Synthesis of gallinamide A analogues as potent falcipain inhibitors and antimalarials

Article abstract of DOI:10.1021/jm501439w

Analogues of the natural product gallinamide A were prepared to elucidate novel inhibitors of the falcipain cysteine proteases. Analogues exhibited potent inhibition of falcipain-2 (FP-2) and falcipain-3 (FP-3) and of the development of Plasmodium falciparum in vitro. Several compounds were equipotent to chloroquine as inhibitors of the 3D7 strain of P. falciparum and maintained potent activity against the chloroquine-resistant Dd2 parasite. These compounds serve as promising leads for the development of novel antimalarial agents.

Full text of DOI:10.1021/jm501439w

Brief Article
pubs.acs.org/jmc
Synthesis of Gallinamide A Analogues as Potent Falcipain Inhibitors
and Antimalarials
Trent Conroy,† Jin T. Guo,‡ Nabiha Elias,† Katie M. Cergol,† Jiri Gut,∥ Jennifer Legac,∥ Lubna Khatoon,‡
Yang Liu,‡ Sheena McGowan,§ Philip J. Rosenthal,∥ Nicholas H. Hunt,‡ and Richard J. Payne*,†
†School of Chemistry, The University of Sydney, Building F11, Sydney, New South Wales 2006, Australia
‡School of Medical Sciences, The University of Sydney, Sydney, New South Wales 2006, Australia
§Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria 3800, Australia
∥Department of Medicine, San Francisco General Hospital, University of California, San Francisco, California 94143, United States
S
* Supporting Information
ABSTRACT: Analogues of the natural product gallinamide A were prepared to
elucidate novel inhibitors of the falcipain cysteine proteases. Analogues exhibited
potent inhibition of falcipain-2 (FP-2) and falcipain-3 (FP-3) and of the development
of Plasmodium falciparum in vitro. Several compounds were equipotent to
chloroquine as inhibitors of the 3D7 strain of P. falciparum and maintained potent
activity against the chloroquine-resistant Dd2 parasite. These compounds serve as
promising leads for the development of novel antimalarial agents.
isolated independently from a Schizothrix species of cyanobac-
teria from the Carribean Coast of Panama13 and from the
Symploca genus in Key Largo, Florida14 (Figure 1). Through
INTRODUCTION
■
Malaria, a mosquito-borne disease caused by infection with
Plasmodium parasites, is the world’s most deadly parasitic
infection.1 Almost half the world’s population live in malaria
endemic areas, and an estimated 1.2 billion people are at high
risk of contracting the disease.2 This has resulted in hundreds of
millions of Plasmodium falciparum infections each year, causing
hundreds of thousands of deaths, primarily in children.2 Nearly
all of these deaths are caused by P. falciparum, the most virulent
human malaria parasite.2 Unfortunately, the introduction of a
highly effective vaccine against malaria has remained elusive3
and, as a consequence, chemotherapy remains central to
control and treatment.4 Natural products and their derivatives,
including quinine, chloroquine (CQ), and artemisinin and its
analogues, have led the way as antimalarial drugs used
clinically.5,6 However, the control of malaria has been severely
compromised in recent years by the widespread resistance of P.
falciparum to nearly all frontline therapeutics used for both
prophylaxis and treatment.7 Of growing concern is recently
discovered resistance to components of artemisinin-based
combination therapies,8 the cornerstone of treatment of
falciparum malaria.9 Consequently, there is an urgent need
for the development of new antimalarials that are structurally
distinct from existing drugs and operate through novel
mechanisms of action.10
Figure 1. Structure of gallinamide A and inhibitory activity against P.
falciparum11 and the falcipains (FPs).15
our synthetic efforts, we have demonstrated that the natural
product exhibits potent activity in vitro against cultured P.
falciparum, with an IC50 of 50 nM. Importantly, gallinamide A
did not exhibit hemolytic activity against red blood cells,11 did
not inhibit the proteasome, and displayed weak or no
detectable activity against mammalian Vero cells, NCI-H460
lung tumor cells, or neuro-2a mouse neuroblastoma cell
lines.13,14
Recently, the putative mode of antimalarial action of
gallinamide A has been revealed in a study by Stolze et al.15
Specifically, the natural product has been shown to inhibit a
We have an interest in utilizing the privileged biological
activity of natural products to elucidate new antimalarial drug
leads. In this area, we have recently reported the efficient total
synthesis11 and stereochemical assignment of the N-terminal
isoleucine residue12 of gallinamide A (also known as
symplostatin 4), a depsipeptide natural product that has been
Received: September 18, 2014
© XXXX American Chemical Society
A
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Journal of Medicinal Chemistry
Brief Article
group of cysteine proteases found in the food vacuole of the
parasite, known as the falcipains (FPs). P. falciparum is known
to possess four falcipains, FP1, FP2, FP2′, and FP3, with the
last three located in the food vacuole of erythrocytic parasites.16
All three food vacuole-associated FPs (FP2, FP2′, and FP3)
were inhibited by gallinamide A at low- to mid-nanomolar
concentrations. The food vacuole FPs are required for the
degradation of hemoglobin and are essential for growth and
survival of the organism.16 Treatment of cultured P. falciparum
with gallinamide A leads to swelling of the food vacuole, which
fills with undegraded hemoglobin; if not halted, falcipain
inhibition leads to parasite death.15,17−19 Over the past decade,
several classes of FP inhibitors have been developed,17,20−28
some of which have shown efficacy in in vivo models of
malaria,27,29 but no falcipain inhibitors have yet progressed into
human clinical trials.30
Gallinamide A possesses a number of unique structural
features, including a dimethyl-terminated aliphatic depsipeptide
backbone, an unusual 4(S)-amino-2-(E)-pentenoyl moiety, and
a C-terminal N-acylpyrrolinone unit. On the basis of these
features, it is likely that gallinamide A is a covalent, irreversible
inhibitor of the FPs via nucleophilic attack by the sulfhydryl
side chain of the active site cysteine of the FPs onto one of the
two Michael acceptor moieties of the natural product. This
hypothesis is supported by a recent study showing that
gallinamide A potently inhibits cathepsin L through a covalent,
irreversible mechanism.31 Given the potent antiplasmodial
activity of gallinamide A, coupled with its general lack of
toxicity against human cell lines, we envisaged the development
of structurally unique gallinamide A analogues as inhibitors of
the food vacuole FPs, which may serve as antimalarial drug
leads. We proposed that the C-terminal pyrrolinone moiety and
N-terminal region of the natural product would be amenable to
significant structural change, thus providing scope for dramatic
alteration and simplification of the structure to provide the first
structure−activity data for this class of natural products.
