts to separate the two diastereomers
using column chromatography, flash chromatography, and pre-
parative TLC were unsuccessful. Moreover, reverse-phase HPLC
using Merck’s RP-8 and RP-18 columns (both 125 and 250 mm)
consistently gave single peaks for each derivative 5b–g (not
shown), indicating that both diastereomers display similar proper-
ties, making their separation extremely difficult to achieve by stan-
dard methods. The same separation problem had already been
observed for precursor compounds 3 and dipeptide derivatives of
primaquine.10,13–16,18
X
X
Me
Me
(iii)
(i) (ii)
R1
1
R2
R3
N
NH
O
H2N
HN
O
R1
4
3
(iv)
X
Me
R2
R3
N
N
X =
O
R4
N
O
NH
R1
MeO
YHN
6, Y = Boc
5, Y = H
(v)
Scheme 1. Synthetic route to PQ-derived imidazolidin-4-ones 5. Reagents and
conditions: (i) 1 equiv BocAAOH, 1.1 equiv DIPCDI, 1.1 equiv HOBt, 1 equiv TEA,
DCM, 0 °C/rt; (ii) TFA, rt, 30% aq Na2CO3, extraction with CHCl3; (iii) R2(C@O)R3, TEA,
refluxing MeOH; (iv) 5 equiv BocAAOH, 5 equiv DIPCDI/HOBt, 3 equiv TEA, DMF,
À10 °C ? rt, N2; (v) TFA/DCM 30%, rt, followed by Na2CO3 aq 30% and extraction
with DCM.
DCCI as coupling agent failed to give compounds 6. Similar difficul-
ties were reported for the solid-phase synthesis of Leu-Enkephalin
peptidomimetics based on the imidazolidin-4-one scaffold, where
resin-bound dipeptide imidazolidin-4-ones containing two methyl
groups at C-2 failed to give the desired N1-acylated products.19
Thus, we decided to optimize the coupling reaction of 3a (R1 = H,
R2 = R3 = Me) with BocGlyOH. Different cocktails of coupling re-
agents in DMF were used, of which the most efficient were DIPCDI
and HOBt (87% yield of 6a; R1 = R4 = H, R2 = R3 = Me).20 The best
coupling method (DIPCDI/HOBt) was then assayed in different sol-
vents, with DMF remaining the best, followed by ACN (76% yield)
and DCM (34%). Coupling did not take place in THF. These results
Imidazolidin-4-ones 5a–g were evaluated against the chloro-
quine-resistant P. falciparum strain W2, and IC50 values obtained
are given in Table 1, together with those previously determined15
for the parent drug, 1, and also for precursors 3a and 3b. IC50 values
for 5 range from 5.5 to 12
lM, with one compound (5g) displaying
activity close to that of primaquine, 1 (3.3
lM). Inspection of the
data in Table 1 shows that antiplasmodial activity is slightly af-
fected by the nature of substituents R4 in the N-terminal amino
acid moiety. For example, the glycyl derivative 5a (R1 = R4 = H) is
equipotent with its leucyl counterpart 5d (R1 = H, R4 = iBu) and
ca. two times more active than its methioninyl counterpart 5f
(R1 = H, R4 = CH2CH2SMe), thus suggesting that large hydrophobic
Table 1
Synthesis data for N1-acyl-imidazolidin-4-one derivatives 5; antiplasmodial activity data of primaquine (1), imidazolidin-4-one derivatives 3a, b and N1-acyl-imidazolidin-4-one
derivatives 5
R1
R2/R3
R4
Yield in %a
P. falciparum W2 IC50
(l
M)
b
Compound
1
—
H
Me
H
H
H
H
H
—
—
—
—
H
—
—
—
3.3c
3a
3b
5a
5b
5c
5d
5e
5f
Me/Me
Me/Me
Me/Me
Me/Me
Me/Me
Me/Me
Me/Me
Me/Me
Me/Me
Me/Me
9.1c
>50c
(87) 86
(71) 93
(69) 89
(37) 81
(69) 98
(68) 91
(58) 84
(1.6d) —
6.7 0.2
8.0 0.5
7.9 0.4
6.3 0.1
10
12
Me
iPr
iBu
Bzl
(CH2)2SMe
H
H
1
1
H
Me
(CH2)2SMe
5g
5h
5.5 0.2
—
a
Yields for Boc removal to obtain 5; in parentheses are the yields for the coupling reaction to obtain 6.
