Anti-Pneumocystis carinii and antiplasmodial activities of primaquine-derived imidazolidin-4-ones

Article abstract of DOI:10.1016/j.bmcl.2007.11.105

A series of primaquine-derived imidazolidin-4-ones were screened for their in vitro activity against Pneumocystis carinii and Plasmodium falciparum W2 strain. Most compounds were active against P. carinii above 10 μg/mL and displayed slight to marked acti

Full text of DOI:10.1016/j.bmcl.2007.11.105

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 485–488
Anti-Pneumocystis carinii and antiplasmodial activities
of primaquine-derived imidazolidin-4-ones
Nuno Vale,a Margaret S. Collins,c,d Jiri Gut,e Ricardo Ferraz,a,b
Philip J. Rosenthal,e Melanie T. Cushion,c,d Rui Moreiraf and Paula Gomesa,*
a
ˆ
CIQUP—Centro de Investigac¸a˜o em Quı´mica da Universidade do Porto, Departamento de Quı´mica, Faculdade de Ciencias,
Universidade do Porto, P-4169-007 Porto, Portugal
bEscola Superior de Tecnologias de Sau´de do Porto, Instituto Polite´cnico do Porto, P-4000-294 Porto, Portugal
cDivision of Infectious Diseases, Department of Internal Medicine, University of Cincinnati, OH 45267-0560, USA
dResearch Services, Veterans Affairs Medical Center, Cincinnati, OH 45220, USA
eDepartment of Medicine, San Francisco General Hospital, University of California, San Francisco, CA 94143-0811, USA
fiMed.UL, CECF, Faculdade de Farma´cia de Lisboa, P-1600-083 Lisboa, Portugal
Received 11 October 2007; revised 22 November 2007; accepted 27 November 2007
Available online 3 December 2007
Abstract—A series of primaquine-derived imidazolidin-4-ones were screened for their in vitro activity against Pneumocystis carinii
and Plasmodium falciparum W2 strain. Most compounds were active against P. carinii above 10 lg/mL and displayed slight to
marked activity. The imidazolidin-4-ones most active against P. carinii were also those most active antiplasmodial agents, in the
lM range. One of the tested imidazolidin-4-ones was slightly more active than the parent primaquine and may represent a lead com-
pound for the development of novel anti-P. carinii 8-aminoquinolines with increased stability and resistance to metabolic
inactivation.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Pneumocystis pneumonia (PCP) is a fungal opportunis-
tic infection caused by Pneumocystis jirovecii (formerly
Pneumocystis carinii1,2) and is one the most frequent
causes of mortality in patients with acquired immunode-
ficiency syndrome (AIDS).3,4 PCP also affects other
immunocompromised individuals such as those under-
going cancer therapy and organ and bone marrow trans-
plants.5 Despite the decline in incidence of PCP in AIDS
patients as a consequence of the highly active antiretro-
viral therapy (HAART), PCP remains the most preva-
lent opportunistic infection found in individuals
infected with the human immunodeficiency virus
(HIV).6,7 P. jirovecii is insensitive to standard antifungal
therapy and thus, the antifolate combination of trimeth-
oprim and sulfamethoxazole (TMP-SMX) has been used
for both its prophylaxis and treatment of PCP.8,9 How-
ever, emerging resistance and allergic reactions against
the sulfa component often lead to the alternative use
of pentamidine and atovaquone.8–10 Unfortunately,
pentamidine is ineffective orally and toxic effects have
been reported for this drug,8 while failure of atovaquone
treatment in AIDS patients with PCP is a major
concern.11
Primaquine (1a), an 8-aminoquinoline antimalarial, is
effective against mild to moderate PCP and is co-admin-
istered with clindamycin to AIDS patients with
comparable efficacy to TMP-SMX.9 Optimization of
8-aminoquinolines to improve their antimalarial activity
as well as to reduce adverse effects such as neutropenia
and methemogobinemia also resulted in an improve-
ment of activity against PCP.8,12,13 This observation
led to the suggestion that such synchrony between the
structure–activity relationships (SARs) for the protozoal
and fungal diseases could be useful to develop novel 8-
aminoquinolines with improved efficacy against PCP
(e.g., 1b, c).14 We recently reported that imidazolidin-
4-one derivatives of primaquine, 2 (Scheme 1), exert po-
tent gametocytocidal activity against Plasmodium berg-
hei infection developed in BalbC mice.15 Such
promising results encouraged us to go on further inves-
tigating the in vitro anti-P. carinii activities of com-
Keywords: AIDS; Antimalarial; Antiplasmodial; HIV; Infection; Ma-
laria; Plasmodium falciparum; Pneumocystis carinii; Primaquine.
*
Corresponding author. Tel.: +351 22 040 2563; fax: +351 22 040
2659; e-mail: pgomes@fc.up.pt
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.105