followed by cleavage from the resin using hexafluoroisopropa-
nol (HFIP) provided tripeptide 5 in excellent yield. From here,
5 was coupled to imide fragments 7 and 8, which were prepared
using a similar protocol to that adopted for the total synthesis
of gallinamide A (see Supporting Information for synthetic
details). The coupling was carried out using HATU at low
temperature to minimize epimerization and afforded analogues
1 and 2, primarily as single diastereoisomers, in good yields. At
this stage, 1 and 2 were subjected to hydrogenation to provide
3 and 4 in 85% and 89% yields, respectively, following HPLC
purification, which also enabled separation of the diaster-
eoisomers of these compounds.
Having prepared the four target gallinamide A analogues, the
compounds were next screened against FP-2 and FP-3 using a
fluorescence-based kinetic assay.26 The compounds were also
screened against the chloroquine sensitive 3D7 strain of P.
falciparum using a [3H]-hypoxanthine incorporation assay
(Table 1).11,12,16 Gallinamide A analogue 1 exhibited potent
Table 1. Inhibition of FP-2, FP-3, and P. falciparum by
Gallinamide A Analogues 1−4 (Errors Are Standard Error of
the Mean of Three Experiments)
IC50 FP-2
[nM]
IC50 FP-3
[nM]
IC50 P. falciparum (3D7)
analogue
[nM]
1
2
3
4
6.78 0.44
2.81 0.39
3710 420
>50000
292 1.3
163 21
>50000
89.3 73
210 80
4375 690
>50000
>50000
inhibitory activity against FP-2 (IC50 = 6.78 nM), FP-3 (IC50
=
292 nM), and P. falciparum in vitro (IC50 = 89.3 nM).
Interestingly, while 1 was equipotent to gallinamide A against
FP-2, the compound exhibited 10-fold weaker inhibition against
FP-3 and an almost 2-fold drop in activity against P. falciparum.
Reduction of the enol moiety in the acyl-pyrrolinone unit in 2
led to a slight improvement in activity against FP-2 and FP-3
but a 2-fold reduction in antiplasmodial activity (IC50 = 210
nM). In contrast, reduction of the olefin in the α,β-unsaturated
imide moiety had a dramatic effect on inhibitory activity.
Specifically, analogue 3 exhibited a 3 orders of magnitude drop
in inhibitory potency against FP-2 (IC50 = 3710 nM) and
demonstrated no measurable inhibition of FP-3. This
compound also showed a marked reduction in activity against
P. falciparum. Removal of both olefinic moieties in analogue 4
led to a loss of measurable inhibitory activity against both the
FPs and the parasite. In addition, when P. falciparum
trophozoites were treated with compounds 1 and 2, both
caused swollen food vacuole morphology, a hallmark of FP
inhibition (see Supporting Information).16,32 In contrast,
compounds 3 and 4 (that were inactive against the FPs) did
not cause swelling of food vacuoles in the parasite (Scheme 1).
Taken together, these studies strongly suggest that the olefinic
functionality within the α,β-unsaturated imide moiety is critical
for inhibitory activity against both the FPs and against P.
falciparum.
RESULTS AND DISCUSSION
■
We were first interested in assessing whether one or both of the
olefinic moieties in gallinamide A were crucial for inhibitory
activity by serving as Michael acceptors for the active site Cys
residue of the FPs. Thus, we initially designed and synthesized
four analogues of gallinamide A with varied degrees of
saturation. This included compound 1 that is structurally
identical to gallinamide A, except that the native ester linkage in
the depsipeptide natural product has been replaced with an
amide bond. We envisioned that this linkage could be formed
en bloc from commercially available amino acids, negating the
use of synthetically challenging preformed amino ester building
blocks (as was required for the total synthesis of the natural
product11,12). The other modification was the incorporation of
a dimethylated valine (Val) residue, which we had previously
shown to be an excellent replacement for the N-terminal
dimethylated isoleucine (Ile) moiety in the natural product (see
Supporting Information). The three other proposed analogues
were 2, where the methoxy-enol moiety in the pyrrolinone ring
of 1 was reduced, 3, where the olefinic component of the 4(S)-
amino-2-(E)-pentenoic acid unit was reduced, and analogue 4,
where both olefinic moieties had been reduced. Preparation of
the proposed analogues began with the synthesis of N-terminal
fragment 5 via Fmoc-strategy SPPS. 2-Cl-Trt-Cl resin (6) was
first loaded with Fmoc-Leu-OH followed by coupling of Fmoc-
Leu-OH and Fmoc-Val-OH. On-resin reductive amination
Having established the importance of unsaturation in the
4(S)-amino-2-(E)-pentenoyl unit for FP inhibition and
antiparasitic activity, we next explored a small library of
gallinamide A analogues (9−16) as second-generation
inhibitors (Scheme 2). The major modification made to these
analogues was substitution of the C-terminal N-acylpyrrolinone
moiety of gallinamide A. Specifically, we proposed that
B
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a
Scheme 1. Synthesis of Gallinamide A Analogues 1−4
Scheme 2. (A) Synthesis of Gallinamide A Analogues 9−14
and (B) Synthesis of Extended Analogues 15 and 16 via a
Solid-Phase Synthesis Approach (NB: Compounds 9−16
a
Were Isolated As the Trifluoroacetate Salts)
a
Reagents and conditions: (i) resin loading: Fmoc-Leu-OH, iPr2EtN,
DMF/CH2Cl2; (ii) Fmoc-SPPS (deprotection, 10 vol % piperidine/
DMF; coupling, 4 equiv Fmoc-AA−OH, 4 equiv PyBOP, 8 equiv
NMM, DMF; capping, 10 vol % Ac2O/pyridine); (iii) HCHO,
NaBH(OAc)3, AcOH, DMF; (iv) 30 vol % HFIP/CH2Cl2; (v) HATU,
NMM, 0 °C for 20 min, rt for 2 h; (vi) H2, Pd/C, MeOH. * indicates
the site of diastereoisomerism in 1 and 2.