Assays were performed as described in Ref. 15.
From Ref. 15.
b
c
d
Compound 6h decomposed in the acidolytic removal of Boc.

4152
N. Vale et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4150–4153
amino acids at the N-terminus of the peptide backbone are some-
what detrimental for antiplasmodial activity. Considering the ef-
fect of R1 substituents at the C-5 position of imidazolidin-4-ones
5 on antiplasmodial activity, the results also suggest that R1 does
not have a marked influence on activity against P. falciparum:
changing H for Me at R1 (i.e., 5a vs 5g) leads only to a marginal in-
crease in activity. Although these SARs are based on diastereomeric
mixtures, it should be noted that the enantiomers of primaquine
are equipotent antimalarials.23
Compounds 5 are generally more active than their N-terminal
imidazolidin-4-one precursors 3, particularly when R1 = Me, as
shown by at least 10-fold difference in IC50 values against P. falci-
parum between the alanine-based imidazolidin-4-one 3b and its
N1-glycyl derivative 5g. This result is consistent with the previ-
ously mentioned relevance of a free primary amino group for anti-
plasmodial activity.17
of 5 could be established, despite the obvious limitations imposed
by the relatively small number of compounds included in this pre-
liminary study. The high aqueous stability displayed by 5 suggests
that incorporation of an imidazolidin-4-one moiety into the C-ter-
minus of a dipeptide derivative of primaquine might be a useful
approach to obtain chemically and enzymatically stable peptidom-
imetic derivatives of 8-aminoquinoline antimalarials. Recent re-
ports indicate that adequate substitution at the C-2, C-4, and C-5
positions of the quinoline moiety can lead to potent 8-aminoquin-
oline blood-schizontocidal antimalarials devoid of significant
blood toxicity.27 Therefore, combination of the imidazolidin-4-
one scaffold with the appropriately substituted quinoline moiety
deserves further attention.
Acknowledgments
The potential of compounds 5a, b, e to prevent the transmission
of malaria was studied using a model consisting of BalbC mice in-
fected with P. berghei and Anopheles stephensi mosquitoes and com-
pared to that of primaquine.14,24 The antimalarial activity was
assessed based on the percentage of mosquitoes with oocysts
and the mean number of oocysts per infected mosquito (Table 2).
Although this model cannot specifically attribute the drug effect
to either gametocytocidal or sporontocidal activity, it can clearly
show if a compound is effective at interrupting the transmission
of the infection to mosquitoes by interference with the cycle in
R.M. and P.G. were supported by Grant POCTI/FCB/39218/2001
from Foundation for Science and Technology (FCT, Portugal). N.V.
thanks FCT for Ph.D. Grant SFRH/BD/17754/2004. P.J.R. was sup-
ported by grants from the National Institutes of Health and Medi-
cines for Malaria Venture.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.05.076.
these insects.25,26 Compounds 5a,
(P < 0.05) the sporogonic development of P. berghei at 10 and
50 mol/kg when compared with the control, though they did
not completely inhibit the production of oocysts at 50 mol/kg.
Compound 5b was inactive at 10 mol/kg.
e
significantly reduced
References and notes
l
l
1. Brueckner, R. P.; Ohrt, C.; Baird, J. K.; Milhous, W. K. 8-Aminoquinolines. In
Antimalarial Chemotherapy; Rosenthal, P. J., Ed.; Humana Press: Totowa, 2001; p
123.
2. Mihaly, G. W.; Ward, S. A.; Edwards, G.; Orme, M. L. E.; Breckenridge, A. M. Br. J.
Clin. Pharmacol. 1984, 17, 441.
3. Baker, J. K.; Bedford, J. A.; Clark, A. M.; McChesney, J. D. Pharm. Res. 1984, 1, 98.
4. Baker, J. K.; Yarber, R. H.; Nanayakkara, N. P. D.; McChesney, J. D.; Homo, F.;
Landau, I. Pharm. Res. 1990, 7, 91.
