Synthesis of macrocyclic trypanosomal cysteine protease inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.06.012

The importance of cysteine proteases in parasites, compounded with the lack of redundancy compared to their mammalian hosts makes proteases attractive targets for the development of new therapeutic agents. The binding mode of K11002 to cruzain, the major cysteine protease of Trypanosoma cruzi was used in the design of conformationally constrained inhibitors. Vinyl sulfone-containing macrocycles were synthesized via olefin ring-closing metathesis and evaluated against cruzain and the closely related cysteine protease, rhodesain.

Full text of DOI:10.1016/j.bmcl.2008.06.012

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5860–5863
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of macrocyclic trypanosomal cysteine protease inhibitors
Yen Ting Chen a,b, Ricardo Lira a,b, Elizabeth Hansell c, James H. McKerrow c, William R. Roush a,b,
*
a Department of Chemistry, Scripps Florida, 5353 Parkside Drive, RE-2, Jupiter, FL 33458, USA
b Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA
c Sander Center for Basic Research in Parasitic Diseases, University of California, San Francisco, CA 94143, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The importance of cysteine proteases in parasites, compounded with the lack of redundancy compared to
their mammalian hosts makes proteases attractive targets for the development of new therapeutic
agents. The binding mode of K11002 to cruzain, the major cysteine protease of Trypanosoma cruzi was
used in the design of conformationally constrained inhibitors. Vinyl sulfone-containing macrocycles were
synthesized via olefin ring-closing metathesis and evaluated against cruzain and the closely related cys-
teine protease, rhodesain.
Received 26 April 2008
Revised 2 June 2008
Accepted 4 June 2008
Available online 10 June 2008
Keywords:
Cysteine protease inhibitors
Macrocyclic dipeptidyl vinyl sulfones
Ó 2008 Elsevier Ltd. All rights reserved.
Trypanosomes are parasitic protozoa responsible for several ne-
glected diseases of global health importance including Chagas’ dis-
ease and sleeping sickness. Chagas’ disease, or American
trypanosomiasis, is a chronic infection caused by the parasite, Try-
panosoma cruzi, and is the leading cause of heart failure in many
Latin American countries.1 T. cruzi is transmitted to humans
through the bite of the triatomine bug or by transfusion of infected
blood. The overall prevalence of human infection is estimated at
16–18 million cases with 13,000 deaths reported each year.2 Try-
panosoma brucei gambiense and Trypanosoma brucei rhodesiense
are the pathogenic agents of human African trypanosomiasis, or
sleeping sickness. These parasites live extracellularly in blood
and tissue fluids of the mammalian host and are transmitted by
the bite of tsetse flies. The disease is endemic in certain regions
of sub-Saharan Africa, covering about 50 million people in 36 coun-
tries. It is estimated that 50,000 to 70,000 people are currently in-
fected; if left untreated, the disease in humans is fatal.3
Current drug therapy for trypanosomal diseases is not always
effective and is often hampered by severe side effects.4 Thus, the
identification of novel targets for trypanocidal agents is needed.
One such target is the major cysteine protease of the parasitic
organisms, which includes cruzain5 in T. cruzi and rhodesain6 in
T. brucei rhodesiense. Both enzymes are clan CA proteases, share
70% similarity in primary structure, and are involved in critical
roles in parasite survival, such as replication, penetration into host
cells, nutrition at the expense of the host, and immunoevasion.7
Selective inhibitors of cruzain have been demonstrated to cure T.