derivatization of a range of scaffolds at the C-terminus (using
simple amides to facilitate more rapid syntheses, R1 in Scheme
2A) would enable investigation of structure−activity relation-
ships for this region of the molecule. Introduction of these
modifications also enabled the rapid construction of analogues
primarily via solid-phase synthesis, without the need for
numerous solution-phase fragment condensation and purifica-
tion steps that were necessary for the synthesis of the natural
product (and of 1−4). It should be noted that, while Stolze et
al. reported that replacement of the C-terminal methylmethoxy-
pyrrolinone moiety in gallinamide A with a C-terminal alanine
methyl ester moiety significantly decreased activity against the
FPs and P. falciparum,15 we were interested in accessing
compounds that possessed greater C-terminal functionalization
so as to explore the effect of modifying the acyl-pyrrolinone on
FP activity and P. falciparum inhibition. We were encouraged by
the fact that reduction of the methoxy-enol moiety in analogue
2 did not have a dramatic effect on FP and P. falciparum
inhibitory activity, suggesting that modifications in this region
may be tolerated. A number of other changes were also
proposed for the N-terminal region, including a range of
aliphatic amino acids at the N-terminus (R2 in Scheme 2A).
Analogues 15 and 16, extended by one L-Ile residue in the
peptide backbone compared to 9−14, were also proposed in
order to probe the importance of the length of the analogues
for inhibition of the FPs and P. falciparum (Scheme 2B).
Synthesis of 9−14 began from 2-Cl-Trt Cl resin preloaded
with Fmoc-Ala (17). Coupling of Fmoc-protected α,β-
unsaturated amino acid 18 (see Supporting Information for
synthesis) followed by elongation via standard Fmoc-strategy
SPPS provided the desired resin-bound peptide sequences.
Following an en bloc reductive methylation of the N-terminus
with formaldehyde and sodium cyanoborohydride, cleavage of
the peptides from the resin using HFIP provided the C-
terminal peptide acids (including the extended peptide
precursor for 15 and 16) in moderate to good yields over
the 10 resin-bound steps following HPLC purification (36−
88%, see Supporting Information for full synthetic details and
yields). Having assembled the C-terminal peptide acids, we next
installed the C-terminal functionality. Benzylamine was coupled
using PyBOP at low temperature and, following purification by
a
Reagents and conditions: (i) 4 equiv 18, 4 equiv PyBOP, 6 equiv
NMM; (ii) Fmoc SPPS (deprotection, 10 vol % piperidine/DMF;
coupling, 4 equiv Fmoc-AA-OH, 4 equiv PyBOP, 8 equiv NMM, DMF;
capping, 10 vol % Ac2O/pyridine); (iii) HCHO, NaBH(OAc)3, AcOH,
DMF; (iv) 30 vol % HFIP/DCM; (v) # R1-NH2, PyBOP, DMF, 0 °C.
* indicates the site of diastereoisomerism in 9−16. # NMM was added
to the coupling reactions for the preparation of inhibitors 10 and 16.
reverse-phase HPLC, the desired inhibitors 9, 11, 13, 14, and
15 were isolated in excellent yields based on the original resin
loading without significant epimerization. 4-(R)-Hydroxy-L-
proline methyl ester was also coupled to the C-terminus of two
peptides, this time with the addition of NMM as a hindered
base, to afford inhibitors 10 and 16 in excellent yields following
HPLC purification. On this occasion, the compounds were
isolated as close to 1:1 mixtures of diastereoisomers, reflecting
the slower coupling rate of hydroxyproline methyl ester to the
C-terminus of the peptides. Finally, aminothiazole was coupled
to the C-terminus of one peptide acid using PyBOP at low
temperature, which provided 12 as a 1:1 mixture of
diastereoisomers in 62% yield following purification based on
the original loading of the resin.
The gallinamide A analogues 9−16 were next screened for
inhibitory activity against FP-2, FP-3, and the 3D7 strain of P.
falciparum in vitro (Table 2). All analogues possessed
significant activity, and all led to the swollen food vacuole
morphology in trophozoites (see Supporting Information).
C
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While the replacement of the C-terminal pyrrolinone unit of
gallinamide with simplified functionalities provided a number of
compounds with activity against FP-2, FP-3, and P. falciparum,
these compounds were significantly less potent than 1 and 2
(vide supra). As such, we chose to reinstate the C-terminal acyl
pyrrolinone moiety in an additional series of analogues to probe
the effect of substitution on the pyrrolinone ring on FP
inhibition and antiparasitic activity. In total, six analogues (19−
24) were proposed possessing the identical peptide backbone
to 1 and 2 but with variation in the side chain on the
pyrrolinone unit and in the substitution of the enol of the
pyrrolinone (Scheme 3). The synthesis of 19−24 began with
Table 2. Inhibition of FP-2, FP-3, and the 3D7 Strain of P.
falciparum by Gallinamide A Analogues 9−16
a
IC50 FP-2
[nM]
IC50 P. falciparum (3D7)
analogue
IC50 FP-3 [nM]
[nM]
9
10500 640
7700 190
11500 190
2500 150
3400 390
6000 160
>50000
>25000
>50000
540 210
1900 620
320 10
10
11
12
13
14
15
16
>25000
34000 460
>25000
1100 800
5400 380
6600 3800
>50000
46000 5400
>50000
>50000
>50000
>50000
a
Errors are standard error of the mean of three experiments.