5. Constantino, L.; Paixão, P.; Moreira, R.; Portela, M. J.; Rosário, V. E.; J. Iley J. Exp.
Toxicol. Pathol. 1999, 51, 299.
l
The decomposition of compounds 5 was studied in non-enzy-
matic and enzymatic conditions using HPLC. No traces of products
resulting from either imidazolidin-4-one ring-opening (i.e., dipep-
tide derivatives of primaquine) or N-terminal amino acid hydroly-
sis (i.e., compounds 3) were detected when incubated in pH 7.4
buffer and 80% human plasma at 37 °C. This result confirms our ini-
tial prediction, that is, N1-acyl imidazolidin-4-ones have negligible
susceptibility to hydrolysis.
6. Umbreit, J. Am. J. Hematol. 2007, 82, 134.
7. Philip, A.; Kepler, J. A.; Johnson, B. H.; Carroll, F. Y. J. Med. Chem. 1988, 31, 870.
8. Borissova, R.; Stjarnkvist, P.; Karlsson, M. O.; Sjoholm, I. J. Pharm. Sci. 1995, 84,
256.
9. Chung, M. C.; Gonçalves, M. F.; Colli, W.; Ferreira, E. I.; Miranda, M. T. M. J.
Pharm. Sci. 1997, 86, 1127.
In conclusion, the synthesis of imidazolidin-4-one derivatives of
primaquine, 5, was attained by solution phase acylation of imidaz-
olidin-4-one precursors 3 with Boc-protected amino acids. Com-
10. Portela, M. J.; Moreira, R.; Valente, E.; Constantino, L.; Iley, J.; Pinto, J.; Rosa, R.;
Cravo, P.; Rosário, V. E. Pharm. Res. 1999, 16, 949.
pounds
5 exhibit moderate activity against a chloroquine-
resistant strain of P. falciparum and inhibit the transmission of
the infection to mosquitoes as efficiently as primaquine. Useful
information concerning the effect of R1 and R4 on the bioactivity
11. Rasmussen, G. J.; Bundgaard, H. Int. J. Pharm. 1991, 71, 45.
12. Bak, A.; Fich, M.; Larsen, B. D.; Frokjaer, S.; Friis, G. J. Eur. J. Pharm. Sci. 1999, 7,
317.
13. Gomes, P.; Araújo, M. J.; Rodrigues, M.; Vale, N.; Azevedo, Z.; Iley, J.; Chambel,
P.; Morais, J.; Moreira, R. Tetrahedron 2004, 60, 5551.
14. Araújo, M. J.; Bom, J.; Capela, R.; Casimiro, C.; Chambel, P.; Gomes, P.; Iley, J.;
Lopes, F.; Morais, J.; Moreira, R.; Oliveira, E.; Rosário, V.; Vale, N. J. Med. Chem.
2005, 48, 888.
15. Vale, N.; Collins, M. S.; Gut, J.; Ferraz, R.; Rosenthal, P. J.; Cushion, M. T.;
Moreira, R.; Gomes, P. Bioorg. Med. Chem. Lett. 2008, 18, 485.
16. Chambel, P.; Capela, R.; Lopes, F.; Iley, J.; Morais, J.; Gouveia, L.; Gomes, J. R. B.;
Gomes, P.; Moreira, R. Tetrahedron 2006, 62, 9883.
17. Nodiff, E. A.; Chatterjee, S.; Musallam, H. A. Prog. Med. Chem. 1991, 28, 1.
18. Ferraz, R.; Gomes, J. R. B.; Oliveira, E.; Moreira, R.; Gomes, P. J. Org. Chem. 2007,
72, 4189.
