Plasmepsin II inhibition and antiplasmodial activity of Primaquine-Statine 'double-drugs'

Article abstract of DOI:10.1016/j.bmcl.2004.03.030

Statine-based inhibitors of Plasmepsin II (PLMII) coupled with Primaquine have been designed using the 'double-drug' approach. The IC₅₀ values for PLMII inhibition ranged from 0.59 to 400nM and the best IC₅₀ value for inhibition of P

Full text of DOI:10.1016/j.bmcl.2004.03.030

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2931–2934
Plasmepsin II inhibition and antiplasmodial activity
of Primaquine–Statine ‘double-drugs’
Sergio Romeo,a,* Mario Dell’Agli,b Silvia Parapini,c Luca Rizzi,a Germana Galli,b
Monica Mondani,c Anna Sparatore,a Donatella Taramellic and Enrica Bosisiob
aInstitute of Medicinal Chemistry, University of Milan, Viale Abruzzi 42, 20131 Milan, Italy
bDepartment of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy
cInstitute of Microbiology, University of Milan, Via Pascal 36, 20133 Milan, Italy
Received 7 January 2004; revised 11 March 2004; accepted 12 March 2004
Abstract—Statine-based inhibitors of Plasmepsin II (PLMII) coupled with Primaquine have been designed using the ‘double-drug’
approach. The IC50 valuesfor PLMII inhibition ranged from 0.59 to 400 nM and the best IC
value for inhibition of Plasmodium
50
falciparum growth in vitro was0.4 lM, which represent a remarkable improvement compared to other statine-based PLMII
inhibitors.
Ó 2004 Elsevier Ltd. All rights reserved.
Malaria is one of the largest parasitic infections in the
world and the incidence of malaria isetsimated to be
300–500 million clinical cases each year, with a mortality
rate of 1.1–2.7 million people, of whom about 1 million
are children under the age of 5 years.1 Drug resistance
has spread and intensified over the last 15–20 years,
leading to a dramatic decline in the efficacy of the most
affordable antimalarial drugs; therefore, there is an
urgent need of new antimalarial therapy.
peptidic character of these molecules is often the cause
of their failure in the cell based assays.
The ‘double-drug’ approach has been successfully used to
improve poor membrane permeation of some HIV pro-
tease inhibitors.4 According to thisapproach a protease
inhibitor unable to permeate cell membrane hasbeen
5
joined to AZT by a cleavable linker, asfor KNI 694,
with a considerable boost in potency in the cell assay.
Plasmepsin II (PLM II) is one of the aspartic proteases
involved in the degradation of haemoglobin during the
intraerythrocytic cycle of Plasmodium falciparum.2 The
inhibition of the enzyme, leading to starvation of the
parasite, is considered a valid drug target. Several PLM
II inhibitors, including molecules with a statine-based
core, have been developed. They possess low Ki values
against the enzyme (nM order), but their effectiveness in
killing the parasite is limited (IC50 range 10–20 lM).3
Differencesof thiskind may be attributed to low bio-
availability, probably because most of these inhibitors
derive from peptides by replacement of the scissile
peptide bond with a transition state analogue.3 The
We report here the synthesis and the inhibition of PLM
II and P. falciparum in vitro of molecules designed based
on the ‘double-drug’ approach, in which a statine-based
inhibitor of PLM II hasbeen linked to the well known
antimalarial Primaquine (PQ).
O
N
HN
NH2
Primaquine
OH
O
O
H
N
O
N
H
N
H
Keywords: P. falciparum; Plasmepsin II; Statine; Primaquine; Double
drug; Chimera drug; In vitro inhibition.
O
R
PS 777621; R=i-Butyl; Ki=180nM (Plasmepsin II)
PS 189863; R=Benzyl; K =110nM (Plasmepsin II)
* Corresponding author. Tel.: +39-02-50317568; fax: +39-02-503175-
65; e-mail: sergio.romeo@unimi.it
i
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.030

