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Malaria
Organic Compounds to Combat Neglected Tropical Diseases 181
N
Cl
Cl
N
HN
Cl
N
O
O
R
2
R
1
N
HN
Cl
N
O
R
2
R
1
N
NH
S
NH
2
H
2
NNH
NH
2
S
N
N
Cl
H
N
N
N
Cl
N
HH
N
H
R
2
R
1
O
X
N
N
Cl
N
N
O
X
R
2
R
1
23, 68-8 9%
24, 68-89%
CH
2
O
1
25
26 X= O, 24-94%
27 X=NNHC(S)NH
2
, 51-86%
(i)
(ii)
Reagents and conditions: (i) EtOH, 45 ºC, 3 h; (ii) EtOH, rt, 3-4 h.
(ii)
3 steps
Scheme 5: Synthesis route of quinoline–ethylene isatin derivatives [14].
3. SYNTHESIS OF INHIBITORS OF A CYSTEINE PROTEASE
(FALCIPAIN)
Cysteine proteases are proteolytic enzymes, which contain a catalytic cysteine
residue in their active site. The cysteine residue is responsible for the hydrolysis
of peptide bonds as shown in Fig. 8 [15, 16].
S
-
O
NH
2
N
+
HN
H
NHR'O
R
NHR'H
S R
O
O
H
H
H
2
O
H
2
NR'
Cys
Gln
His
S
O
NH
2
N
HN
Cys
Gln
His
R
O
O
NH
2
N
HN
Cys
Gln
His
Figure 8: Schematic representation of the active site of a cysteine protease, illustrating the
proposed mechanism for the enzyme-catalysed hydrolysis of peptide bonds [16].
As mentioned in section 2.2, falcipain is a P. falicparum cysteine protease
responsible for hemoglobin degradation within the parasite food vacuole during
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182 Organic Compounds to Combat Neglected Tropical Diseases de Oliveira et al.
erythrocyte infection and has been validated as promising drug target. Plasmodium
parasites are highly dependent on hemoglobin hydrolysis to s upply the amino acids
for protein synthesis and to maintain the osmotic stability [17, 18].
To date, four subtypes of falcipain have been identified, falcipain-1, falcipain-2,
falcipain-2’ and falcipain-3. Falcipain-2 and falcipain-3 are the major
hemoglobinases of P. falciparum [19].
Considerable efforts have been made to find inhibitors of falcipain-2 for use as
therapeutic agents in treatment of malaria, leading to the discovery of different
classes of inhibitors that include peptides, peptidomimetics, chalcones,
isoquinolines, and thiosemicarbazones.
3.1. Peptides
Since falcipain is a proteolytic enzyme, most inhibitors were designed from
peptide lead compound. Typically, these inhibitors possess a peptide chain having
an electrophilic α,β-unsaturated moiety, such as vinyl ketone, vinyl sulfones, vinyl
amide, vinyl ester and vinyl nitrile, acting as a Michael acceptor for the
nucleophilic sulfhydryl groups of catalytic cysteine residue [20]. Representative
examples of this class of inhibitors are shown in Fig. 9.
Figure 9: Examples of peptide inhibitors of falcipain.
The vinyl sulfone group has found wide use in the design of irreversible cysteine
protease inhibitors, with the advantage of being less reactive against other
proteases such as serine proteases [20].
In general, peptidyl vinyl sulfones and derivatives can be synthesized using an
amino acid aldehyde as building block as shown in Scheme 6. Commercially
Malaria
Organic Compounds to Combat Neglected Tropical Diseases 183
available Boc-protected amino acid derivatives 29 are converted, in two steps,
into amino acid aldehyde by formation of N,O-dimethylhydroxamate derivative
30, followed by reduction with LiAlH
4
. Horner–Wadsworth–Emmons reaction of
the corresponding aldehydes 31 with an alkyl-sulfonylmethylphosphonate,
afforded the vinyl sulfones 32 in 65-87% yield. Completion of the synthesis
involves removal of the N-Boc protecting group and subsequent coupling of the
resulting vinyl sulfone with different N-protected amino acids or peptide
derivatives, functionalized or not, using common peptide coupling reagents like 1-
hydroxybenzotriazole (HOBt) and 1-ethyl-3-(3-dime thy lam inopropyl)carbodiimide)
hydrochloride (EDC) (Scheme 6) [21-23].
