Imidazolidin-4-one derivatives of primaquine as novel transmission-blocking antimalarials

Article abstract of DOI:10.1021/jm0494624

Imidazolidin-4-one derivatives of primaquine were synthesized as potential double prodrugs of the parent drug. The title compounds inhibit the development of the sporogonic cycle of Plasmodium berghei, affecting the appearance of oocysts in the midguts of the mosquitoes. The imidazolidin-4-ones are very stable, both in human plasma and in pH 7.4 buffer, indicating that they are active per se. Thus, imidazolidin-4-ones derived from 8-aminoquinolines represent a new entry in antimalarial structure - activity relationships.

Full text of DOI:10.1021/jm0494624

888
J. Med. Chem. 2005, 48, 888-892
Imidazolidin-4-one Derivatives of Primaquine as Novel Transmission-Blocking
Antimalarials
Maria Joa˜o Arau´jo,† Joana Bom,‡ Rita Capela,§ Catarina Casimiro,‡ Paula Chambel,| Paula Gomes,† Jim Iley,
Francisca Lopes,§ Jose´ Morais,| Rui Moreira,*,§ Eliandre de Oliveira,# Virg´ılio do Rosa´rio,‡ and Nuno Vale†
CECF, Faculty of Pharmacy, University of Lisbon, Av. Forc¸as Armadas, 1649-019 Lisboa, Portugal, UCTM,
Faculty of Pharmacy, University of Lisbon, Av. Forc¸as Armadas, 1649-019 Lisboa, Portugal, CIQUP, Department of Chemistry,
Faculty of Sciences, University of Oporto, Rua do Campo Alegre 687, 4169-007 Porto, Portugal, Department of Chemistry,
The Open University, Milton Keynes, MK7 6AA, U.K., CMDT, Instituto de Higiene e Medicina Tropical, Rua da Junqueira, 67,
1000 Lisboa, Portugal, and Proteomics Platform, Barcelona Science Park, C/Josep Samitier, 1-5, E-08028 Barcelona, Spain
Received July 6, 2004
Imidazolidin-4-one derivatives of primaquine were synthesized as potential double prodrugs
of the parent drug. The title compounds inhibit the development of the sporogonic cycle of
Plasmodium berghei, affecting the appearance of oocysts in the midguts of the mosquitoes.
The imidazolidin-4-ones are very stable, both in human plasma and in pH 7.4 buffer, indicating
that they are active per se. Thus, imidazolidin-4-ones derived from 8-aminoquinolines represent
a new entry in antimalarial structure-activity relationships.
Introduction
Malaria is the major life-threatening parasitic disease
in tropical and subtropical regions. Worldwide, there are
at least 300 million acute cases of malaria and more
than 1-2 million deaths each year, mostly young
children infected with Plasmodium falciparum.1 Most
of the drugs actually used in antimalarial chemotherapy
are particularly active against the asexual blood forms
of the parasite, which are responsible for the clinical
symptoms of the disease. With the rapid spread of drug-
resistant P. falciparum strains, the development of safe
and effective antimalarials that prevent transmission,
in addition to curing patients, has become an important
strategy toward achieving an effective control of ma-
laria.2
In contrast to the asexual blood forms of Plasmodium,
the sexual form of the parasite is a much unexplored
life-cycle target. Currently, primaquine, 1, is the only
available transmission-blocking antimalarial displaying
a marked activity against gametocytes from all species
of parasite causing human malaria, including chloro-
quine-resistant P. falciparum.3 However, the use of
primaquine is limited by its extensive conversion to
carboxyprimaquine, 2, and by its toxic effects, among
them hemolytic anemia, particularly in patients who are
deficient in glucose-6-phosphate dehydrogenase.4-6
Several peptide and amino acid derivatives of pri-
maquine and other 8-aminoquinoline antimalarials have
been synthesized to reduce the metabolic oxidative
deamination pathway, as well as to reduce toxicity of
the parent drug.7-10 Such derivatives display improved
activity/toxicity ratios when compared to primaquine,
which can be ascribed either to a reduction in metabolic
inactivation8,9 or to a selective hydrolysis inside the
parasite,7 leading to a higher intracellular drug con-
centration. However, we, and others, have shown that
amino acid and peptide derivatives of primaquine are
rapidly hydrolyzed to primaquine by aminopeptidases
and endopeptidases,9,11 suggesting that they might
undergo extensive hydrolysis to the parent drug in the
GI tract when given orally. One approach to enhance
the enzymatic stability of amino acid or peptide deriva-
tives of primaquine toward proteolytic degradation at
the mucosal absorption barrier or in the blood is the
development of a double prodrug.12 To this end, imid-
azolidin-4-one formation was introduced as a useful
prodrug approach to protect the N-terminal amino acid
residue of di- to pentapeptides against aminopeptidase-
catalyzed hydrolysis.13-16 Usually, peptide imidazolidin-
4-one derivatives are quantitatively hydrolyzed to the
parent peptide in pH 7.4 buffer at 37 °C with half-lives
ranging from 1 to 30 h, depending on the N-terminal
dipeptide sequence and on the imidazolidinone sub-
stituents.13-16 Therefore, we reasoned that imidazolidin-
4-one derivatives, 4, of primaquine (see Scheme 1) would
release the corresponding amino acid derivative via a
nonenzymatic reaction, which, in turn, could be enzy-
matically hydrolyzed to primaquine. In this study, we
report the reactivity in human plasma and gametocyto-
cidal activity of imidazolidin-4-ones, 4.