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S. Romeo et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2931–2934
R
O
1
2
O
O
O
O
O
O
O
O
OH
N
H
N
H
N
HN
3
4
N
H
N
H
N
H
R
N
H
O
O
O
O
NH
O
2
O
O
O
5
O
Several statine-based inhibitors, such as PS777621 and
PS189863, have been developed.6 These compounds
showed inhibition of PLM II at nanomolar concentra-
tions, but the IC50 valuesagainst P. falciparum growth
in vitro were approximately 10 lM.6
compounds 1–5 that incorporate linkersderived from
dicarboxylic acidsthat hs ould mimic the P
PS777621.
residue of
3
Compounds 1–5 were prepared starting from Boc-
Sta(3S,4S)OH by coupling with butylamine, followed by
removal of the Boc protecting group and coupling with
BocIleOH. The dicarboxylic linkerswere introduced as
monobenzylesters using standard HBTU coupling after
Boc deprotection (Scheme 1). Benzylesters 6–10 and
CbzLeuLys(Boc)Primaquine8 were hydrogenated and
the crude material wascoupled using HBTU/HOBt. Boc
removal by TFA treatment followed by precipitation of
the phosphate salt afforded final products 1–5 (Scheme
1).
Primaquine ishighly active againts the gametocytesof
all malaria species infecting humans and against hypno-
zoitesof the relapisng malaria cauesd by
P. vivax and
P. ovale, leading to the eradication of the infection.
Concern about toxicity of PQ haslimited itsues, but
recently PQ hasbeen revaluated asa safe and effective
agent for the prophylaxisof malaria due to both P. fal-
ciparum and P. vivax.7 It wasshown that the toxicity of
PQ isdiminihsed and the activity isincreaesd by the
conversion of PQ to peptide prodrugs, that is HVal-Leu-
Lys-PQ, which, following cleavage by plasmin, releases
the Lys-PQ fragment.8
Compounds 1–5 were evaluated on PLMII9;10 and
human red blood cellsinfected with both chloroquine-
sensitive (D10) and chloroquine-resistant (W2) strains of
P. falciparum.11;12 All compoundsinhibited PLM II and
parasite growth in a dose-dependent manner and the
IC50 valuesare reported in Table 1.
We reasoned that the linkage of the peptide derivative
HLeuLysPQ to the Ile residue of PS777621 would pro-
vide a potential ‘double-drug’. We have thussynthesized
OH
O
BocHN
O
OH
OH
OH
O
H
N
H
N
a,b,c
b,d
BocHN
N
H
BnO
HN
N
H
R
O
O
6 -10
O
OH
O
e, f ,g
O
N
H
N
H
N
HN
N
H
N
H
N
H
N
H
R
O
O
2H PO
3
4
1 - 5
NH
2
Scheme 1. Reagentsand conditions: (a) C 4H9NH2, HBTU, HOBt; (b) HCl, dioxane; (c) BocIleOH, HBTU, HOBt; (d) BnOROH, HBTU, HOBt;
(e) H2, Pd–C; (f) H-Leu-Lys(Boc)-PQ, HBTU, HOBt; (g) TFA, H3PO4.

S. Romeo et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2931–2934
2933
Table 1
O
O
O
OH
N
H
N
H
N
HN
N
H
N
H
N
H
R
N
H
O
O
NH
2
R
PLM II IC50 SD (nM)
Plasmodium falciparum IC50 SD (lM)
D10
W2
O
1
2
3
396 25
135 4.4
1.51 0.23
20% at 9 lM
5.5 1.8
40% at 9 lM
4.2 0.2
O
O
O
O
6.2 0.8
4.7 0.3
O
O
O
4
5
0.59 0.02
9.6 2.1
0.4 0.1
3.3 0.3
0.7 0.4
1.0 0.5
O
O
O
O
O
Compound 1 with the linker derived from succinic acid
was the least active of the series in both assays, in
agreement with the literature that indicatesan aromatic
substituent as preferred in P3 for PLMII inhibition.6 The
introduction of an aromatic ring in compound 2 caused
an increase in potency, as expected. Compound 3 with a
naphthyl linker wasconisderably more active in the
enzyme assay, but the activity against the parasite did
not improve. Compound 4 derived from 4,40-oxy-
bis(benzoic acid) was remarkably active in inhibiting
both the enzyme and the parasite growth with IC50
valuesof 0.59 nM and 0.4 lM, respectively. These IC50
values are substantially superior compared to the IC50 of
3. In order to ameliorate the activity of the naphthyl
linker, by adding more conformational freedom and
increasing the distance between PQ and statine, com-
pound 5, with an alkyl chain and an aromatic moiety
joined by an ester bond, was synthesized. This com-
pound was less active against the enzyme, but slightly
more active against P. falciparum compared to the
naphthyl derivative 3.
To verify the influence of PQ in the in vitro assay, and
the possibility that the LysLeu peptide bond of com-
pound 4 could undergo hydrolysis by plasmin, the
peptide HLysPQ 11 and the carboxylic acid 12 were
prepared.
Compound 11 was found substantially inactive both
against PLM II and P. falciparum: (IC50 > 1 lM and
approx. 20 lM, respectively). Compound 12, compared
to 4, showed a drastic reduction in inhibition both of
PLM II (IC50: 123 nM) and of parasite growth (IC50
approx. 17 lM) indicating that compound 4 isactive in
the cell assay without being cleaved. In conclusion, these
novel PQ–Statine ‘double-drugs’ have achieved, in
comparison with other peptidomimetic inhibitors,3;6
a
significant improvement in the inhibition of both of
PLM II activity and P. falciparum growth in vitro,
including drug-resistant strains. Moreover, a direct
correlation wasfound between the inhibition of PLM II
and the antiplasmodial activity, suggesting that PQ–
Statine ‘double-drugs’ kill the parasites mainly by
NH
2
O
H
N
O
O
H
N
H
N
H
N
HN
NH
2
N
H
HO
N
O
OH
O
O
O
O
11
12

