Diverted total synthesis of falcitidin acyl tetrapeptides as new antimalarial leads

Article abstract of DOI:10.1016/j.tetlet.2014.02.008

We report not only the convergent total synthesis of falcitidin, a natural inhibitor of falcipain-2 from myxobacterium Chitinophaga, but also its diversification into a new antimalarial class of N-acyl tetrapeptides (Acyl-His-Ile-Val-Pro-NH₂). Despite the lack of whole-cell activity of falcitidin itself, our study led to the identification of a trifluoromethyl (CF₃) analogue displaying sub-micromolar IC₅₀ activity against Plasmodium falciparum 3D7 in a standard blood-cell assay, but only when N-tritylated on its histidine (imidazole) residue.

Full text of DOI:10.1016/j.tetlet.2014.02.008

Tetrahedron Letters 55 (2014) 1949–1951
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Diverted total synthesis of falcitidin acyl tetrapeptides as new
antimalarial leads
Santosh R. Kotturi a, Brinda Somanadhan b,c, Jun-Hong Ch’ng d, Kevin S.-W. Tan d, Mark S. Butler a,b,e
,
Martin J. Lear a,f,
⇑
a Department of Chemistry and Medicinal Chemistry Program, 3 Science Drive 3, National University of Singapore, Singapore 117543, Singapore
b MerLion Pharmaceuticals, 41 Science Park Road, #04-03B The Gemini, Singapore Science Park II, Singapore 117610, Singapore
c Takeda Vaccines Pte Ltd, #04-10 Capricorn, 1 Science Park Road, Singapore Science Park II, Singapore 117528, Singapore
d Department of Microbiology, Yong Loo Lin School of Medicine, 5 Science Drive 2, Singapore 11759, Singapore
e Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, University of Queensland, St Lucia 4072, Queensland, Australia
f Department of Chemistry, Graduate School of Science, Tohoku University, Aza Aramaki, Aoba-ku, Sendai 980-8578, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We report not only the convergent total synthesis of falcitidin, a natural inhibitor of falcipain-2 from
myxobacterium Chitinophaga, but also its diversification into new antimalarial class of N-acyl
tetrapeptides (Acyl-His-Ile-Val-Pro-NH2). Despite the lack of whole-cell activity of falcitidin itself, our
study led to the identification of a trifluoromethyl (CF3) analogue displaying sub-micromolar IC50 activity
against Plasmodium falciparum 3D7 in a standard blood-cell assay, but only when N-tritylated on its
histidine (imidazole) residue.
Received 8 January 2014
Revised 30 January 2014
Accepted 4 February 2014
Available online 13 February 2014
a
Keywords:
Falcitidin
Ó 2014 Elsevier Ltd. All rights reserved.
Tetrapeptide
Trifluoromethyl
Natural products
Antimalarial agents
Due to the ever evolving resistance mechanisms of Plasmodium
parasites to available drugs, especially to the quinolines and
artemesinins,1 malaria continues to be a threat to both mammals
and humans alike.2 It thus remains important that new
compounds, new targets and new pathways are translated into
new antimalarial regimes.3,4 As an apt starting-point, Nature
continues to be a diverse source of bio-relevant leads and we have
recently described the enzyme-guided discovery of sub-milligram
amounts of falcitidin (1) from a myxobacterium Chitinophaga sp.
strain Y23.5 Through a linear total synthesis, we characterized
Immediately after determining the stereochemistry of the
natural tetrapeptide,5 we were keen to determine if the in vitro
enzyme-activity of falcitidin (1) could translate into our standard
whole-cell antimalarial assay.4e,6 We thus devised a convergent to-
tal synthesis of 1 that could be diversified via a common tripeptide
intermediate 3, so as to provide a set of N -acylated, all-L ana-
a
logues 2 (Fig. 1). Our convergent route to naturally configured 1 be-
gan with the four readily available building blocks 4, 5, 6 and 7
(Scheme 1).
First, commercial Nim-trityl-D-histidine 5 was converted into its
the natural product to be isovaleric acid-
D
-His-
L
-Ile-
L
-Val-
L
-Pro-
corresponding bis-TMS ether with TMSCl/Et3N7 and coupled imme-
NH2 with an IC50 value of 6 M against the falcipain-2 cysteine
l
diately with the mixed anhydride of isovaleric acid 4 by using ethyl
protease of Plasmodium falciparum in an in vitro enzyme-based
screening assay (Fig. 1). Herein, we describe a convergent and
diverted total synthesis of 1 in order to identify preliminary
analogues with the base structure 2 with improved whole-cell
activities in a standard antimalarial blood assay.6
chloroformate/NMM.8 This afforded the trityl protected N-acyl-
D-
histidine 8. In parallel, the N-Boc proline amide 7 was deprotected
with TFA and the crude amine salt was coupled immediately with
the known dipeptide N-Boc-
This gave the key tripeptide intermediate N-Boc-
L-Ile-L
-Val 65 by using HATU/HOAt.9
L-Ile-L-Val-L-Pro-
NH2 3, which was also used later for SAR diversification purposes.
The use of EDC, HOBt and NMM types of amide coupling protocols
did not give better yields.10 Lastly, the Boc group of tripeptide 3 was
⇑
Corresponding author. Tel.: +81 22 795 6565; fax: +81 22 795 6566.
E-mail address: Martin.Lear@m.tohoku.ac.jp (M.J. Lear).
Previous address.
cleaved using TFA and coupled to N-acyl-D-histidine 8 using HATU/
HOAt to give the Nim-trityl protected tetrapeptide 9 in good yield.
http://dx.doi.org/10.1016/j.tetlet.2014.02.008
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.

