Linear and cyclic dipeptides with antimalarial activity

Article abstract of DOI:10.1016/j.bmcl.2012.09.094

Several natural and synthetic polypeptides possess important antimalarial activity. Shorter peptides with potent antimalarial activity have also been described, among them linear di-, tri-, tetra- and pentapeptides and their cyclic analogs. In an attempt

Full text of DOI:10.1016/j.bmcl.2012.09.094

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7048–7051
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Linear and cyclic dipeptides with antimalarial activity
Lemuel Pérez-Picaso a,b, Horacio F. Olivo c, Rocío Argotte-Ramos d,
María Rodríguez-Gutiérrez d, María Yolanda Rios a,
⇑
a Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Col. Chamilpa, Cuernavaca, Morelos 62209, Mexico
b Universidad del Papaloapan, Campus Tuxtepec, Circuito Central 200, Col. Parque Industrial, Tuxtepec, Oaxaca 68301, Mexico
c Medicinal and Natural Products Chemistry, The University of Iowa, Iowa City, IA 52242, USA
d Centro de Investigación sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Avenida Universidad 655, Col. Santa María Ahuacatitlán, Cuernavaca, Morelos
62508, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
Several natural and synthetic polypeptides possess important antimalarial activity. Shorter peptides with
potent antimalarial activity have also been described, among them linear di-, tri-, tetra- and pentapep-
tides and their cyclic analogs. In an attempt to find dipeptides with antimalarial activities we show that
linear and cyclic dipeptides, the latter known as diketopiperazines, still retain the fundamental core to
preserve antimalarial activity. Thirteen linear dipeptides and ten diketopiperazines were investigated.
Received 24 August 2012
Revised 20 September 2012
Accepted 25 September 2012
Available online 2 October 2012
Eight linear dipeptides showed IC50 values between 2.78 and 7.07 lM, while eight diketopiperazines
Keywords:
Dipeptides
Diketopiperazines
Malaria
were also active with IC50 values between 2.26 and 4.26
l
M on Plasmodium berghei schizont cultures.
Ó 2012 Elsevier Ltd. All rights reserved.
Plasmodium berghei schizonts
Malaria is a parasitic disease expanding quickly around the
world. An estimated 3.3 billion people were at risk of malaria in
2010 and about 216 million episodes of malaria occurred that year.
About 655 thousand malaria deaths occurred in 2010, with 86% of
them being children under five years of age.1 In humans, malaria is
caused by protozoan parasites belonging to five Plasmodium
species: Plasmodium falciparum, Plasmodium vivax, Plasmodium
malariae, Plasmodium ovale and Plasmodium knowlesi. The
most serious infections among these species are caused by
P. falciparum.2,3 Uncomplicated P. falciparum malaria should be
treated with an artemisinin-based combination therapy. P. vivax
malaria should be treated with chloroquine or with artemisinin-
based combination therapy when P. vivax resistance to chloroquine
has been documented.1 In recent years, the social impact of malaria
has increased due to the lower abundance and high cost of
artemisinin and related products, and also because of the
emergence of resistant strains of P. falciparum and P. vivax to
chloroquine, mefloquine and pyrimethamine.4 The number of
artemisinin-based combination treatment courses procured by
the public sector increased greatly from 11.2 million in 2005 to
76 million in 2006, and reached from 181 million in 2010 to 287
million treatment courses in 2011. International funding for
malaria control has continued to rise, to a peak of $2 billion USD
in 2011.1 For these reasons, there is an increasing need for new
chemical pharmacophores which may prove effective therapeutic
antimalarial agents.
Several antimalarial peptides have been isolated from natural
sources and also obtained synthetically.3,5–15 Linear antimalarial
peptides with fifteen or more amino acid residues, such as leuki-
nostatin A, efrapeptin, zervamicin and antiamoebin have been iso-
lated. These fungal peptides kill P. falciparum in culture with IC50 in
the micromolar range.16 Also shorter peptides with similar range of
potency have been described, among them linear di-, tri-, tetra-
and pentapeptides and their cyclic analogs.17–22
Diketopiperazines are a class of cyclic dipeptides found in nat-
ure showing all advantages of cyclic peptides.23 These compounds
show reduction in conformational freedom and extra conforma-
tional restrictions, resulting in a wide spectrum of therapeutic
activities such as antibacterial and antifungal,24 antitumoral,25
antiviral,26 etc. Structurally, diketopiperazines have two accep-
tor–donator hydrogen bridge places, making it possible to have
higher receptor binding affinities, showing high potential as
specific targets to inhibit or activate binding sites of enzymes
and proteins.27 Some diketopiperazines have been described as
antimalarial agent, such as 1-demethylhyalodendrin tetrasulfide
(IC50 2.5 l
g/ml)28 and brevicompanine B (IC50 35 mg/ml) (Fig. 1).29
Two strategies for linear and cyclic peptides syntheses have
been developed: solution and solid phase peptide syntheses. Both
strategies needed protection, activation, coupling and deprotection
reactions as key steps. Today, solid phase peptide synthesis is the
best strategy for production of long peptides; however, linear
⇑
Corresponding author. Tel.: +52 777 329 7000/6024; fax: +52 777 329 7997.