Scheme 3. Synthesis of Second-Generation Gallinamide A
a
Analogues 19−24
However, in general, the replacement of the N-acyl pyrrolinone
moiety in gallinamide A (and analogue 1) with different C-
terminal groups was detrimental to activity against both the FPs
and the parasite. Analogues 9, 11, and 13, bearing a C-terminal
benzylamide moiety, all exhibited similar activity: low micro-
molar inhibition of FP-2 (IC50 = 3.4−11.5 μM), no measurable
inhibition of FP-3 at 25 μM, and nanomolar inhibitory activity
against P. falciparum (IC50 = 320−5400 nM). Interestingly,
introduction of an N-methylproline functionality at the N-
terminus of the peptide, while retaining the C-terminal
benzylamide in 14, led to inhibition of both FP-2 (IC50 = 6
μM) and FP-3 (IC50 = 46 μM) but exhibited less potent
antiparasitic activity (IC50 = 6.6 μM). The loss of activity was
particularly striking for 11, which possesses an identical
structure to analogue 1 (Table 1, IC50 FP-2 = 6.78 nM, IC50
FP-3 = 292 nM, IC50 3D7 = 89.3 nM), with the exception that
the C-terminal acyl pyrrolinone unit has been replaced by a C-
terminal benzylamide. The 3 orders of magnitude drop in
activity against the FPs and order of magnitude decrease in
antiparasitic activity from 1 to 11 suggests that the N-acyl
pyrrolinone unit is important for activity. Introduction of a
more highly functionalized and flexible hydroxyproline methyl
ester to the C-terminus in 10 provided similar inhibitory
activity to the benzylamide-derived compounds against FP-2
and P. falciparum. C-terminal functionalization as a thiazole
amide in 12 led to moderate inhibitory activity against both FP-
2 (IC50 = 2.5 μM) and FP-3 (IC50 = 34 μM) as well as low
micromolar antiparasitic activity (IC50 = 1.1 μM). Taken
together, these data suggest that the N-acyl pyrrolinone moiety
is critical for potent FP inhibition and in vitro antimalarial
activity. The importance of this functionality may lie in the
ability of the aminopentenoyl imide motif to serve as a better
Michael acceptor for the active site Cys residue in the FPs
(Cys42 in FP-2 and Cys51 in FP-3)33 when compared to the
corresponding amide. Specifically, replacement of the imide
moiety with the corresponding amide in analogues 9−14
reduces the propensity of the α,β-unsaturated amide unit to
serve as a Michael acceptor, presumably due to the less electron
withdrawing nature of the amide compared to the imide in the
native acyl-pyrrolinone linkage. Finally, extension of the
analogues through the insertion of an additional Val residue
in the peptide backbone in 15 and 16 abolished enzyme and
parasite inhibitory activity. It is possible that these compounds
can no longer be accommodated into the active site of the FPs,
as the Leu residue that would be expected to be positioned in
the S2 site of the protease33 has been replaced by a Val or the
double bond must be positioned at a suitable distance from the
N-terminus to exhibit inhibitory activity.
a
Reagents and conditions: (i) (1) Meldrum’s acid, EDC, DMAP,
CH2Cl2, 0 °C to rt; (ii) EtOAc, 77 °C; (iii) R2-OH, DIAD, PPh3,
CH2Cl2, 0 °C to rt; (iv) 1:4 v/v piperidine/MeCN, rt, 15 min; (v)
CF3COOC6F5, pyridine, DMF, 0 °C; (vi) 25−30, n-BuLi, THF, −78
°C; (vii) 1:1 v/v TFA/CH2Cl2; (viii) tripeptide 5, HATU, NMM,
DMF, 0 °C. ND = not determined due to co-contamination with
DIAD by-product. * indicates the site of diastereoisomerism in 19, 21,
23, and 24.
the preparation of the requisite pyrrolinones 25−30 from
commercially available Fmoc-protected amino acids 31−34.
Coupling of Meldrum’s acid to amino acids 31−34 with EDC
in the presence of DMAP followed by reflux of the Meldrum’s
adduct in ethyl acetate to effect cyclization-condensation
provided the corresponding Fmoc-protected pyrrolinones
35−38 in good yield over the two steps. These were each
D
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Journal of Medicinal Chemistry
Brief Article
reacted with methanol under Mitsunobu conditions using
DIAD and triphenylphosphine to provide O-methylated
pyrrolinones 39−42 in 48−79% yields. In addition, 35 and
36 were treated with benzyl alcohol under the same conditions
to provide 43 and 44. All that remained for the synthesis of the
target pyrrolinones was removal of the Fmoc group, which was
effected smoothly by treatment with piperidine in acetonitrile
to provide 25−30 in excellent yields. With each of the required
pyrrolinone building blocks in hand, attention turned to the
modular assembly of the proposed gallinamide A analogues. To
this end, Boc-protected amino acid 4511 was activated as the
corresponding pentafluorophenyl ester by treatment with
pentafluorophenyl trifluoroacetate in the presence of pyridine.
Separately, pyrrolinones 25−30 were deprotonated with n-
butyllithium at low temperature before addition of the
pentafluorophenyl esters to afford imides 46−51 in moderate
yields (36−59%). Unfortunately, despite efforts to improve the
reactions by modifying the temperature and base, these yields
could not be improved. From here, acidolysis of the Boc group
from 46−51 followed by coupling to the N-terminal tripeptide
5 using HATU as the coupling reagent and NMM as the base
(at low temperature to minimize epimerization), furnished the
desired gallinamide A analogues 19−24 in good yields
following HPLC purification. As with the synthesis of
gallinamide A and analogues 1−4, epimerization occurred
during the final fragment condensation reaction. The minor
epimer could be separated by preparative HPLC for analogues
20 and 22, while 19, 21, 23, and 24 were isolated as
predominantly one diastereoisomer.
side chains onto the pyrrolinone ring in 21 and 22 did not lead
to a change in activity against FP-2 but, as with 20, led to a
marked increase in inhibitory activity against FP-3 and P.
falciparum. Compound 22, bearing an indole side chain on the
pyrrolinone ring, proved to be the most potent inhibitor of FP-
3 (IC50 = 66.7 nM) and P. falciparum (IC50 = 9.7 nM) in this
series of compounds.