1950
S. R. Kotturi et al. / Tetrahedron Letters 55 (2014) 1949–1951
O
N
O
CONH2
H
H
D
N
N
N
N
H
O
O
N
Falcitidin (1)
H
Nα-(isovaleroyl)-D-His-L-Ile-L-Val-L-Pro-NH2
Diversify via
Boc-L-Ile-L-Val-L-Pro-NH2 (3)
O
N
O
CONH2
H
H
R1
N
N
L
N
N
H
O
O
N
all-L, acyl tetrapeptides (2)
R2
N
α-(R1CO)-L-His-
N
(
im R2)-L-Ile-L-Val-L-Pro-NH2
Figure 1. Falcitidin-derived (1) acylpeptide lead compound template 2 to explore
preliminary structure–activity relationships (SAR) via tripeptide 3.
Scheme 2. Diverted synthesis of all-L, N-acyl tetrapeptide analogues 2 of falcitidin
(1) via the common tripeptide N-Boc-L-Ile-L-Val-L-Pro-NH2 3.
O
H2N
OH
O
H
CONH2
O
N
BocHN
OH
BocN
N
Table 1
OH
N
O
Yields of falcitidin acylated analogues 2a–h (from amino-peptide 13) and their IC50
values against P. falciparum 3D7 in a whole-cell assay (standardized with commercial
Tr
4
5
6
7
chloroquine; IC50 0.020 0.002 l
M)4e,6
Entry
R1C(O)
R2
Yield (%)
IC50 (lM)
(ii) 4, EtOCOCl, NMM
THF, 0 °C to rt
76% (2 steps)
(ii) 6, HATU, HOAt, DIPEA
DMAP (cat.), CH2Cl2
0 °C to rt, 62% (2 steps)
(i) 5, TMSCl
Et3N, CH2Cl2
(i) 7, TFA
CH2Cl2, rt
O
2a
2b
Tr
H
79 (from 13)
80 (from 2a)
1.6
1.2
O
H
N
HO
H
OH
O
CONH2
H
2c
Tr
H
67 (from 13)
20 (from 2c)
1.4
O
N
BocHN
N
O
N
N
O
2d
>10
Tr
8
3
O
O
2e
H
37 (from 13)
1.3
OMe
(ii) 8, HATU, HOAt DIPEA
DMAP, CH2Cl2, 79% (2 steps)
(i) 3, TFA
CH2Cl2
O
2f
H
33 (from 13)
3.3
N
H
O
H
O
CONH2
H
CF3
MeO
N
N
N
N
2g
2h
Tr
H
89 (from 13)
70 (from 2g)
0.14
2.4
H
O
O
O
N
N
R2
9, R2= Tr (IC50 0.5 μM)
TFA, CH2Cl2
iPr3SiH, 80%
1, R2 = H (IC50 > 10 μM)
configuration of the histidine amino acid in protected and
unprotected form, and (ii) replacement of the isovaleric acid unit
with differing hydrogen bonding acyl acceptors–donors that may
possess potential antimalarial pharmacophoric properties.
Scheme 1. Convergent total synthesis of falcitidin (1) via the key tripeptide
intermediate 3 and the whole-cell active N-trityl analogue 9.
Under our initial consideration, the histidine amino acid was
omitted altogether. We thus synthesized the truncated isovaleric
analogue 11 by following standard peptide coupling methods from
tripeptide 3 via the TFA-amine salt 10 (Scheme 2). As anticipated,
The final peptide 1 was obtained by removal of the trityl group of 9
using TFA in the presence of triisopropylsilane.11 The NMR and
HPLC profiles of synthetic material matched closely to those of
the natural product 1 as reported previously.5
11 showed a lack of activity (IC50 >10 lM). Next, 3 was diverted to
With synthetic material in hand, falcitidin 1 was soon found to
display poor whole-cell activity against chloroquine-sensitive P.
the all-L analogues 2 through a short, but linear amidation–acyla-
tion sequence via the all-L Fmoc-tetrapeptide 12. Fmoc N-terminus
protection was selected over typical Boc-protection so that N-trity-
lated imidazole analogues 2 could be obtained and biologically
falciparum 3D7 (IC50 >10
in vitro falcipain-2 enzyme activity (IC50 6
of cell permeability, we tested the trityl precursor 9, which dis-
played good whole cell activity (IC50 0.5 M). With the aim of
l
M) in spite of possessing acceptable
l
M).5 Suspecting issues
tested en route. Thus, N-Fmoc-L-His-Nim(Tr)-L-Ile-L-Val-L-Pro-NH2
l
12 was prepared by coupling 10 with commercial N -Fmoc-
a
exploring further antimalarial relationships, we thus synthesized
various all-L analogues 2a–h of the falcipain-2 inhibitor 1 by diver-
sifying the common tripeptide fragment 3 (Scheme 2, Table 1).
Here, our preliminary design of falcitidin analogues 2a–h was
based on two main considerations: (i) the importance and
Nim-trityl-L
-histidine. Fmoc deprotection of 12 with piperidine12
gave the key amino-tetrapeptide 13, which was diversified with
various carboxylic acids (R1CO2H) under HATU/HOAt amide
coupling conditions9 to afford the trityl analogues 2 (R2 = Tr). These
were also de-tritylated to 2 (R2 = H) for biological comparison.