E-mail address: myolanda@uaem.mx (M.Y. Rios).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.094

L. Pérez-Picaso et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7048–7051
7049
R2
N
R1
O
CH3
O
H
R4
N
S4
N
H
O
BocHN
O
NH
R3
O
N
OH
O
N
H
H
O
1 R1=(S)-Pr-NHCbz; R2=H; R3=Bn; R4=tBu
2 R1=(S)-Pr-NHCbz; R2=H; R3=Bn; R4=Me
1-demethylhyalodendrin tetrasulfide
brevicompanine B
3 R1=(S)-Pr-NHCbz; R2=H; R3=iPr; R4=tBu
4 R1=(R)-Pr-NHCbz; R2=H; R3=iPr; R4=Me
5 R1=(R)-Pr-NHCbz; R2=H; R3=iPr; R4=tBu
Figure 1. Antimalarial diketopiperazines 1-demethylhyalodendrin tetrasulfide and
brevicompanine B.
6 R1=R3=(S)-Bn; R2=H; R4=tBu
7 R1=(S)-Bn; R2=H; R3=iPr; R4=tBu
8 R1=(S)-Bn; R2=CH3; R3=H; R4=Me
9 R1=(S)-Bn; R2=CH3; R3=H; R4=tBu
10 R1=R3=(S)-iPr; R2=H; R4=tBu
11 R1=(S)-iPr; R2=H; R3=Bn; R4=tBu
12 R1=R2=H; R3=iPr; R4=tBu
dipeptides can be obtained using solution synthesis with excellent
yields. On the other hand, the use of suitable reagents to carry out
an effective activation of the C-terminus of the protected peptide
and of suitable nucleophilic amines make the solution peptide syn-
thesis also more convenient and quicker than solid phase peptide
synthesis for production of cyclic peptides.30
13 R1=R2=H; R3=Bn; R4=tBu
Cyclization of dipeptides to diketopiperazines implies a trans-cis
isomerization of the linear precursor, followed by nucleophilic at-
tack of an amino group on the C-terminal carbonyl group.31,32
Specifically, 2,5-diketopiperazine synthesis in solution could be
carry out using five main strategies: intramolecular formation of
N1–C2, intramolecular formation of N1–C6, tandem formation of
N1–C2/C3–N4, tandem formation of N1–C2/N4–C5 and tandem
formation of C2–N1–C6, being the first one the most commonly
employed method.33
R2
R3
N
O
R1
O
N
H
14 R1=(S)-Pr-NHCbz; R2=H; R3=Bn
15 R1=(S)-Pr-NHCbz; R2=H; R3=iPr
16 R1=(R)-Pr-NHCbz; R2=H; R3=iPr
17 R1=(S)-Bu-NHCbz; R2=H; R3=iPr
18 R1=R3=(S)-Bn; R2=H
In an attempt to find ultra-short peptides with antimalarial
activities, we have previously shown that tetrapeptides with the
sequence Xdd-Xcc-Phe-Orn have antiplasmodial activities against
19 R1=(S)-iPr; R2=H; R3=Bn
20 R1=(S)-Bn; R2=CH3; R3=H
21 R1=R3=(S)-iPr; R2=H
22 R1=R2=H; R3=Bn
23 R1=R2=H; R3=iPr
Plasmodium berguei (IC50 ranging between 2.5 and 3.3 l
M).34 In
this Letter, we report the antiplasmodial activities of protected lin-
ear dipeptides 1–13 and their cyclic analogs, 2,5-diketopiperazines
14–23, which were synthesized using intramolecular formation of
N1–C2 strategy (Fig. 2).