19. Rinnová, M.; Nefzi, A.; Houghten, R. A. Tetrahedron Lett. 2002, 43, 2343.
20. Synthesis of compound 6a was carried out as follows: compound 3a (1 mmol)
was suspended in solvent (20 mL), TEA (3 equiv) and the mixture was stirred at
À10 °C for 20 min, under inert atmosphere. After addition of BocGlyOH (5
equiv) plus DIPCDI (5 equiv), the mixture was kept at À10 °C for further 4 h,
under stirring. The temperature was then increased to 10 °C and thus
maintained till the end of reaction (24 h by TLC). The solid was removed by
suction filtration, the liquid phase was evaporated at 90 °C in vacuum to
dryness and the resulting residue was dissolved in 40 mL of DCM. This solution
was washed three times with 15 mL portions of 10% aq NaHCO3 and the
organic layer dried over anhydrous MgSO4 and evaporated to dryness. The
residue was submitted to column chromatography on silica using DCM/
acetone. The product was isolated as yellow-orange oil and identified as 6a; dH,
8.51 (1H, dd, J = 4.20, 1.38); 7.92 (1H, dd, J = 8.26, 1.42 Hz); 7.30 (1H, dd,
Table 2
Effect of compounds 5a, 5b, and 5e, and primaquine, 1, on the sporogonic
development of Plasmodium berghei ANKA in Anopheles stephensi mosquitoes
Compound
Dose/
l
mol kgÀ1
% Infected mosquitoesa
Mean no. of oocysts/
mosquito ( SEMb)
1
0
65.4
41.7
42.9
66.1
42.0
40.5
66.1
67.9
40.1
65.4
43.8
40.0
16.3 3.85
2.00 1.05
0.95 0.35
9.10 1.45
1.08 0.23
0.79 0.27
9.10 1.45
9.71 2.49
1.34 0.31
16.3 3.85
3.69 1.17
2.20 0.77
10
50
0
10
50
0
10
50
0
10
50
5a
5b
5e
a
Counting of oocysts was carried out at day 10 post-feed.
Mean standard error.
b

N. Vale et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4150–4153
4153
J = 8.28, 4.20); 6.34 (1H, d, J = 2.45); 6.28 (1H, d, J = 2.43); 6.01 (1H, d, J = 8.22);
5.35 (1H, br s); 3.95 (2H, s); 3.89 (3H, s); 3.81 (2H, d, J = 4.31); 3.69–3.63 (1H,
m); 3.30–3.18 (2H, m); 1.84–1.72 (4H, m); 1.60(4) and 1.59(9) (6H, s+s); 1.44
(9H, s); 1.31 (3H, d, J = 6.30). dC, 166.03; 165.36; 159.50; 155.84; 145.03;
144.43; 135.42; 134.89; 129.99; 121.99; 96.90; 91.88; 80.91; 79.99; 55.33;
47.92; 47.16; 43.80; 39.91; 34.13; 28.42; 25.84; 24.60; 24.52; 20.81. m/z
(M+H+) = 514.21 (calcd, 514.30). The different coupling methods assayed for
the preparation of compounds 6 and the corresponding yields are in the
Supporting Information, together with spectroscopic data.
evaporated to dryness. Chromatographically homogeneous yellow-orange oils
were obtained and identified as the target structures
Information).
5 (cf. Supporting
22. See Supporting Information for detailed spectroscopic data.
23. Brossi, A.; Millet, P.; Landau, I.; Bembenek, M. E.; Abell, C. W. FEBS Lett. 1987,
214, 291.
24. Coleman, R. E.; Vaughan, J. A.; Hayes, D. O.; Hollingdale, M. R.; Do Rosário, V. E.
Acta Leiden. 1988, 57, 61.
25. Coleman, R. E.; Clavin, A. M.; Milhous, W. K. Am. J. Trop. Med. Hyg. 1992, 46, 169.
26. Coleman, R. E.; Nath, A. K.; Schneider, I.; Song, G.-H.; Klein, T. A.; Milhous, W. K.
Am. J. Trop. Med. Hyg. 1994, 50, 646.
21. Acidolytic removal of Boc: compounds 6 were dissolved in TFA at 30% in DCM
and the reaction was allowed to proceed for 2 h at room temperature. Excess
TFA was neutralized by dropwise addition of 30% aq Na2CO3 until pH 10; the
supernatant oily layer formed was extracted six times with 10 mL portions of
chloroform and the organic layers pooled, dried over anhydrous MgSO4 and
27. Vangapandu, S.; Sachdeva, S.; Jain, R.; Jain, M.; Singh, S.; Singh, P. P.; Kaul, C. L.;
Jain, R. Bioorg. Med. Chem. 2004, 12, 239.