cruzi infection both in cell culture screens and in mouse models
of Chagas’ disease.8 In a recent report, a cruzain inhibitor was also
found to be effective in treating Chagas’ disease in beagle dogs.9
A large number of cysteine protease inhibitors have been re-
ported to date, several classes of which are potent, irreversible
inhibitors.10,11 Based on the pioneering studies by Hanzlik and
Liu12 and Palmer et al.,13 our group has developed peptidyl vinyl
sulfone inhibitors of parasitic cysteine proteases.14,15 The vinyl
sulfones serve as Michael acceptors for the nucleophilic active site
cysteine, and the peptidic backbone contains several hydrogen
bond acceptors that interact with complementary residues in the
active site. Several cruzain-inhibitor complexes have been solved
by X-ray crystallography, which displayed the active site Cys25
of cruzain covalently bound to the vinyl sulfone unit of the
inhibitor.16
With the aim to improve upon the lead compounds from previ-
ous studies and to develop an inhibitor with a broad spectrum of
activity against a variety of parasitic hosts, we were interested in
the design of conformationally constrained vinyl sulfones. Limiting
conformational flexibility of the inhibitor or ligand is a well-estab-
lished strategy to improve binding energies by decreasing the
entropic barrier to binding of a particular conformation. Hence,
in principle, by tethering distal segments of the inhibitors to form
a rigid structure with a conformation favorable to binding, selectiv-
ity and/or potency of the inhibitor can be improved. This approach
has been implemented in the design of various biologically active
molecules such as aspartyl protease inhibitors,17 and Grb2 SH2 do-
main-binding ligands.18
The crystal structures of cruzain with bound vinyl sulfonyl
inhibitors are instrumental in elucidating the key factors that con-
tribute to inhibitor binding. Examination of the crystal structures
of cruzain with vinyl sulfones such as K11002 (1) reveal several
highly conserved binding interactions.16 These include hydrogen
* Corresponding author. Tel.: +1 561 799 8880.
E-mail address: roush@scripps.edu (W.R. Roush).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.012

Y. T. Chen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5860–5863
5861
bonding between the side chains of Gln19, His159, and Trp177
with the sulfonyl oxygen atoms, a hydrogen bond between the P1
nitrogen with the Asp158 peptide carbonyl, a hydrogen bond be-
tween the P2 carbonyl and the Gly66 amide, and a hydrogen bond
between the P2 amide nitrogen with the Gly66 carbonyl (Fig. 1b).16
The S2 pocket is the primary recognition element for cruzain and
all other enzymes in the papain class. The Phe side chain of
K11002 and other similar inhibitors is deeply buried in the well-
O
O
H
N
MeO
MeO
O
N
NH
O
N
NH
O
4, 5
Me
O
Me
O
OH
n
Ot-Bu
n
7
6
O
MeO
NH2
OH
defined S2 pocket. However, the S0 , S1, and S3 pockets are very shal-
N
Me
1
MeO
Ot-Bu
low and poorly defined, therefore the P0 , P1, and P3 groups are
+
+
NH2
n
1
O
O
highly solvent exposed. In addition, the urea carbonyl does not par-
ticipate in any interactions with the enzyme and thus was assumed
to be non-essential to inhibitor binding.
10
11
8, n = 1
9, n = 2
In designing a conformationally rigid inhibitor scaffold, we
aimed to preserve the geometry of the peptide backbone and all
of the critical hydrogen bonding interactions identified in Figure
1b. Since the P1 side chain and the urea carbonyl are not involved
in specific binding interactions to the enzyme, they were selected
as the sites to form a conformationally restricted linkage. The dis-
Figure 2. Disconnective analysis of inhibitors 4 and 5.
the synthesis of two members of this class of compounds (4 and
5) and their inhibitory potency against cruzain and rhodesain.
As illustrated in Figure 2, we sought to assemble inhibitors 4
and 5 from scaffold 6, which could be accessible from diene 7 via
ring closing olefin metathesis. Diene 7, in turn, could be prepared
from either allylglycine or homoallylglycine which serves as the
tance between the urea carbonyl carbon and the c-carbon of hom-
oPhe in 1 when bound to cruzain is 4.51 Å, therefore we reasoned
that a 10- or 11-membered macrocycle would provide the optimal
ring size to preserve the bioactive peptide backbone conformation.