In order to identify more effective peptide-based falcipain inhibitors, Shenai et al.
prepared different vinyl sulfones derivatives by replacing the alkyl substituent in
the sulfonyl phosphonate moiety, resulting in the formation of phenyl vinyl
sulfones 34, vinyl sulfonate esters 35 and vinyl sulfonamides 36 (Scheme 7). The
phenyl vinyl sulfones 34 were synthesized according to the route depicted in
Scheme 6. Vinyl sulfonate esters 35 and vinyl sulfonamides 36 were prepared via
the vinyl sulfonyl chlorides 40. The sulfonate ester 39 was dealkylated with
tetrabutylammonium iodide and converted to the sulfonyl chloride 40 by
treatment with triphosgene in the presence of catalytic N,N-dimethylformamide
(DMF). The vinyl sulfonyl chloride 40 was then subjected to reaction with
different phenols (R = H, OMe, F) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
to form the vinyl sulfonate esters 35. The vinyl sulfonamides 36 were prepared by
reacting 40 with O-benzylhydroxylamine in presence of 2,6-lutidine [21].
Based on the results of the in vitro biological assessment, vinyl sulfonamides 36
and vinyl sulfones 34 tend to be more active than the vinyl sulfonate esters 35
against the P. falciparum. However, an opposite order with respect to the activity
against falcipain-2 was observed, being sulfonate esters 35 more active than
corresponding vinyl sulfonamides 36 and vinyl sulfones 34 [21].
Despite its potent activity as an inhibitor of cysteine protease, peptidyl inhibitors
are potentially problematic due to poor pharmacokinetic properties (i.e. poor
absorption, metabolic susceptibility, rapid excretion, etc). Such limitations are
mainly related to high molecular weight, poor water solubility and presence of
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184 Organic Compounds to Combat Neglected Tropical Diseases de Oliveira et al.
amide bonds that are susceptible to cleavage by proteases. Therefore, nonpeptidic
compounds have been designed to overcome these limitations.
Scheme 6: General route of synthesis of vinyl sulfones 33 [21-23].
EtO
PSO
2
Ph
O
EtO
EtO
PSO
3
Et
O
EtO
SO
2
ClBocHN
Pep
SO
2
Ph
Pep
BocHN
S
N
H
O
OO
BocHN
Pep
S
O
OO
BocHN
Pep
R
34, 65-87%
35
38
aaa
aaa
36, 60-67%
SO
3
Et
Pep
BocHN
39, 65-87%
37
Reagent and conditions: (i) NaH, THF, aaa = amino acid aldehyde; (ii) a) Bu
4
NI, acetone b) triphosgen
e
DMF, CH
2
Cl
2
; (iii) substituted phenol, DBU, CH
2
Cl
2
; (iv) BnONH
2
, 2,6-lutidine, CH
2
Cl
2
. Pep= coupled peptidyl
c
hain.
(iii)
(iv)
(ii)
40, 70-78%
(i)
(i)
a) Obtention of phenyl
v
inyl sulfones
b) Obtention of vinyl sulfonamides and vynil sulfonate esters
Scheme 7: Route of synthesis of phenyl vinyl sulfones 34, vinyl sulfonate esters 35 and vinyl
sulfonamides 36 [21].
3.2. Peptidomimetics
A strategy used to reduce the peptide character of falcipain-2 inhibitors is the
incorporation of nonpeptidic scaffold or unnatural amino acids into the amino acid
sequence of the inhibitor. The benzo[1,4]diazepin-2-one is a commonly used
Malaria
Organic Compounds to Combat Neglected Tropical Diseases 185
scaffold, introduced internally to a peptide sequence, which mimics the fragment
D-Ser-Gly [24] (Fig. 10).
N
H
O
O
O
N
N
O
O
H
N
O
F
3
C
Cl
N
H
O
H
N
HN
O
HO
Gly
Ser
vinyl ester inhibitor Ser-Gly pept ide fragme nt
benzo[1,4]diazepin-2-one fragment
Figure 10: Examples of scaffold mimetics of peptides.