* To whom correspondence should be adressed. Tel: +351 217946477.
Fax: +351 217946470. E-mail: rmoreira@ff.ul.pt.
† CIQUP, Department of Chemistry, University of Oporto.
‡ CMDT, Instituto de Higiene e Medicina Tropical.
§ CECF, Faculty of Pharmacy, University of Lisbon.
| UCTM, Faculty of Pharmacy, University of Lisbon.
Department of Chemistry, The Open University.
# Proteomics Platform, Barcelona Science Park.
Results and Discussion
Chemistry. The synthesis and characterization of
imidazolidin-4-ones 4 has been described elsewhere.17
In short, compounds 4 can be synthesized in good yields
from the corresponding amino acid derivatives AA-PQ,
10.1021/jm0494624 CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/14/2005

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 889
Scheme 1a
a
(i) Me2CO or C5, C6, or C7 cyclic ketones in refluxing MeOH,
TEA, molecular sieves.
Table 1. Percentage of Hydrolysis of Imidazolidin-4-ones 4 to
the Corresponding Amino Acid Products 3, in 80% Human
Plasma and in pH 7.4 Phosphate Buffer at 37 °C after 3 Days
of Incubation, with Half-Lives for the Hydrolysis in pH 7.4
Buffer in Parentheses
% hydrolysis to 3
Figure 1. Time courses for compounds 4c (O), Ala-PQ (b),
and primaquine (9) when 4c (open symbols) and Ala-PQ
(closed symbols) were incubated in 80% human plasma at 37
°C.
after 3 days
80% human pH 7.4 buffer
compd
R1
R2
R3
plasma
(t1/2/d)
4a
4b
4c
4d
4e
4f
4g
4h
4i
H
(CH2)5
stablea
11 (18)d
19 (9.8)c
16 (12)c
10 (20)d
17 (11)d
28 (6.4)c
6 (31)c
> 4) occurs by an SN1-type mechanism which involves
the departure of an amide leaving group (Scheme 2).13
The amides resulting from the rate-limiting ring open-
ing of 4 are much poorer leaving groups than those from
dipeptide imidazolidin-4-ones (the difference in pKa is
about 3.3 units18). Assuming that the pH-independent
hydrolysis of imidazolidin-4-ones has the same suscep-
tibility to the leaving group effect as the analogous
acyclic N-Mannich bases,19 i.e. with a Bro¨nsted âlg value
of ca. -1, then it would be expected that compounds 4
would hydrolyze ca. 103 times slower20 than their
counterparts derived from dipeptides. The smaller dif-
ferences reported herein might be attributed to the fact
that the amino acid chain affects both the amide leaving
group ability and the ability of the imidazolidin-4-one
N1 amino nitrogen atom to expel the amide. A remark
should be made on the higher stability of 4 in human
plasma when compared to pH 7.4 phosphate buffer. This
might be ascribed to the binding to plasma proteins. A
decrease in reactivity in human plasma when compared
to pH 7.4 buffers has also been reported for imidazoli-
din-4-ones derived from dipeptides and pentapetides.14,15
In Vivo Gametocytocidal Studies. The potential
of compounds 4 to prevent the transmission of malaria
was studied using a model consisting of BalbC mice
infected with Plasmodium berghei and Anopheles
stephensi mosquitoes. The two criteria used to assess
the antimalarial activity of each compound were (i) the
percentage of mosquitoes with oocysts and (ii) the mean
number of oocysts per infected mosquito. These in vivo
screening assays are of major support in analyzing the
effect of drugs in the sporogonic cycle, since no equiva-
lent in vitro assays exist.21 Although this model cannot
specifically attribute the drug effect to either gameto-
cytocidal activity (i.e. affects parasite development by
killing gametocytes) or sporontocidal activity (i.e. affects
directly the development of oocysts on the stomach wall
of the mosquitoes), it can clearly show if a compound is
effective at interrupting the transmission of the infec-
tion to mosquitoes by interference with the cycle in these
insects.22,23
H
Me Me
(CH2)5
16b
Me
9b
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CHMe2
CHMe2
CHMe2
CHMe2
(CH2)6
stablea
stablea
3b
(CH2)5
(CH2)4
Me Me
(CH2)6
stablea
nde
nde
(CH2)5
stablea
5b
8 (26)c
4j
4k
4l
(CH2)4
21 (8.8)c
16 (12)c
10 (20)d
Me Me
stablea
stablea
CH2CHMe2 Me Me
a No degradation after 3 days of incubation. b Percentage of
hydrolysis after 3 days of incubation. c From ref 17. d This study.