Having elucidated a number of gallinamide A analogues as
potent FP and P. falciparum inhibitors, we were next interested
in investigating whether selected compounds were capable of
maintaining activity against a CQ-resistant (Dd2) strain of P.
falciparum and whether the compounds exhibited selective
killing of parasites over human cells by screening against a
HEK298 cell line (Table 4). Finally, we were interested in
Table 4. Inhibition of the CQ-Resistant Dd2 Strain of P.
falciparum, HEK298 Cells, and P. falciparum
Aminopeptidase M1, M17, and M18 (AP M1, M17, and
a
M18) by Gallinamide A Analogues
IC50 P. falciparum
(Dd2) [nM]
AP M1, M17, and
M18 [nM]
analogue
HEK298 [nM]
1
302 126
421 152
378 20
14 200 1050
>50000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
9
11
13
19
20
21
22
>50000
170 68
>50000
419 185
165 68.0
67.0 30.0
29.0 16.0
16 300 1700
9650 1480
18 900 110
8500 124
With the small library of pyrrolinone-modified analogues in
hand, the compounds were next screened against FP-2, FP-3,
and the 3D7 strain of P. falciparum (Table 3). Reintroduction
a
Errors are standard error of the mean of three experiments.
investigating the selectivity of the compounds in inhibiting the
FPs over other parasitic proteases. To this end, the compounds
were screened against three aminopeptidase (AP) enzymes
from P. falciparum, namely AP M1, AP M17, and AP M18. The
compounds selected included the potent N-acyl-pyrrolinone-
containing analogues 1 and 19−22 as well as the C-terminal
amide derivatives, 9, 11, and 13, which exhibited IC50 values
<600 nM against the 3D7 strain of P. falciparum (see Table 2).
All of the tested compounds (1, 9, 11, 13, and 19−22)
exhibited potent inhibition of the CQ-resistant Dd2 strain of P.
falciparum (IC50 = 29.0−421 nM). Despite exhibiting potent
inhibitory activity against human cathepsin, the compounds
were selective inhibitors of P. falciparum over HEK298 cells,
with 9, 11, and 13 showing no measurable inhibition of this cell
line at a concentration of 50 μM (see Supporting Information).
In addition, none of the analogues displayed any inhibitory
activity against the metalloproteases AP M1, AP M17, or AP
M18 from P. falciparum (see Supporting Information). Taken
together, these data suggest that structural analogues of
gallinamide A are promising leads for the pursuit of potent
food vacuole FP inhibitors as antimalarial compounds.
Table 3. Inhibition of FP-2, FP-3, and the 3D7 Strain of P.
falciparum by Gallinamide A Analogues 1 and 19−24
a
IC50 FP-2
[nM]
IC50 FP-3
[nM]
IC50 P. falciparum (3D7)
analogue
[nM]
1
6.78 0.44
24.8 22.9
9.52 0.14
5.25 2.06
12.0 3.19
9.59 0.21
6.86 2.53
292 1.30
225 26.5
131 43.6
81.4 7.73
66.7 25.4
196 7.00
182 15.8
89.3 73
57.3 26.0
16.6 9.0
20.0 8.0
9.7 2.0
19
20
21
22
23
24
CQ
96.0 74.0
62.0 47.0
17.3 3.0
a
Errors are standard error of the mean of three experiments.
of the C-terminal N-acyl-pyrrolinone in 19−24 led to
reinstatement of potent inhibitory activity against the FPs
and P. falciparum, as observed for gallinamide A and analogue 1.
All compounds also showed swollen food vacuole morphology
at a concentration of less than 5 nM, supporting potent
inhibition of the FPs as the mode of antiplasmodial action (see
Supporting Information). Compound 19, bearing no sub-
stitution on the pyrrolinone moiety, exhibited similar inhibitory
activity to 1 against FP-2 (IC50 = 24.8 nM), FP-3 (IC50 = 225
nM), and P. falciparum (IC50 = 57.3 nM). Incorporation of a
more hydrophobic substituent on the pyrrolinone ring in 20 led
to improvement in inhibitory activity against FP-3 (IC50 = 131
nM) and P. falciparum (IC50 = 16.6 nM). Indeed, 20 proved to
be equipotent to the antimalarial drug CQ against the CQ-
sensitive 3D7 strain of the parasite. Introduction of aromatic
CONCLUSIONS
■
In summary, a number of potent new inhibitors of the food
vacuole FPs, FP-2 and FP-3, were discovered based on the
structure of the cyanobacterium-derived natural product
gallinamide A. The importance of the α,β-unsaturated imide
moiety of the natural product for inhibitory activity was initially
demonstrated through the synthesis of selectively reduced
analogues and through the synthesis and evaluation of several
derivatives bearing a C-terminal amide in place of the imide
E
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Journal of Medicinal Chemistry
Brief Article
1673, 1621, 1515, 1455, 1365, 1323 cm−1. 1H NMR (400 MHz,
CDCl3) δ 7.39 (dd, 1H, J 15.8, 1.6 Hz, CH), 7.02 (dd, 1H, J 15.8, 4.4
Hz, CH), 5.08 (s, 1H, CH), 4.69−4.61 (m, 2H, NH, CH), 4.45 (m,
1H, CH), 3.84 (s, 3H, OCH3), 2.55 (m, 1H, CH), 1.44 (s, 9H, 3 ×
CH3), 1.29 (d, 3H, J 7.2 Hz, CH3), 1.10 (d, 3H, J 7.2 Hz, CH3), 0.74
(d, 3H, J 7.2 Hz, CH3). 13C NMR (100 MHz, CDCl3) δ 179.8, 175.7,
170.7, 164.6, 149.5, 122.1, 94.8, 79.8, 64.1, 58.6, 47.5, 28.5, 20.5, 18.9,
15.5; MS (ESI): m/z 375 [(M + Na)+, 100%]. HRMS: calcd for
C18H28N2O5Na M + Na+, 375.1896; found M + Na+, 375.1891.