486
N. Vale et al. / Bioorg. Med. Chem. Lett. 18 (2008) 485–488
MeO
MeO
(i)
N
N
O
(ii)
HN
HN
NH2
*
*
*
NH2
NH
R1
Me
Me
1a
3
(iii)
MeO
O
*
N
HN
*
N
R1
Me
*
R2
N
H
R3
2
Scheme 1. Reagents and conditions: (i) Na-Boc-protected amino acid, diisopropyl- or dicyclohexyl-carbodiimide, 1-hydroxybenzotriazole,
dichloromethane; rt, 120 h; (ii) a—neat TFA, rt, 2 h; b—30% aq Na2CO3 + extraction with chloroform; (iii) molecular sieves, refluxing methanol,
72 h.16–18
pounds 2, which are now herein reported. Since most of
the SARs available for 8-aminoquinolines refer to liver
or blood schizontocidal activities, we also decided to
screen the activity of primaquine imidazolidin-4-ones,
2, against the chloroquine-resistant Plasmodium falciparum
strain W2.
Imidazolidin-4-ones 2a–m were evaluated19 against P.
carinii in an ATP measurement assay based on a lucif-
erin–luciferase mediated reaction20 and against chloro-
quine-resistant P. falciparum strain W2; the results
obtained are given in Table 1.
Inspection of Table 1 shows that imidazolidin-4-ones
2a–m exhibit a twofold variation in anti-P. carinii activ-
ities, ranging from practically insignificant (2b) to
marked (2j), in this case even slightly higher (in lM)
than that of the parent PQ (1a). Considering the effect
of substituents R2 and R3 at imidazolidin-4-one C-2
on the activity, those compounds derived from propa-
none (2a–d) are among the less active of the set, whereas
the top-three compounds are those derived from cyclo-
heptanone (2g, 2j–k). The substituents R2 and R3
brought by the carbonyl reactant seem to play a major
role in the modulation of the anti-P. carinii activity of
compounds 2, with larger and more hydrophobic groups
being apparently preferable. This is illustrated by analy-
sis of the (S)-Val series, where the activity increases
2c < 2e ffi 2f < 2g. Imidazolidin-4-ones 2 are hydrolyzed
very slowly to the corresponding amino acid derivatives,
3, in pH 7.4 buffer with half-lives ranging from ca. 10–30
days in pH 7.4 buffer at 37 ꢁC.15 In the present study, we
assessed the stability of compound 2g, which presents a
half-life higher than 10 days in the same reaction media.
This result is consistent with the observation that imi-
dazolidin-4-ones 2 containing a seven-membered ring
and derived from amino acids containing large a-sub-
stituents are stable in aqueous solutions,15 thus suggest-
ing that 2g is active against P. carinii per se. Finally,
comparison of data from 2m1, 2m2 and 2m3+2m4 shows
that (i) when R2 = H, the activities are identical to those
Y
X
MeO
N
HN
NH2
Me
1a, X = Y = H
1b, X = Me,Y = OAryl
1c, X = Me, Y = OAlkyl
Imidazolidin-4-ones, 2, were obtained from condensa-
tion of racemic primaquine-derived a-aminoamides, 3,
with symmetrical ketones and o-methylbenzaldehyde
(Scheme 1).16,17 Compounds 2 resulting from the reac-
tion of 3 with propanone and cyclic ketones were iso-
lated as mixtures of two diastereomers (enantiomers,
in the case of glycine derivatives 2a, i). In contrast, three
chromatographic distinct fractions were obtained for the
(S)-Phe-derived imidazolidin-4-one 2m, synthesized by
reacting the (S)-Phe derivative 3 and o-methylbenzalde-
hyde: two were isolated as pure diastereomers (2m1 and
2m2) and one as the unresolved diastereomeric mixture
of the remaining two (2m3+2m4).16,17