e Not determined due to solubility problems.
3, by refluxing with an excess of the appropriate ketone
in methanol in the presence of triethylamine (TEA) and
4 Å molecular sieves.
In Vitro Stability Studies. The hydrolyses of imid-
azolidin-4-ones 4 in 80% human plasma were monitored
by HPLC for the simultaneous disappearance of sub-
strate and formation of the amino acid derivative 3 and
primaquine. With the exception of compound 4b, imid-
azolidin-4-ones 4 display unusually high stability when
incubated in 80% human plasma (Table 1), with no
significant disappearance of the starting material over
3 days of incubation. The stability of 4 in human plasma
is not significantly affected either by the nature of the
amino acid R1 substituent or by the R2 and R3 substit-
uents in the imidazolidin-4-one moiety. This contrasts
with the behavior of the corresponding amino acid
intermediates, 3, which are hydrolyzed quantitatively
to primaquine with rates depending on the nature of
the amino acid side chain (Figure 1: 4c versus Ala-PQ).
Thus, while simple amino acid derivatives behave as
prodrugs of primaquine, the corresponding imidazolidin-
4-ones 4 are too stable to be considered prodrugs.
In contrast to their behavior in plasma, the imid-
azolidin-4-ones 4 hydrolyze to the corresponding amino
acid derivatives in pH 7.4 buffer with half-lives ranging
from 9 to 30 days (Table 1). Moreover, compounds 4 are
hydrolyzed 50-100 times slower than the imidazolidin-
4-one counterparts derived from dipeptides or penta-
peptides.13-16 We were surprised by such large differ-
ences in reactivity, though one possible explanation
might lie in the mechanism of hydrolysis. The pH-
independent hydrolysis of imidazolidin-4-ones (i.e. at pH
The antimalarial activity data are presented in Table
2, and from these the following observations can be
made. First, primaquine (PQ) and the imidazolidin-4-
one derivatives 4a-g (i.e. those derived from Gly-PQ,
Ala-PQ, and Phe-PQ) completely inhibited the produc-

890 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Brief Articles
Scheme 2
Table 2. Effect of Compounds 4 and Primaquine on the Sporogonic Development of Plasmodium berghei ANKA in Anopheles
stephensi Mosquitoes
dose/
% infected
mean no. of oocysts/
dose/
% infected
mean no. of oocysts/
compd
µmol‚ kg-1
mosquitoesa
mosquito ((SE)
compd
4h
µmol‚kg-1
mosquitoesa
mosquito ((SE)
4a
0
10
50
0
10
50
0
10
50
0
10
50
0
10
50
0
10
50
0
65
25
0
65
21
0
93
20
0
100
90
0
93
83
0
80
65
0
80
76
0
105 (22)
7.4 (9.3)
0
105 (22)
6.1 (3.5)
0
83.2 (7.1)
0.8 (1.1)
0
120 (8.0)
78.9 (6.6)*
0
83.2 (7.1)
71.0 (6.5)*
0
66.1 (9.1)
51.6 (8.8)*
0
66.1 (9.1)
61.2 (7.5)*
0
0
10
50
0
10
50
0
10
50
0
10
50
0
100
65
120 (8.0)
17.7 (6.5)
4b
4c
4d
4e
4f
4i
100
85
25
100
35
15
65
25
5
100
30
20
93
28
0
51.0 (9.1)
61.1 (6.7)* b
0.4
4j
51.0 (9.1)
4.2 (3.3)
0.5 (0.2)
105 (22)
5.0 (2.5)
0.9 (0.5)
120 (8.0)
13.7 (10.9)
1.0 (0.9)
83.2 (7.1)
2.6 (2.0)
0
4k
4l
10
50
0
10
50
primaquine
4g
10
50
a Counting of oocysts was carried out at day 10 post-feed. b (*) P > 0.05 versus control, by Student’s t test.