functionality found in the natural product. A number of potent
inhibitors of FP-2 and FP-3 were elucidated through variation
of the side chain on the pyrrolinone ring. Several of these
compounds also demonstrated potent inhibition of the CQ-
sensitive 3D7 strain of P. falciparum, with a number of these
proving similarly potent to CQ. Gratifyingly, these analogues
maintained potent activity against the CQ-resistant Dd2 strain
of P. falciparum and did not possess noteworthy toxicity to
HEK298 cells. These compounds serve as promising leads for
assessing in vivo efficacy of this class of inhibitors as well as for
crystallographic studies which will aid in the development of
second-generation natural-product-based FP inhibitors for the
discovery of potential antimalarials. These studies will be the
focus of ongoing research in our laboratories.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures and analytical data for all novel
1
compounds including H and 13C NMR and analytical HPLC.
This material is available free of charge via the Internet at
http://pubs.acs.org.
EXPERIMENTAL SECTION
■
Final inhibitors 19−24 were synthesized as detailed in the
representative example for compound 20 below. The purity of all
final compounds was determined to be ≥95% by NMR and HPLC-MS
analysis. General methods, full experimental details, and original NMR
spectra for all analogues can be found in the Supporting Information.
Me2-Val-Leu-Leu-Apa-pyVal-OMe·TFA (20). Imide 47 (20 mg,
57 μmol) was dissolved in 1:1 v/v TFA/CH2Cl2 (2 mL) and the
reaction was stirred for 15 min before it was concentrated in vacuo,
and the residue was redissolved in DMF (0.3 mL) and cooled to 0 °C.
N,N-Dimethyltripeptide 5 (46.5 mg, 95 μmol) and HATU (36.1 mg,
95 μmol) were added, followed by NMM (21 μL, 190 μmol), and the
reaction was stirred for 20 min at 0 °C, then 2 h at room temperature.
The reaction was subsequently quenched with TFA (20 μL), diluted
with 1:1 v/v MeCN/H2O (4 mL), and purified by preparative reverse
phase HPLC (gradient: 0−60% MeCN over 40 min) to afford 20 as a
white amorphous solid and a single diastereomer (27 mg, 65%). Rt
[0−100% MeCN over 30 min] = 20.1 min; [α]D = 30.2 (c = 0.4,
MeOH). IR (thin film) νmax = 3295, 3073, 2963, 1725, 1670, 1646,
1625, 1550, 1464, 1350 cm−1. 1H NMR (500 MHz, CDCl3) δ 8.95 (d,
1H, J 8.0 Hz, NH), 8.26 (s, 1H, NH), 7.37 (d, 1H, J 15.5 Hz, CH),
7.35 (s, 1H, NH), 6.95 (dd, 1H, J 15.5, 5.0 Hz, CH), 5.09 (s, 1H, CH),
4.83−4.70 (m, 3H, 3 × CH), 4.60 (d, 1H, J 2.5 Hz, CH), 3.87 (d, 1H,
J 8.5 Hz, CH), 3.84 (s, 3H, CH3), 3.00 (s, 3H, NCH3), 2.86 (s, 3H,
CH3), 2.54 (m, 1H, CH), 2.26 (m, 1H, CH), 1.71−1.53 (m, 6H, 2 ×
CH2, 2 × CH), 1.26 (d, 3H, J 7.0 Hz, CH3), 1.12−1.07 (m, 6H, 2 ×
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +61 2 9351 5745. Fax: +61 2 9351 3329. E-mail:
richard.payne@sydney.edu.au.
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Ian Luck, School of Chemistry, University of
Sydney, for assistance with NMR, the John Lamberton
Research Scholarship and the Australian Postgraduate Award
Programs (T.C. and N.E.), and a National Health and Medical
Research Council of Australia (NHMRC) Postgraduate
Scholarship (J.G.) for Ph.D. support. R.J.P. and S.M. are
supported by Australian Research Council Future Fellowships
(R.J.P., FT130100150; S.M., FT100100690). This research was
funded by a NHMRC project grant 1062216.
CH3), 0.93−0.82 (m, 15H, 5 × CH3), 0.73 (d, 3H, J 6.5 Hz, CH3). 13
C
ABBREVIATIONS USED
NMR (125 MHz, CDCl3) δ 180.0, 172.1, 172.0, 170.7, 166.6, 164.5,
148.5, 122.7, 94.7, 71.7, 64.1, 58.7, 51.7, 51.7, 46.4, 41.7, 41.6, 28.9,
28.1, 25.1, 25.0, 23.2, 23.1, 22.2, 21.7, 19.7, 19.3, 18.9, 18.8, 15.4. MS
(ESI): m/z 629 [(M + Na)+, 100%]. HRMS calcd for C32H56N5O6 M
+ H+, 606.4230; found M + H+, 606.4225.
■
CQ, chloroquine; FP, falcipain; NMM, N-methylmorpholine;
EDC, 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide;
DIAD, diisopropyl azodicarboxylate; PyBOP, (benzotriazol-1-
yloxy)tripyrrolidinophosphon-ium hexafluorophosphate;
DMAP, 4-dimethylaminopyridine; HATU, 1-[bis-
(dimethylamino) methylene]-1H-1,2,3-triazolo[4,5-b]-
pyridinium 3-oxid hexafluorophosphate
Boc-Apa-pyVal-OMe (47). To a solution of amino acid 4511 (101
mg, 396 μmol) in DMF (2 mL) at 0 °C was added pentafluorophenyl
trifluoroacetate (89 μL, 0.51 mmol), followed by pyridine (32 μL, 396
mmol), and the reaction was allowed to warm to room temperature.