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S. Romeo et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2931–2934
3. Boss, C.; Richard-Bildstein, S.; Weller, T.; Fischli, W.;
Meyer, S.; Binkert, C. Curr. Med. Chem. 2003, 10, 883–
907.
4. Matsumoto, H.; Kimura, T.; Hamawaki, T.; Kumagai, A.;
Goto, T.; Sano, K.; Hayashi, Y.; Kiso, Y. Bioorg. Med.
Chem. 2001, 9, 1589–1600.
inhibiting PLM II and consequently the digestion of
haemoglobin, that is essential for the survival of P. fal-
ciparum.
5. Matsumoto, H.; Matsuda, T.; Nakata, S.; Mitoguchi, T.;
Kimura, T.; Hayashi, Y.; Kiso, Y. Bioorg. Med. Chem.
2001, 9, 417–430.
Acknowledgements
6. Carroll, C. D.; Patel, H.; Johnson, T. O.; Guo, T.;
Orlowski, M.; He, Z. M.; Cavallaro, C. L.; Guo, J.;
Oksman, A.; Gluzman, I. Y.; Connelly, J.; Chelsky, D.;
Goldberg, D. E.; Dolle, R. E. Bioorg. Med. Chem. Lett.
1998, 8, 2315–2320.
7. Fryauff, D. J.; Baird, J. K.; Basri, H.; Sumawinata, I.;
Purnomo; Richie, T. L.; Ohrt, C. K.; Mouzin, E.; Church,
C. J.; Richards, A. L.; Subianto, B.; Sandjaja, B.; Wignall,
F. S.; Hoffman, S. L. Lancet 1995, 346, 1190–1193.
8. Philip, A.; Kepler, J. A.; Johnson, B. H.; Carroll, F. I.
J. Med. Chem. 1988, 31, 870–874.
We would like to thank undergraduate students, Ric-
cardo Villa, Daniele De Zani, Federico Secchi and
Valentino Mandelli for helpfully collaborating to this
project. The pro-plasmespin II cDNA was a kind gift of
Dr. Berry, Cardiff University. This work was supported
by the Ministry of University and Research (MIUR)
PRIN 2001–2003 and the University of Milan FIRST
2002 and FIRST 2003.
9. Hill, J.; Tyas, L.; Phylip, L. H.; Kay, J.; Dunn, B. M.;
Berry, C. FEBS Lett. 1994, 352, 155–158.
10. Dell’Agli, M.; Parapini, S.; Basilico, N.; Verotta, L.;
Taramelli, D.; Berry, C.; Bosisio, E. Planta Med. 2003, 69,
162–164.
References and notes
1. World Health Organization WHO Expert Committee on
Malaria: Twentieth Report: Geneva, 2000; Vol. 892.
2. Banerjee, R.; Liu, J.; Beatty, W.; Pelosof, L.; Klemba, M.;
Goldberg, D. E. Proc. Natl. Acad. Sci. U.S.A. 2002, 99,
990–995.
11. Makler, M. T.; Ries, J. M.; Williams, J. A.; Bancroft, J. E.;
Piper, R. C.; Gibbins, B. L.; Hinrichs, D. J. Am. J. Trop.
Med. Hyg. 1993, 48, 739–741.
12. Monti, D.; Basilico, N.; Parapini, S.; Pasini, E.; Olliaro,
P.; Taramelli, D. FEBS Lett. 2002, 522, 3–5.