tion of oocysts at a dose of 50 µmol/kg. Second, imid-
azolidin-4-ones derived from Val-PQ and Leu-PQ, 4i-
l, although significantly (P < 0.05) affecting the sporogon-
ic development of P. berghei, did not completely inhibit
the production of oocysts at 50 µmol/kg. Third, the mean
number of oocysts was also significantly affected by
compounds 4a-c and 4j-l at a dose of 10 µmol/kg.
Primaquine was similarly effective at this dose level.
In contrast, at a dose of 10 µmol/kg, derivatives 4d-g
(derived from Phe-PQ) and 4h,i (derived from Val-PQ)
did not significantly (P > 0.05) reduce the oocyst
production when compared with the control.
From these results it can be concluded that the
imidazolidin-4-ones derived from Gly-PQ, 4a,b, and Ala-
PQ, 4c, are the most effective gametocytocidal agents,
displaying an antimalarial activity comparable to that
of primaquine. The activity of 4a and 4b compares to
the activity reported previously for Gly-PQ, thus sug-
gesting that incorporation of the imidazolidin-4-one
scaffold does not alter substantially the antimalarial
activity. In contrast, imidazolidin-4-ones 4 derived from
the more lipophilic amino acids phenylalanine, valine,
and leucine were less active when compared to pri-
maquine. A similar observation has been reported for
dipeptide derivatives of primaquine, AA-Gly-PQ,9 for
which it was found that the most active compound was
AA ) Gly, while those containing the bulky and lipo-
philic amino acids (R)-Phe and (S)-Phe were less active.
More recently, Vangapandu et al. reported that the
attachment of a hydrophobic amino acid to the terminal
amino group of primaquine analogues results in de-
creased blood-schizontocidal antimalarial activity.10
Taken together, these suggest that hydrophobic amino
acid side chains have a detrimental effect on the activity
of 8-aminoquinoline derivatives against both blood
schizonts and gametocytes.
The effect of the substituents at the C-2 position of
imidazolidin-4-ones 4 on the gametocytocidal activity is
less clear. For example, there is no difference in activity
between the imidazolidin-4-ones prepared from Phe-PQ
and cycloheptanone, 4d, cyclohexanone, 4e, cyclopen-
tanone, 4f, and acetone, 4g, at 10 or 50 µmol/kg.
Similarly, no difference in activity is observed between
the Gly-PQ derivatives 4a and 4b. In contrast, the
cycloheptanone, cyclopentanone, and acetone deriva-
tives of Val-PQ (4h, 4j, and 4k, respectively) are active
at 10 µmol/kg, while their cyclohexanone counterpart
4i is inactive at this dose level. This warrants further
studies to disclose the effect of the imidazolidin-4-one
C-2 substituents on antimalarial activity.
The imidazolidin-4-one N1 nitrogen atom of 4 is
substantially less basic (pKa ca. 3)13,24 than that of
primaquine (pKa 10) or its amino acid or peptide
derivatives (pKa 8-8.5). Consequently, a major finding
that emerges from this study is that the presence of a
terminal, strongly basic amino group, as found in
primaquine or its amino acid and peptide derivatives,
is not a necessary requirement for gametocytocidal
activity.
Conclusion
The reported imidazolidin-4-ones prepared from amino
acid derivatives of primaquine exhibit potent gameto-
cytocidal activity against P. berghei. In general, those
derivatives 4 containing small amino acid chains (Gly
and (S)-Ala) are superior to those containing bulky/
hydrophobic side chains ((S)-Phe, (S)-Val, and (S)-Leu).

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 891
Supporting Information Available: HPLC chromato-
grams of incubation mixtures in 80% human plasma and in
pH 7.4 buffer for compounds 4f and 4j. This material is
available free of charge via the Internet at http://pubs.acs.org.