The reaction was subsequently stirred for 1 h before diluting with 1:1
v/v Et2O/EtOAc (20 mL) and washing with 0.2 M aqueous HCl (5
mL), saturated aqueous NaHCO3 solution (5 mL) and brine. The
organic phase was then dried (MgSO4) before concentrating in vacuo
to afford the pentafluorophenyl ester as a pale-yellow oil which was
used immediately in the following reaction. To a solution of
pyrrolinone 26 (28.0 mg, 183 μmol) in THF (1.5 mL) at −78 °C
was added 2.41 M n-butyllithium in hexane (76 μL, 183 μmol), and
the reaction was stirred for 10 min. A solution of the freshly prepared
pentafluorophenyl ester (99 mg, 235 mmol) in THF (0.5 mL) was
subsequently added dropwise over 15 min, and the reaction was
allowed to stir for a further hour at −60 °C. The reaction was
subsequently quenched with AcOH (50 μL) and concentrated in
vacuo, and the residue was purified by column chromatography
(eluent: 2:1 v/v hexane/EtOAc) to afford imide 47 as a colorless oil
(24 mg, 37%). Rf [1:1 v/v hexane/EtOAc] = 0.65; [α]D = +69 (c = 1.0,
CHCl3). IR (thin film) νmax = 3343, 3102, 2971, 2934, 2877, 1719,
REFERENCES
■
(1) Persidis, A. Malaria. Nature Biotechnol. 2000, 18, 111−112.
(2) Malaria Report; World Health Organisation: Geneva, 2012;
http://www.who.int/malaria/publications/world_malaria_report_
2012/.
(3) Birkett, A. J.; Moorthy, V. S.; Loucq, C.; Chitnis, C. E.; Kaslow,
D. C. Malaria vaccine R&D in the decade of vaccines: Breakthroughs,
challenges and opportunities. Vaccine 2013, 31, B233−243.
(4) Kokwaro, G. Ongoing challenges in the management of malaria.
Malar. J. 2009, 8, S2.
(5) Newman, D. J.; Cragg, G. M.; Snader, K. M. The influence of
natural products upon drug discovery. Nat. Prod. Rep. 2000, 17, 215−
234.
(6) Rosenthal, P. J. Antimalarial drug discovery: old and new
approaches. J. Exp. Biol. 2003, 206, 3735−3744.
F
dx.doi.org/10.1021/jm501439w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
(7) Fidock, D. A.; Rosenthal, P. J.; Croft, S. L.; Brun, R.; Nwaka, S.
Antimalarial drug discovery: efficacy models for compound screening.
Nature Rev. Drug Discovery 2004, 3, 509−520.
(24) Shah, F.; Mukherjee, P.; Gut, J.; Legac, J.; Rosenthal, P. J.;
Tekwani, B. L.; Avery, M. A. Identification of novel malarial cysteine
protease inhibitors using structure-based virtual screening of a focused
cysteine protease inhibitor library. J. Chem. Inf. Model. 2011, 51, 852−
864.
(25) Shah, F.; Gut, J.; Legac, J.; Shivakumar, D.; Sherman, W.;
Rosenthal, P. J.; Avery, M. A. Computer-aided drug design of falcipain
inhibitors: virtual screening, structure−activity relationships, hydration
site thermodynamics, and reactivity analysis. J. Chem. Inf. Model. 2012,
52, 696−710.
(26) Rosenthal, P. J.; Olson, J. E.; Lee, G. K.; Palmer, J. T.; Klaus, J.
L.; Rasnick, D. Antimalarial effects of vinyl sulfone cysteine proteinase
inhibitors. Antimicrob. Agents Chemother. 1996, 40, 1600−1603.
(27) Olson, J. E.; Lee, G. K.; Semenov, A.; Rosenthal, P. J.
Antimalarial effects in mice of orally administered peptidyl cysteine
protease inhibitors. Bioorg. Med. Chem. 1999, 7, 633−638.
(28) Shenai, B. R.; Lee, B. J.; Alvarez-Hernandez, A.; Chong, P. Y.;
Emal, C. D.; Neitz, R. J.; Roush, W. R.; Rosenthal, P. J. Structure−
activity relationships for inhibition of cysteine protease activity and
development of Plasmodium falciparum by peptidyl vinyl sulfones.
Antimicrob. Agents Chemother. 2003, 47, 154−160.
(8) Dondorp, A. M.; Nosten, F.; Yi, P.; Das, D.; Phyo, A. P.; Tarning,
J.; Lwin, K. M.; Ariey, F.; Hanpithakpong, W.; Lee, S. J.; Ringwald, P.;
Silamut, K.; Imwong, M.; Chotivanich, K.; Lim, P.; Herdman, T.; An,
S. S.; Yeung, S.; Singhasivanon, P.; Day, N. P.; Lindegardh, N.;
Socheat, D.; White, N. J. Artemisinin resistance in Plasmodium
falciparum malaria. N. Engl. J. Med. 2009, 361, 455−467.
(9) Nosten, F.; White, N. J. Artemisinin-based combination
treatment of falciparum malaria. Am. J. Trop. Med. Hyg. 2007, 77,
181−192.
(10) Guiguemde, W. A.; Shelat, A. A.; Garcia-Bustos, J. F.; Diagana,
T. T.; Gamo, F. J.; Guy, R. K. Global phenotypic screening for
antimalarials. Chem. Biol. 2012, 19, 116−129.
(11) Conroy, T.; Guo, J. T.; Hunt, N. H.; Payne, R. J. Total synthesis
and antimalarial activity of symplostatin 4. Org. Lett. 2010, 12, 5576−
5579.