Figure 3. Linear dipeptides 1–13 and diketopiperazines 14–23.
synthesized35 was proved unambiguously using a single crystal
X-ray structure for the first time (colorless crystals, Fig. 4).38
Plasmodium berghei is a unicellular parasite that does not affect
humans, therefore is commonly used as a model to look for
antimalarial activity.39 We used sarcosine (Sar) and ornithine
(Orn) to investigate the effect of non-proteinogenic residues,
phenylalanine and glycine for the aromatic effects, and lysine
and valine for the aliphatic/hydrophobic effects of linear dipeptides
and diketopiperazines on antimalarial activity. The chloroquine-
sensitive strain ANKA 2.34 of P. berghei was used to test the
antimalarial activity of dipeptides 1–23. Table 1 shows IC50 values
of peptides 1–13 and diketopiperazines 14–23 on P. berghei
schizont cultures.40
We have previously reported the syntheses of dipeptides 1–3
and 5–13 and of diketopiperazines 14–16 and 18–23 (Fig. 3) and
unambiguously assigned their NMR data.35 Dipeptide 4 and
diketopiperazine 17 were synthesized in aqueous solution using
microwave-assisted methodology.35
The chemical structures for the newly synthesized dipeptide 4
and diketopiperazine 17 were confirmed on the basis of their spec-
tral IR and NMR data.36,37 Dipeptide 4 showed IR absorptions at
3337 (NH-amide stretching), 1709 (CO carbamate and ester) and
1675 (CO amide) cmꢀ1; ester and amide carbonyls and Cbz and
Boc carbamate groups showed 13C NMR resonance signals at d
172.48, 172.33, 156.82 and 155.88, respectively. Valine residue
was evidenced by methine and methyl groups signals at dH 4.49
(H
fragment showed signals at dH 4.29 (H
(Hb and H ). Diketopiperazine 17 showed IR absorptions at 3347
a), 2.13 (Hb) and 0.92 and 0.88 (H
c
) at 1H NMR, while ornithine
), 3.19 (Hd) and 1.91–1.50
The linear dipeptides possessing the non-proteinogenic
Boc-Orn(Z) fragment at the N-terminal residue (compounds 1
a
c
and 2) were found inactive to P. berghei (IC50 >200
lM). Compound
(NH stretching), 1676 (CO amide), 1530 (trans-NH in plane), 1454
(cis-NH in plane) and 1344 (CN stretching amide) cmꢀ1. Carbonyl
amides and Nd-Cbz-carbamate groups showed 13C NMR resonance
signals at d 170.89, 170.60 and 159.80. The 1H NMR spectrum for
3 showed moderate antimalarial activity with an IC50 = 33.88
however its analogues Boc-D-Orn(Z) compounds 4 and 5 were
the most active dipeptides of this series with an IC50 = 3.05 and
2.78 lM, respectively. On the other hand, those series possessing
the non-proteinogenic residue sarcosine, dipeptides 8 and 9 and
diketopiperazine 20 showed also very good antimalarial activity
l
M,
compound 17 showed Ha-valine signal at dH 3.82 and Ha-lysine
at dH 3.96. The structure of cyclic peptide 20 previously
with an IC50 = 3.63, 3.38 and 2.26 lM, respectively.
Four dipeptides with one aromatic amino acid (Phe derivatives)
at the N-terminal residue were evaluated (compounds 6–9). The
first dipeptide corresponds to the replacement of Boc-Orn(Z) frag-
ment on compound 1 by Boc-Phe residue, leading to the inactive
dipeptide 6. The second dipeptide corresponds to the same ex-
change in compound 3 to yield dipeptide 7, which shows very good
R2
N
R2
N
R3
R3
O
O
H2O
R1
R1
MW
O
NH
Boc R4
O
N
H
250 oC, 250 W, 150 psi
activity against P. berghei with an IC50 = 3.23 lM. In same series,
introduction of Sar amino acid as initial residue yielded active
Figure 2. Intramolecular N1–C2 strategy for 2,5-diketopiperazines synthesis.
dipeptides 8 and 9 (vide supra).

7050
L. Pérez-Picaso et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7048–7051
proteinogenic amino acids such as Orn and Sar, are excellent resi-
dues to ensure antimalarial activity (compounds 4, 5, 7–10 and 12).
Dipeptide 7 showed excellent activity with an IC50 = 3.23 M, how-
ever the inverse sequence dipeptide 11 has approximately half the
l
activity of compound 7 with an IC50 = 7.07 lM. Finally, the nature
of the protecting group at the initial amino acid residue (OtBu or
OMe) seems not to be significant on the activity.
Diketopiperazines 14–23 were efficiently prepared in aqueous
solution using microwave-assisted methodology as previously de-
scribed.35 In this manner, cyclization of dipeptides 1 (or 2) and 3
afforded the inactive diketopiperazines 14 and 15, respectively.