N. Vale et al. / Bioorg. Med. Chem. Lett. 18 (2008) 485–488
487
Table 1. Anti-P. carinii activity and cytotoxicity of PQ (1) and its imidazolidin-4-one derivatives 2a–ma
Compound
R1
R2
R3
IC50
P. carinii in lM (in lg/mL)b,c
P. falciparum W2 in lMd
1
2a
2.5 (0.6)
23 (8.3)
226 (83.7)
40 (16)
32 (14)
23 (9.7)
26 (11)
6.0 (2.7)
12 (5.7)
43 (17)
1.9 (0.8)
4.1 (1.9)
23 (9.8)
21 (11)
13 (6.7)
20 (10)
3.34
9.08
>50
>50
>50
>50
>50
8.89
>50
>50
2.42
2.63
ND
ND
ND
ND
H
Me
Me
Me
Me
Me
Me
Me
Me
2b
2c
Me
iPr
Bzl
iPr
iPr
iPr
Bzl
H
2d
2e
(CH2)4
(CH2)5
(CH2)6
(CH2)4
(CH2)5
(CH2)6
(CH2)6
2f
2g
2h
2i
2j
2k
Me
iBu
Me
Bzl
Bzl
Bzl
2l
[4-Me](CH2)5
2m1
2m2
2m3+2m4
H
H
H
(o-Me)Ph
(o-Me)Ph
(o-Me)Ph
a All biological activities resulted from the average of at least three determinations.
b Drug activity scale: highly active, <0.01 lg/mL; very marked, <0.1 lg/mL; marked, 0.1–0.9 lg/mL; moderate, 1.0–9.9 lg/mL; slight, 10.0–49.9 lg/
mL.21
c 72-h incubation assays.
d Assays of parasite development were performed as described earlier.22
derived from propanone and cyclopentanone (e.g., 2m
vs 2d and 2h) and (ii) there are no marked differences be-
tween diastereomers, even though 2m2 is slightly more
active than 2m1.
In conclusion, the reported imidazolidin-4-ones pre-
pared from amino acid derivatives of primaquine exhibit
potent activity against P. carinii. The present screening
of compounds 2 allowed the selection of 2j as a potential
lead structure for the future development of effective
anti-PCP agents. Although P. jirovecii is the causative
agent of PCP in humans, in vitro drug screening systems
using organisms derived from rodent models have pro-
vided investigators with the only method to identify
and screen new candidate anti-Pneumocystis com-
pounds. However, in vivo studies have shown that there
is a high degree of correlation between animal models
and human beings.23 In general, those derivatives 2 con-
taining larger and more hydrophobic R2 and R3 substit-
uents at C-2 are superior to those containing small
substituents. Another interesting finding is that the imi-
dazolidin-4-one weakly basic amino group (pKa ca. 4) is
not detrimental to the anti-P. carinii activity, thus sug-
gesting that the strongly basic primary amino group
present in primaquine and other 8-aminoquinoline side
chains is not a major requirement for anti-PCP activity.
Taking into account that acylation of primary amino
group blocks cytochrome P450- and MAO-catalyzed
oxidative deamination of primaquine,24 imidazolidin-4-
ones 2 might offer a useful approach to overcome meta-
bolic inactivation of primaquine into carboxyprimaqu-
ine. Finally, the antiplasmodial activity seems to be a
useful indicator of the anti-P. carinii activity.
When analyzing the effect of the amino acid side chain,
that is, the R1 substituents at the C-5 position of imidaz-
olidin-4-ones 2 on the anti-P. carinii activity, the results
suggest that R1 has not a well-defined or marked influ-
ence on the anti-P. carinii activity. If the three most ac-
tive compounds (2g, 2j, and 2k) are considered, the IC50
values follow the order 2g, (S)-Val >2k, (S)-Leu >2j,
(S)-Ala, but differ at the most by 3-fold (2g vs 2j). On
the other hand, if we consider the propanone-derived
subset 2a–d, the highest IC50 is displayed by the (S)-
Ala derivative (2b), differing from the second highest va-
lue (for 2c) by as much as 55-fold. Interestingly, the
anti-P. carinii SAR herein described is not entirely coin-
cidental with that for the gametocytocidal activity of 2,
as in this case small amino acid side chains significantly
improved the gametocytocidal activity of those com-
pounds against P. berghei, whereas bulky/hydrophobic
amino acids had detrimental effects.15 In contrast, the
reported influence of R2 and R3 on the gametocytocidal
activity of compounds 2 is not marked,15 which suggests
that the stereoelectronic requisites for the fine-tuning of
2 as gametocytocidals do not exactly match those for
optimal anti-P. carinii activity. However, if the antiplas-
modial activities of 2 against the chloroquine-resistant
P. falciparum strain W2 are considered (Table 1), one
observes that (i) the most active antiplasmodial agents
are also those most active against P. carinii (i.e., 2g,
2j–k) and (ii) excepting propanone derivative 2a, all
remaining compounds 2 are inactive against P. falcipa-
rum in this screen. Thus, the presence of larger and more
hydrophobic groups, such as a seven-membered ring, at
C-2 of the imidazolidin-4-one moiety is also a major
requirement for antiplasmodial activity.
Acknowledgments
ˆ
N.V. thanks Fundac¸a˜o para a Ciencia e Tecnologia
(FCT, Portugal) for Ph.D. Grant SFRH/BD/17754/
2004. P.G. and R.M. thank FCT for financial support
to CIQUP and CECF, respectively. P.J.R. was sup-
ported by grants from the National Institutes of Health
and Medicines for Malaria Venture. M.T.C. and M.S.C.