In addition, introduction of a linking unit between these two units
was expected not to interfere with enzyme binding, since the P1
homoPhe residue is highly solvent exposed in the available crystal
structures.16 We anticipated that 2 would not be chemically stable,
due to the acid labile aminal moiety. This was addressed by replac-
‘P1’ fragment (8 or 9), leucine methyl ester (10), and
ter 11.
a-hydroxyes-
The synthesis of the ‘P1’ fragments of 8 and 9 is illustrated in
Scheme 1. Weinreb amide 13 was prepared by functionalization
of allylglycine (12) as described by Borzilleri et al.19 Deprotection
of the Boc group afforded amine 8 as a TFA salt. The synthesis of
the Weinreb amide of homoallylglycine (9) began with asymmetric
alkylation of pseudoephedrine glycinamide (14)20 with 4-bromo-
1-butene (15) to provide 16. Removal of the chiral auxiliary fol-
lowed by Boc protection of the amine and coupling of the resulting
acid with N,O-dimethylhydroxylamine21 gave Weinreb amide 18.
Finally, deprotection of the Boc group provided homoallylglycine 9.
Allylboration of t-butyl glyoxylate 1922 with (S,S)-2023 gave
ing the aminal unit with an a-amino amide as shown in 3. Finally,
we also elected to use leucine as the P2 residue as shown in 4 and 5
to facilitate the extension of this strategy to the synthesis of inhib-
itors that might be effective against other parasitic CA proteases
that are much more specific at P2 than cruzain. We herein report
a
-hydroxyester 11 with 74% ee24 (Scheme 2). This material was
carried onto the next two steps without further enantiomeric
enrichment. The alcohol was converted to the corresponding tri-
flate and this was used to alkylate leucine methyl ester (10) which
provided 21 as a ca. 5:1 diastereomeric mixture which was sepa-
rated by silica column chromatography. Amine 21 was protected
Scheme 1. Reagents and conditions: (a) (i) Boc2O, NaHCO3, H2O, THF, 0–23 °C, 12 h;
(ii) CH3(CH3O)NHHCl, HOBT, NMM, EDC, CH2Cl2, 0–23 °C, 12 h, 84% (two steps); (b)
TFA, CH2Cl2, quant.; (c) LDA, LiCl, THF, 0 °C, 29 h, 62% (de > 20:1); (d) (i) NaOH, H2O,
reflux, 3 h; (ii) Boc2O, NaOH, H2O, dioxane, 0–23 °C, 16 h, 82% (two steps); (e)
CH3(CH3O)NHHCl, HOBT, NMM, EDC, CHCl3, 0–23 °C, 14 h, 88%; (f) TFA, CH2Cl2, 0–
23 °C, 1 h, quant.
Figure 1. (a) K11002 and its 3D structure when bound to cruzain; (b) design
rationale for conformationally constrained inhibitors 4 and 5.

5862
Y. T. Chen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5860–5863
Scheme 2. Reagents and conditions: (a) 16, toluene, 4 Å mol. sieves, ꢀ78 °C, 3 h, 70% (74% ee); (b) (i) Tf2O, pyridine, CH2Cl2, 0–23 °C, 30 min; (ii) 10, proton sponge, CHCl3,
ꢀ78 to 23 °C, 18 h, 70% (two steps); (c) CbzCl, n-BuLi, Et2O, ꢀ78 to 23 °C, 72 h, 55%; (d) (i) NaOH, THF, MeOH, 0 °C; (ii) 8 or 9, EDC, HOBT, NMM, CH2Cl2, 0–23 °C, 14 h (two
steps), 23 (77%), 24 (68%); (e) 25, 1,2-dichloroethane, 95 °C, 1 h; (f) H2, 10% Pd/C, EtOAc, EtOH, 36 h, 26 (65% from 23), 27 (67% from 24); (g) (i) TFA, CH2Cl2, 6 h; (ii)
benzylamine, EDC, HOBT, NMM, CH2Cl2, 0–23 °C, 14 h (two steps), 28 (89%), 29 (73%); (h) (i) LiAlH4, THF, ꢀ10 °C 30 min; (ii) 30, NaH, THF, 0–23 °C; 4–8 h (two steps), 4 (58%),
5 (51%).