The synthesis of inhibitors based on benzodiazepine nucleus can be achieved by
preparation of the key intermediate, the oxazolidine derivative 43, as described in
Scheme 8. The synthesis starts with the protection of the amino group of the
starting material D-serine methyl ester hydrochloride 41 with Boc-anhydride,
followed by formation of N,O-acetal upon treatment with 2,2-dimethoxypropane
(2,2-DMP) in presence of BF
3
.Et
2
O. Lithium hydroxide hydrolysis of the methyl
ester 42 leads to free carboxylic acid 43, which is activated as mixed anhydride
and reacted with 2-aminobenzophenone to generate amide 44. Removal of Boc
protective group leads to concomitant cyclization of the resulting intermediate
amino, yielding the 3-hydroxymethyl-1,4-benzodiazepine 45. The hydroxyl group
is then protected with tert-butyldimethylsilyl chloride (TBSCl) and the N-
alkylation of the lactam leads to the introduction of the side-chain of the
benzodiazepine nucleus [25].
The presence of hydroxyl (after deprotection) and carboxylic acid groups on the
side-chains of the 1,4-benzodiazepine 47 provides access to a variety of
analogues. Several pharmacophores can be coupled to the benzo[1,4]diazepin-2-
one scaffold, such as the aspartyl aldehyde building block [26], vinyl sulfone [27],
vinyl ester [20, 24] and vinyl ketone [20, 28] groups, which are able to form a
covalent bond with the thiol group of the cysteine active site. For instance, Ettari
et al., based on chemical structure of 47, designed and synthesized the
peptidomimetic 50 displaying high antiplasmodial activity (IC
50
= 9.1 µM). The
synthesis of 50 is illustrated in Scheme 9 [27].
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186 Organic Compounds to Combat Neglected Tropical Diseases de Oliveira et al.
Scheme 8: Route of synthesis of inhibitors based on benzodiazepine nucleus [25].
N
TBSO
N
O
COOH
H
2
N
(CH
2
)
2
Ph
N
TBSO
N
O
O
N
H
(CH
2
)
2
Ph
N
O
N
O
O
N
H
(CH
2
)
2
Ph
H
N
O
F
3
C
Cl
N
O
N
O
O
N
H
(CH
2
)
2
Ph
H
N
O
F
3
C
Cl
SO
2
Et
SO
2
Et
F
3
C
Cl
NCO
47
Reagent and conditions: (i) HATU, CH
2
Cl
2
, rt, 12 h; (ii) TBAF, THF, rt, 5 h; (iii) 4-chloro-2-trifluoromethylphenyl
isocyanate, rt, 12 h; (iv) Hoveyda catalyst, CH
2
Cl
2
, 100 ºC, microwave, 2 h.
(i)
(ii), (iii)
(iv)
48, 90%
49, 74%
50, 50%
Scheme 9: Route of synthesis of peptidomimetic 50 [27].
The carboxylic acid 47 was coupled to (S)-1-phenethyl-allylamine using O-(7-
azabenzotriazol-1-yl)-N,N,N,N′-tetramethyluronium hexafluorophosphate (HATU)
to afford the amide 48 in high yield (90%). Desilylation of 48 using tetrabutyl-
ammonium fluoride (TBAF), followed by reaction with 4-chloro-2-trifluoro-
methylphenyl isocyanate, gave 49. Cross metathesis of 49 with ethyl vinyl sulfone
HO
COOMe
NH
2
. HCl
NBoc
O
OMe
O
NBoc
O
H
N
O
O
N
HO
N
H
O
N
TBSO
N
H
O
N
TBSO
N
O
COOH
NBoc
O
OH
O
41
Reagent and conditions: (i) a) NaHCO
3(aq)
/THF 2:8, (Boc)
2
O, rt, 12 h, b) 2,2-DMP, BF
3
.