488
N. Vale et al. / Bioorg. Med. Chem. Lett. 18 (2008) 485–488
As an example, compound 2g was prepared by refluxing
3g (R1 = iPr, 1 mol equiv) with cycloheptanone (2 mol
equiv) in dry methanol for 72 h. The crude mixture was
separated by column chromatography on silica, using
dichloromethane/tetrahydrofuran (5:1 v/v) as eluant, to
isolate 2g as a yellow oil (72% yield) with the following
spectroscopic data: dH (CDCl3, 300 MHz) 8.51 (dd, 1H,
J = 3.90, 1.35 Hz); 7.91 (dd, 1H, J = 8.25, 1.35 Hz); 7.29
(dd, 1H, J = 8.25, 3.90 Hz); 6.32 (d, 1H, J = 2.70 Hz);
6.29 + 6.28 (d + d, 1H, J = 2.70 Hz), 6.01 (dd, 1H,
J = 7.95, 3.15 Hz); 3.88 (s, 3H); 3.64 (m, 1H); 3.41 (m,
1H); 3.32 (d, 1H, J = 4.50 Hz); 3.01 (m, 1H); 2.12 (m, 1H);
1.89 (m, 1H); 1.71–1.34 (m, 16H); 1.30 (d, 3H,
J = 6.60 Hz); 1.01 (d, 3H, J = 6.90 Hz); 0.89 (d, 3H,
J = 6.30 Hz). dC (CDCl3, 75 MHz) 174.3; 174.2; 159.5;
159.4; 145.1; 145.0; 144.3; 144.2; 135.4; 134.7; 129.9; 121.8;
96.8; 96.7; 91.6; 80.9; 80.8; 77.3; 62.3; 55.2; 47.9; 47.5; 41.0;
40.9; 40.2; 40.1; 38.2; 38.1; 34.0; 30.3; 29.5; 29.4; 29.3; 29.2;
26.2; 26.1; 25.6; 22.5; 22.4; 22.1; 21.9; 20.7; 20.6; 19.3; 19.2;
17.1; 17.0. m/z (MWmonoisotopic, M+) = 452.3847 (Calcd,
452.3151).
acknowledge the support of Public Health Service Grant
N01 AI75319 from the National Institutes of Allergy
and Infectious Diseases and the Medical Research Ser-
vice Department of the Veterans Affairs, USA.
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˚
in the presence of triethylamine and 4 A molecular sieves.