S. R. Kotturi et al. / Tetrahedron Letters 55 (2014) 1949–1951
1951
Table 1 summarizes the yields of formation (from amino-tetrapeptide
13) and the whole-cell antimalarial activities of purified analogues
2a–h against P. falciparum 3D7 (with commercial chloroquine as a
standard; IC50 of 20 2 nM).4e
for initial antimalarial tests. Latterly, this work was supported by
the National Medical Research Council (NMRC/1310/2011) of
Singapore (to K.S.W.T., J.H.C. and M.J.L.).
Under our second consideration, the natural product like, but
Supplementary data
all-L isovaleric analogues 2a,b (R1 = iBu; R2 = Tr or H) and
a-hydro-
xy phenyl acylated derivatives 2c,d (R1 = (S)-PhCH(OH); R2 = Tr or
H) were synthesized from 13 (Scheme 2, Table 1). Interestingly,
the all-L analogues 2a,b of falcitidin showed similar whole-cell
antimalarial activities, whether tritylated or not (IC50 range of
Supplementary data (experimental procedures and character-
isation data for all new compounds)associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
j.tetlet.2014.02.008.
1.3–1.6
l
M). This is in contrast to the Nim-tritylated derivative
M over its
2c, which showed a significant IC50 activity of 1.4
l
References and notes
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a
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Tetrapeptides 2a–h possessing a hydrophobic N-trityl group on
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its tritylated form (IC50 of 0.14 lM). Importantly, this focused set
of falcitidin-derived compounds (except 2e) lack irreversible cys-
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Acknowledgments
We thank the Ministry of Education of Singapore for initial
funding (AcRF Tier-2 Grant T206B1112) and a Ph.D. graduate
scholarship (to S.R.K.). We appreciate the help of Alvin Choong