On the other hand, dipeptides 4 (or 5), 7 (or 11), 8 (or 9), 10 and
12 yielded diketopiperazines 16, 19, 20, 21 and 23, respectively,
all of them showing practically the same activity than their
respective open chain dipeptides with an IC50 = 3.09, 3.89, 2.26,
2.45 and 3.23
rendered diketopiperazines 18 and 22, both increasing the activity
largely with an IC50 = 2.54 and 4.26 M, respectively. Finally,
diketopiperazine 17 also showed important activity against
P. berghei schizont with an IC50 = 3.12 M (Table 1).
lM, respectively. Additionally, dipeptides 6 and 13
l
l
In both cases, linear dipeptides and diketopiperazines, the
change of configuration of the Orn residue (L to D, compounds
3–5 and 15–16) dramatically increased antimalarial activity.
The ability to suppress the development of P. berghei schizonts
in culture of dipeptides 1–23 (chemosuppression) after 16 h of
exposure are expressed as percentages in Table S1.48 These data
is in agreement with the IC50 value obtained for each compound,
being 8, 9, 17, 19, 20 and 23 the most effective compounds reduc-
ing the schizont number.
In conclusion, twenty three compounds were screened to assess
their in vitro antiprotozoal activity. Linear dipeptides 4–5 and 7–12
and their corresponding cyclic derivatives diketopiperazines 16–23
are active compounds against P. berghei schizont cultures. To our
knowledge, these dipeptides are the simplest diketopiperazines
described until today with only two stereogenic centers including
the pharmacophore possessing antimalarial activity. These
peptides are candidates for subsequent in vitro assays against
P. falciparum and P. vivax and for antimalarial in vivo assays.
Figure 4. ORTEP drawing of diketopiperazine 20.
Table 1
IC50
(l
M) values of dipeptides 1–23 on P. berghei schizonts
Compound (200
lM)
IC50 (lM)
Boc-Orn(Z)-Phe-OtBu (1)35
Boc-Orn(Z)-Phe-OMe (2)35
Boc-Orn(Z)-Val-OtBu (3)35
Boc-D-Orn(Z)-Val-OMe (4)36
Boc-D-Orn(Z)-Val-OtBu (5)35
Boc-Phe–Phe-OtBu (6)41
Boc-Phe-Val-OtBu (7)35
Boc-Phe-Sar-OMe (8)35
Boc-Phe-Sar-OtBu (9)35
Boc-Val-Val-OtBu (10)35
Boc-Val-Phe-OtBu (11)35
Boc-Gly-Val-OtBu (12)35
Boc-Gly-Phe-OtBu (13)35
Cyclo[Phe-Orn(Z)] (14)35
Cyclo[Val-Orn(Z)] (15)35
Cyclo[Val-D-Orn(Z)] (16)35
Cyclo[Val-Lys(Z)] (17)37
Cyclo(Phe–Phe) (18)42
Cyclo(Val-Phe) (19)43,44
Cyclo(Sar-Phe) (20)35
>200
>200
33.88
3.05
2.78
112.20
3.23
3.63
3.38
2.81
7.07
Acknowledgments
This work was financially supported by CONACyT (Grant num-
ber 79584-Q). M.Y.R. thanks CONACyT for a sabbatical fellowship
(Grant number 178520). We are grateful to Enrique Salazar-Leyva,
Perla P. Bravo, Diana G. Vargas, Victoria Labastida and María
Medina-Pastor for technical assistance.
3.16
164.05
123.02
66.02
3.09
3.12
2.54
3.89
2.26
2.45
4.26
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.09.
094. These data include MOL files and InChiKeys of the most
important compounds described in this article.