These imidazolidin-4-ones 4 are very stable both in
chemical and in enzymatic conditions, suggesting that
they are active per se. Thus, the imidazolidin-4-ones 4
can be considered as a novel type of 8-aminoquinoline
antimalarial. Recent reports indicate that adequate
substitution at the C-2, C-4, and C-5 positions of the
quinoline moiety can lead to potent 8-aminoquinoline
blood-schizontocidal antimalarials devoid of significant
blood toxicity. Therefore, combination of the imidazoli-
din-4-one scaffold with the appropriately substituted
quinoline moiety deserves further attention.
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Experimental Section
HPLC Analysis. High-performance liquid chromatography
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analytical column. A LiChrospher 100 RP-8 5 µm (Merck,
Germany) was employed as precolumn. The solvent system
used was a gradient of sodium acetate buffer (pH 4.75; 0.05
M) (A) and acetonitrile (B); 10-3 M triethylamine was added
to the aqueous mobile phase in order to improve peak shape.
The gradient was as follows: 0 min, 50% B; 4.5 min, 50% B;
5.0 min, 10% B; 20 min, 10% B. For the imidazolidin-4-one
derivatives of valine a second gradient was developed: 0 min,
40% B; 5.5 min, 40% B; 6.0 min, 10% B; 20 min, 10% B. Elution
was performed at a solvent flow rate of 1 mL/min, and a 15
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Hydrolysis in Human Plasma. The compounds 4 were
incubated at 37 °C in human plasma (from heparinized blood
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phosphate buffer. At appropriate intervals, aliquots were
added to acetonitrile to quench the reaction and precipitate
plasma proteins. These samples were centrifuged and the
supernatant was analyzed by the HPLC method described
above for the presence of substrate and products.
Hydrolysis in Aqueous Solution. The rates of hydrolyses
of compounds 4 were determined in pH 7.4 phosphate saline
buffer, at 37 °C. Usually, a 10 µL aliquot of a 10-2 M stock
solution of substrate in acetonitrile was added to 10 mL of the
appropriate thermostated buffer solution. At regular intervals,
samples of the reaction mixture were analyzed by HPLC. All
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In Vivo Gametocytocidal Activity. BalbC mice were
infected by intraperitoneal inoculations of 107 erythrocytes
parasitized with P. berghei ANKA. After 4 days, when the
presence of gametocytes and exflagellation was observed by
microscopic observation of Giemsa stained blood films, mice
were randomly separated into five different groups of six
animals. Each group was treated by intraperitoneal admin-
istration with one single dose of each compound 4 and
primaquine (10 and 50 µmol/kg in inoculation volumes of 0.1-
0.2 mL; controls consisted of mice given a PBS solution). Two
hours after administration, mice were anesthetized and placed
on top of individual cages containing ca. 50 glucose-starved
Anopheles stephensi female mosquitoes, which were allowed
to feed for 2 h. After the blood meal, unfed females mosquitoes
were removed from each cage. Ten days after the blood meal,
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Acknowledgment. The authors thank Fundac¸a˜o
para a Cieˆncia e Tecnologia (Portugal) for financial
support through Research Project POCTI/FCB/39218/
2001, FEDER, and Ph.D. grant to P.C.

892 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
(20) Assuming that the equation log k ) -pKa + C holds for
Brief Articles
(23) Coleman, R. E.; Nath, A. K.; Schneider, I.; Song, G.-H.; Klein T.
A.; Milhous, W. K. Prevention of sporogony of Plasmodium
falciparum and P. berghei in Anopheles stephensi mosquitoes by
transmission-blocking antimalarials. Am. J. Trop. Med. Hyg.
1994, 50, 646-653.
(24) For example, the pKa value for the derivative 4f, kinetically
determined from a pH-rate profile, is 3.5 (data not shown).
2
1
compounds 4, then log(k1/k2) ) pKa - pKa
.
(21) Coleman, R. E.; Vaughan, J. A.; Hayes, D. O.; Hollingdale, M.
R.; Do Rosa´rio, V. E. Effect of mefloquine and artemisinin on
the sporogonic cycle of Plasmodium berghei ANKA in Anopheles
stephensi mosquitoes. Acta Leiden. 1988, 57, 61-74.
(22) Coleman, R. E.; Clavin, A. M.; Milhous, W. K. Gametocytocidal
and sporontocidal activity of antimalarials against Plasmodium
berghei ANKA in ICR mice and Anopheles stephensi mosquitoes.
Am. J. Trop. Med. Hyg. 1992, 46, 169-182.
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