Et
2
O, acetone, rt, 2
h; (ii) LiOH 1 M, methanol, 0 ºC
rt, 6 h; (iii) i-BuOCOCl, N-methylmorpholine, CH
2
Cl
2
, 0 ºC
rt, 30 min,
b) 2-aminobenzophenone, reflux for 20 min
rt, 13 h; (iv) HCl 5%/MeOH 1:5, reflux, 7 h; (v) TBSCl,
imidazole, CH
2
Cl
2
, 0 ºC
rt, 12 h; (vi) a) NaH, DMF, 0 ºC
rt, 30 min, then
BrCH
2
COOEt, 0 ºC
rt, 5 h, b)
LiOH 1 M, methanol, 0 ºC
rt, 6 h.
(i)
(iii)
(iv)
(vi)
(v)
42, 95%
44, 79%
45, 85%
46, 87%
47, 64%
43, 86%
(ii)
Malaria
Organic Compounds to Combat Neglected Tropical Diseases 187
using second generation Hoveyda–Grubbs catalyst provided peptidomimetic 50
[27].
Another rigid nonpeptidic scaffold described in literature is the pyridone ring,
which replaces the leucine residue present in various peptide inhibitors. Verissimo
et al. prepared a series of falcipain inhibitors with different Michael acceptor
groups attached to the pyridone ring, as shown in Schemes 10 and 11. Initially,
the key intermediate 58 was synthesized by a convergent route in six steps. First,
the pyridone 53 was obtained by reduction of the nitro group of the commercially
available 3-nitropyrid-2-one, followed by protection of the amino group in 52
with benzyloxycarbonyl chloride (Cbz-Cl). The triflate 57 was prepared from
commercially available Boc-D-phenylalanine (R = CH
2
Ph) and Boc-D-
homophenylalanine (R = C
2
H
4
Ph) using the following sequence of reaction: a)
Boc-deprotection, b) nitrous acid deamination, c) esterification under acid
conditions and d) reaction of alcohols with trifluoromethanesulfonic anhydride in
the presence of 2,6-lutidine. Finally, triflate 57 was reacted with pyridone 53, in a
S
N
2 type reaction, to give the key intermediate 58 [29].
Scheme 10: Route of synthesis of key intermediates 58 [29].
From intermediate 58, a series of 2-pyridone peptidomimetic falcipain inhibitors
was prepared as illustrated in Scheme 11. Reduction of esters 58 to corresponding
NH
O
O
2
N
NH
O
H
2
N
NH
O
N
H
Cbz
ON
H
OH
O
O
R
HO
OH
O
R
HO
O
O
R
CH
3
O
O
O
R
CH
3
S
F
3
C
OO
N
O
N
H
Cbz
O
O
CH
3
R
H
2
OCl
O
(CF
3
SO
2
)
2
O
Pd/C
(i)
(iii)
(iv)
(v)
(vi)
52, 98%
53, 50%
55, 64-69%
56, 100%
57, 53-59%
58, 67-76%
51
Reagents and conditions: (i) ethanol, rt, 8 h; (iii) HCI, dioxane, hexane, then H
2
SO
4
, NaNO
2(aq)
; (iv) HCl, methanol,
rt, overnight; (v) 2,6-lutidine, 0 ºC, 1 h; (vi) NaH, THF, rt, 2 h.
(ii)
R = CH
2
Ph or C
2
H
4
Ph
54
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188 Organic Compounds to Combat Neglected Tropical Diseases de Oliveira et al.
alcohols using NaBH
4
, followed by Swern oxidation delivered the aldehyde 60,
which underwent olefination employing Wittig or Horner-Wadsworth-Emmons
conditions to furnish the desired products 61 and 62, respectively. These
peptidomimetic derivatives displayed in vitro antimalarial inhibition, with IC
50
ranging from 6 to 40 M [29].
Scheme 11: Route of synthesis of 2-pyridone peptidomimetic falcipain inhibitors [29].
3.3. Chalcones
Chalcones represent an important class of natural products with a wide range of
biological activity. Chemically they consist of two aromatic rings (A and B)
linked by an enone moiety (Fig. 11a). It is well known the ability of chalcones to
inhibit the falcipain-2, acting as a Michael acceptor due the presence of an α, β-
unsaturated carbonyl system in their structure. This effect can be intensified by
protonation of the carbonyl oxygen in the acidic environment, in which the
falcipain-2 is found. Licochalcone A (Fig. 11b), a naturally occurring product, is a
promising antimalarial compound of this family. Chalcones are readily
synthesized and a series of analogues with a variety of ring substitution patterns
can be accessed [13, 30].