(12) Conroy, T.; Guo, J. T.; Linington, R. G.; Hunt, N. H.; Payne, R.
J. Total synthesis, stereochemical assignment, and antimalarial activity
of gallinamide A. Chem.Eur. J. 2011, 17, 13544−13552.
(13) Linington, R. G.; Clark, B. R.; Trimble, E. E.; Almanza, A.;
(29) Rosenthal, P. J.; Lee, G. K.; Smith, R. E. Inhibition of a
Plasmodium vinckei cysteine proteinase cures murine malaria. J. Clin.
Invest. 1993, 91, 1052−1056.
Urena, L.-D.; Kyle, D. E.; Gerwick, W. H. Antimalarial peptides from
̃
marine cyanobacteria: isolation and structural elucidation of
gallinamide A. J. Nat. Prod. 2009, 72, 14−17.
̀
(30) Ettari, R.; Bova, F.; Zappala, M.; Grasso, S.; Micale, N.
Falcipain-2 inhibitors. Med. Res. Rev. 2010, 30, 136−167.
(31) Miller, B.; Friedman, A. J.; Choi, H.; Hogan, J.; McCammon, J.
A.; Hook, V.; Gerwick, W. H. The marine cyanobacterial metabolite
gallinamide A is a potent and selective inhibitor of human cathepsin L.
J. Nat. Prod. 2014, 77, 92−99.
(14) Taori, K.; Liu, Y.; Paul, V. J.; Luesch, H. Combinatorial
strategies by marine cyanobacteria: symplostatin 4, an antimitotic
natural dolastatin 10/15 hybrid that synergizes with the coproduced
HDAC inhibitor largazole. ChemBioChem 2009, 10, 1634−1639.
(15) Stolze, S. C.; Deu, E.; Kaschani, F.; Li, N.; Florea, B. I.; Richau,
K. H.; Colby, T.; van der Hoorn, R. A. L.; Overkleeft, H. S.; Bogyo,
M.; Kaiser, M. The antimalarial natural product symplostatin 4 is a
nanomolar inhibitor of the food vacuole falcipains. Chem. Biol. 2012,
19, 1546−1555.
(32) Rosenthal, P. J.; McKerrow, J. H.; Aikawa, M.; Nagasawa, H.;
Leech, J. H. A malarial cysteine proteinase is necessary for hemoglobin
degradation by Plasmodium falciparum. J. Clin. Invest. 1988, 82, 1560−
1566.
(33) Kerr, I. D.; Lee, J. H.; Pandey, K. C.; Harrison, A.; Sajid, M.;
Rosenthal, P. J.; Brinen, L. S. Structures of falcipain-2 and falcipain-3
bound to small molecule inhibitors: implications for substrate
specificity. J. Med. Chem. 2009, 52, 852−857.
(16) Rosenthal, P. J. In Cysteine Proteases of Pathogenic Organisms;
Robinson, M. W., Dalton, J. P., Eds.; Springer-Verlag: Berlin, 2011;
Vol. 712, p 30.
(17) Rosenthal, P. J.; Sijwali, P. S.; Singh, A.; Shenai, B. R. Cysteine
proteases of malaria parasites: targets for chemotherapy. Curr. Pharm.
Des. 2002, 8, 1659−1672.
(18) Sijwali, P. S.; Rosenthal, P. J. Gene disruption confirms a critical
role for the cysteine protease falcipain-2 in hemoglobin hydrolysis by
Plasmodium falciparum. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 4384−
4389.
(19) Sijwali, P. S.; Koo, J.; Singh, N.; Rosenthal, P. J. Gene
disruptions demonstrate independent roles for the four falcipain
cysteine proteases of Plasmodium falciparum. Mol. Biochem. Parasitol.
2006, 150, 96−106.
(20) Verissimo, E.; Berry, N.; Gibbons, P.; Cristiano, M. L. S.;
Rosenthal, P. J.; Gut, J.; Ward, S. A.; O’Neill, P. M. Design and
synthesis of novel 2-pyridone peptidomimetic falcipain 2/3 inhibitors.
Bioorg. Med. Chem. Lett. 2008, 18, 4210−4214.
(21) Kerr, I. D.; Lee, J. H.; Farady, C. J.; Marion, R.; Rickert, M.;
Sajid, M.; Pandey, K. C.; Caffrey, C. R.; Legac, J.; Hansell, E.;
McKerrow, J. H.; Craik, C. S.; Rosenthal, P. J.; Brinen, L. S. Vinyl
sulfones as antiparasitic agents and a structural basis for drug design. J.
Biol. Chem. 2009, 284, 25697−25703.
(22) Coteron
́
, J. M.; Catterick, D.; Castro, J.; Chaparro, M. A. J.;
dez, E.; Ferrer, S.; Gamo, F. J.; Gordo, M.; Gut, J.; de
Díaz, B.; Fernan
́
las Heras, L.; Legac, J.; Marco, M.; Miguel, J.; Munoz, V.; Porras, E.; de
̃
la Rosa, J. C.; Ruiz, J. R.; Sandoval, E.; Ventosa, P.; Rosenthal, P. J.;
Fiandor, J. M. Falcipain inhibitors: Optimization studies of the 2-
pyrimidinecarbonitrile lead series. J. Med. Chem. 2010, 53, 6129−6152.
(23) Breuning, A.; Degel, B.; Schulz, F.; Buchold, C.; Stempka, M.;
̈
Machon, U.; Heppner, S.; Gelhaus, C.; Leippe, M.; Leyh, M.; Kisker,
C.; Rath, J.; Stich, A.; Gut, J.; Rosenthal, P. J.; Schmuck, C.;
Schirmeister, T. Michael acceptor based antiplasmodial and anti-
trypanosomal cysteine protease inhibitors with unusual amino acids. J.
Med. Chem. 2010, 53, 1951−1963.
G
dx.doi.org/10.1021/jm501439w | J. Med. Chem. XXXX, XXX, XXX−XXX