Cyclo(Val-Val) (21)35
Cyclo(Phe-Gly) (22)45
Cyclo(Val-Gly) (23)46,47
Chloroquine
3.23
0.06
References and notes
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According to IC50 values shown in Table 1, linear protected
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inactive (compounds 1, 2, 6 and 13) or only moderately active
(compound 11). Hydrophobic amino acid such as Val, and non-

L. Pérez-Picaso et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7048–7051
7051
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172.33 (s, CO-Orn), 156.82 (s, CO-Cbz), 155.88 (s, CO-Boc), 136.69 (s, CAr),
128.49 (d, CAr), 128.06 (d, CAr), 80.09 (s, Boc), 66.55 (t, CH2-Cbz), 57.21 (d, C
Val), 53.75 (d, C -Orn), 52.15 (q, OMe), 40.23 (t, Cd-Orn), 31.08 (d, Cb-Val),
29.96 (t, C -Orn), 28.35 (q, CH3-Boc), 26.19 (t, Cb-Orn), 19.04 (q, C -Val), 17.82
(q, C
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(100) [C7H8]+, 72 (22), 57 (26). HRMS (FAB+): m/z 480.2759 (Calcd for
a-
a
c
c
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3206, 3093, 3058, 2962, 2936, 2874, 1676, 1530, 1454, 1344, 1251, 1139, 1045,
836, 739, 697. 1H NMR (400 MHz, CD3OD): d 7.47–7.28 (5H, m, HAr), 5.06 (2H, s,
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a-Lys), 3.82 (1H, dd, J = 4.0,1.2 Hz, Ha-Val), 3.13 (2H, t,
J = 6.8 Hz, H -Lys), 2.26 (1H, m, Hb-Val), 1.83 (2H, m, Hb-Lys), 1.53 (2H, m, Hd-
e
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CO-Val), 159.8 (s, CO-Cbz), 138.8 (s, CAr), 129.62 (d, CAr), 129.10 (d, CAr), 128.94
(d, CAr), 67.48 (t, CH2-Cbz), 61.51 (d, C
a-Val), 55.97 (d, C
a-Lys), 41.60 (t, C
e-
Lys), 35.43 (t, Cb-Lys), 33.51 (d, Cb-Val), 30.65 (t, Cd-Lys), 23.54 (C
c
-Lys), 19.40
(q, Cc
-Val), 17.78 (q, Cc0-Val). FAB+MS m/z: 384 (78) [M+Na]+, 362 (100)
[M+H]+, 338 (21), 318 (42), 254 (55). HRMS (FAB+): m/z 362.2106 [M+H]+
(Calcd for C19H27N3O4 362.2080).
38. CCDC 863172 contains the supplementary crystallographic data for this paper.
These data can be obtained free of charge from The Cambridge Crystallographic
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structure for compound 20 shows the aromatic ring folded on diketopiperazine
ring, in a boat conformation, with an important interaction of diketopiperazine
and aromatic rings, probably due to a dipole-dipole interaction between both
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amide groups and the
was observed also in solution at 1H NMR, where only one of the H
methylene protons was observed upfield [d 3.48 (H
) vs 2.79 (Ha0)].
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40. Chloroquine-sensitive cultured plasmodium berghei schizonts (ANKA 2.34)
were used to assess the antimalarial activity of compounds 1–23. Peptides 1–
23 were dissolved in PBS containing DMSO 0.1% and 5% ethanol. Parasites were
incubated for 16 h at 37 °C. Chloroquine (C6628 sigma chloroquine
diphosphate salt solid, P98% purity) was used as positive control. Samples
of 0.5 mL of each culture were taken to prepare smears and used to count
numbers of schizonts in 2000 erythrocytes. The IC50 values of each compound
p
electrons of the aromatic ring. Evidence of this folding
a
sarcosine
a
30. Katsara, M.; Tselios, T.; Deraos, S.; Deraos, G.; Matsoukas, M. T.; Lazoura, E.;
Matsoukas, J.; Apostolopoulos, V. Curr. Med. Chem. 2006, 13, 2221.
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were determined using concentrations of 5, 50, 100, and 200 lM and by
extrapolation from the concentration response curve. The IC50 value
represents the drug concentration producing a 50% reduction in the number
of P. berghei schizonts (compared to drug-free control cultures). For each
assay, each drug dilution was analyzed in duplicate, and the results were
averaged in each case.
32. Davies, J. S. J. Pept. Sci. 2003, 9, 471.
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36. Boc-D-Orn(Cbz)-Val-OMe (4). 88% yield; Colorless oil. IR (KBr, cmꢀ1): 3337,
2969, 2881, 1709, 1529, 1456, 1369, 1255, 1167, 1020, 865, 743, 699, 608. 1H
NMR (400 MHz, CDCl3): d 7.32–7.29 (5H, m, HAr), 7.16 (1H, br s, NH-Val), 5.51
(1H, br s, NH–Boc), 5.43 (1H, br s, NH-Cbz), 5.10 and 5.09 (1H each, d,
41. Dineen, T. A.; Zajac, M. A.; Myers, A. G. J. Am. Chem. Soc. 2006, 128, 16406.
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48. see Supplementary data.
J = 14.2 Hz, CH2-Cbz), 4.49 (1H, dd, J = 8.1,5.1 Hz, H
Orn), 3.69 (3H, s, OMe), 3.19 (2H, m, Hd-Orn), 2.13 (1H, m, Hb-Val), 1.91–1.50
(4H, m, Hb,H -Orn), 1.42 (9H, s, CH3-Boc), 0.92 (3H, d, J = 7.2 Hz, H -Val), 0.88
a-Val), 4.29 (1H, m, Ha-
c
c