Chalcones can be readily prepared via Claisen-Schmidt condensation by reacting
an aromatic methylketones with the appropriate aromatic aldehydes under basic
conditions, as demonstrated by Tadigoppula and co-workers (Scheme 12) [30].
Malaria
Organic Compounds to Combat Neglected Tropical Diseases 189
Figure 11: (a) general structure of chalcones, (b) structure of Licochalcone A.
O
O
H
R'
O
RR
R'
(i)
63
Reagents and conditions: (i) NaH, THF, rt, 9 h or KOH
(aq)
, ethanol, rt, 48 h.
64
Scheme 12: Synthesis of chalcones via Claisen-Schmidt condensation [30].
Domínguez et al. reported the synthesis of phenylurenyl chalcones with good
inhibitory properties against falcipain-2. The best inhibitor of this series, the
chalcone 67, displayed an IC
50
value of 1.8 µM against falcipain-2, associated
with a significant inhibition of P. falciparum growth in vitro (IC
50
value of 3 µM).
The synthesis of 67 was based on Claisen-Schmidt condensation as previously
described (Scheme 12). The first step of synthesis consisted in the reaction of p-
amino-acetophenone and p-chlorophenyl isocyanate 65 to obtain the desired
urenylacetophenone 66 in 65% yield. In the next step, 66 was submitted to
Claisen-Schmidt condensation with 3,4,5-trimethoxybenzaldehyde in presence of
sodium hydroxide, yielding the chalcone 67 (Scheme 13) [31].
Cl
N
C
O
CH
3
O
H
2
N
Cl
N
H
N
H
O
O
OCH
3
OCH
3
OCH
3
Cl
N
H
N
H
O
O
H
O
OCH
3
OCH
3
OCH
3
(i)
(ii)
65
Reagents and conditions: (i) acetone, rt; (ii) methanol, NaOH, rt.
66, 65%
67, 82%
Scheme 13: Route of synthesis of phenylurenyl chalcone 67 [31].
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190 Organic Compounds to Combat Neglected Tropical Diseases de Oliveira et al.
3.4. Isoquinolines
Isoquinolines derivatives, such as compound 68, were first designed as falcipain-2
inhibitors from modeling studies that demonstrated the importance of the interaction
of 1-(4-hydroxy-phenyl) group of the isoquinoline with the Asp234 in the active site
of falcipain-2. Further, compounds bearing a bulky p-benzyloxyphenyl group (Fig.
12) were found to be more potent in inhibiting the falcipain-2 than their
corresponding hydroxyl analogs, suggesting the presence of a pocket in the active
site of the enzyme, capable of accommodating hydrophobic groups [13, 32].
In the search for more potent analogues, Micale et al. synthesized a series of new 1-
aryl-6,7-disubstituted-2H-isoquinolin-3-ones to explore the effects of structural
variations at the positions 2 and 3 of the isoquinoline ring. These derivatives
exhibited inhibitory activity against falcipain-2 and against in vitro culture of P.
falciparum, although they have displayed no selectivity against human cysteine
proteases. General procedure for the preparation of the 2-substituted isoquinolin-3-
ones is outlined in Scheme 14. The 2-substituted isoquinolin-3-ones were prepared
in six steps from commercially available methyl phenylacetates 72. The first step
consisted in the acylation of methyl phenylacetates with 4-methoxybenzoic acid in
presence of phosphorous pentoxide. The hydrazides 75 was prepared from ester 73
via lithium hydroxide hydrolysis and subsequent treatment with N-Boc-hydrazine
and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) hydrochloride (EDC).
Cyclization of 75 in presence of sodium hydride in THF gave the N-substituted
isoquinolin-3-ones 76. Removal of the N-Boc protecting group followed by reaction
with ethyl or with aryl isocyanates provided the corresponding 2H-isoquinolin-3-
ones 78 [33].
Figure 12: Chemical structure and IC
50
of isoquinoline derivatives falcipain-2 inhibitors.
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