with a Cbz group using n-butyllithium as the base, since attempted
ring-closing metathesis of the amine corresponding to 24 was
unsuccessful presumably due to catalyst poisoning.25 The require-
ment for a strong base in this step is most likely due to the poor
accessibility of the highly hindered amine proton. Selective hydro-
lysis of the methyl ester in compound 22, followed by coupling
with Weinreb amide 8 or 9 gave dienes 23 and 24, respectively,
which set the stage for macrocyclization via ring-closing olefin
metathesis.26 Macrocycle formation was achieved by treatment
of either 23 or 24 with 20 mol % of second generation Grubbs’ cat-
alyst 2527 in 1,2-dichloroethane (0.8 mM substrate concentra-
tion).28 Use of other solvents and higher reaction concentration
led to substantial amounts of oligomers. The resulting alkene unit
and Cbz group were removed by hydrogenation to yield 26 and 27.
Cleavage of the t-butyl ester by treatment with TFA, followed by
coupling of the resulting carboxylic acid with benzyl amine gave
28 and 29. Finally, the vinyl sulfone group was introduced by
reduction of the Weinreb amide to the corresponding aldehyde,
followed by Horner–Wadsworth–Emmons olefination with phos-
phonate 30.29 This yielded the targeted macrocyclic cysteine prote-
ase inhibitors 4 and 5 in 51% and 58% yield, respectively.
The activities of vinyl sulfones 4 and 5 as cysteine protease
inhibitors were tested against cruzain and rhodesain. Recombi-
nantly expressed cruzain or rhodesain was incubated with the
inhibitor in sequential dilution followed by addition of Z-Phe-
Arg-AMC as a fluorescent substrate. The increase in fluorescence
produced by cleavage of the substrate allows determination of pro-
tease inhibition. IC50 values were determined in the liner portion of
a plot of inhibition versus log of inhibitor concentration.
The results are summarized in Table 1. For comparison,
K117778 was also assayed under similar conditions. The conform-
ationally constrained compounds were substantially less active as
inhibitors of cruzain and rhodesain compared to the acyclic vinyl
sulfone. Owing to the very weak inhibitor potency, full kinetic
analyses of these two inhibitors were not determined. Of the two
macrocyclic inhibitors, 5 was more effective, which suggests that
Table 1
Inhibition of cruzain and rhodesain by vinyl sulfone derivatives
Compounds
IC50 (lM)
Rhodesain
Cruzain
4
5
10
6
0.1
>10
2
0.004
K11777
the 11-membered macrocycle may have more flexibility to adopt
a geometry that is more favorable for binding than the 10-mem-
bered ring scaffold of 4. Our current hypothesis is that P3 unit in
4 and 5 is suboptimal compared to the P3 units in K11002 and
other dipeptidyl vinylsulfonyl inhibitors.16 It is possible that the
free amine in 4 or 5, which will be protonated under conditions
of the enzyme assay, may negatively impact the binding of these
inhibitors to the enzyme targets. It is also possible that the urea
units of acyclic cysteine protease inhibitors (cf. K11002) are much
more important to inhibitor binding than initially assumed. Stud-
ies addressing these issues via the synthesis and evaluation of a
second-generation series of conformationally constrained vinyl-
sulfonyl cysteine protease inhibitors will be reported in due course.
Acknowledgment
This research was supported by a grant from the National Insti-
tutes of Allergy and Infectious Diseases (Program Project Grant AI
35707).

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Supplementary data associated with this article can be found, in
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