Dipeptidyl-α,β-epoxyesters as potent irreversible inhibitors of the cysteine proteases cruzain and rhodesain

Article abstract of DOI:10.1016/j.bmcl.2007.10.056

The dipeptidyl epoxyesters 3 and 4 are potent, irreversible inhibitors of cruzain and rhodesain.

Full text of DOI:10.1016/j.bmcl.2007.10.056

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6697–6700
Dipeptidyl-a,b-epoxyesters as potent irreversible inhibitors
of the cysteine proteases cruzain and rhodesain
a,
a
a
*
´
´
Florenci V. Gonzalez, Javier Izquierdo, Santiago Rodrıguez,
James H. McKerrowb and Elizabeth Hansellb
a
`
`
´
Departament de Qu´ımica Inorganica i Organica, Universitat Jaume I, 12071 Castello, Spain
bSandler Center for Basic Research in Parasitic Diseases, Box 2550, University of California, San Francisco,
San Francisco, CA 94143-2550, USA
Received 5 September 2007; revised 15 October 2007; accepted 16 October 2007
Available online 22 October 2007
Abstract—The dipeptidyl epoxyesters 3 and 4 are potent, irreversible inhibitors of cruzain and rhodesain.
Ó 2007 Elsevier Ltd. All rights reserved.
Cysteine proteases are an important class of enzymes
involved in the hydrolysis of peptides and proteins.1,2
The papain family of cysteine proteases includes
cathepsins, calpains, and the parasitic cysteine prote-
ases cruzain and rhodesain which are essential for
the development and survival of the protozoan Try-
panosoma cruzi and Trypanosoma brucei, respectively.3
T. cruzi causes Chagas’ disease in humans in South
and Central America,4,5 whereas T. brucei causes
sleeping sickness in humans in large areas of central
and southern Africa.6,7 Consequently, inhibition of
cysteine proteases has emerged as an important strat-
egy for the treatment of these diseases.
3 and 4, that combine the reactivity of the E-64c9,10
epoxide electrophile with the Cbz-Phe-HPhe sequence
already known as a selective moiety for effective cruzain
inhibition.11–13
Although the stereochemistry of the epoxide in E-64c is
2(S),3(S) and precedents8 of epoxy inhibitors pointed to
2(S) as the best selection, we carried out a synthesis of
both isomeric epoxides 3 and 4.
The preparation of these inhibitors involved an enantio-
selective anti aldol14 reaction using thiazolidinethione 5
(prepared by acylation of thiazolidinethione derived
from L-phenylalanine), ethyl 4-oxo butenoate (prepared
from furfural),15 and MgBr2 as a catalyst (Scheme 1).
The crude mixture of protected aldols was then treated
with 1 M aq HCl and separated by chromatography,
affording a 4:1 mixture of free aldols 6 and 7. Then pro-
tection and removal of the chiral auxiliary in 6 with
H2O2 and LiOH gave carboxylic acid 8 which was sub-
mitted to Curtius reaction16 using DPPA and Et3N
affording isocyanate 9. Compound 9 was then directly
In 1998 dipeptidyl a,b-epoxy ketones 1 and 2 were re-
ported as cysteine protease inhibitors.8 For improved
potency and selectivity, we envisioned a new class of
inhibitors, represented by structures 3 and 4, which
incorporated the epoxyester unit.
We report herein the design and synthesis of a new class
of cysteine protease inhibitors, represented by structures
O
Ph
3
O
Ph
3
O
H
O
H
O
2
H
O
2
N
CO2H
Ph
R
O
N
R
Ph
O
N
N
N
N
H
H
H
O
O
O
O
O
Ph
Ph
E-64c
2, R=H
4, R=CO2Et
1, R=H
3, R=CO2Et
Keywords: Cysteine proteases inhibitors; Epoxides; Epoxyesters; Dipeptides.
*
Corresponding author. Tel.: +34 964728241; fax: +34 964728214; e-mail: fgonzale@qio.uji.es
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.056

´
F. V. Gonzalez et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6697–6700
6698
S
S
O
OH
O
OH
S
O
1. MgBr2·Et2O
TMSCl, AcOEt
2. HCl
CO2Et
S
N
CO2Et
+
S
N
CO2Et
+
S
N
OHC
Ph
Ph
Bn
Bn
Bn
7
6
4 : 1
5
Ph
OTBS
Ph
OTBS
HO2C
1. TBSOTf, 2,6-lutidine
2. H2O2, LiOH
Cbz-Phe
DPPA, Et3N
tol.
6
OCN
CO2Et
9
CO2Et
DMAP
Ph
8
Ph
Ph
O
OTBS
O
OH
H
H
1. TBAF
2. TBPLi
D-M
N
N
3
Ph
O
N
CO2Et
Ph
O
N
CO2Et
O
H
H
O
Ph
O
Ph
10
11
syn:anti (7:3)
OTBS
OTBS
Cbz-Phe
DMAP
1. TBSOTf, 2,6-lutidine
2. H2O2, LiOH
OCN
7
DPPA, Et3N
tol.
HO2C
CO2Et
CO2Et
12
Ph
O
13
Ph
Ph
Ph
O
OTBS
O
OH
H
H
1. TBAF
2. TBPLi
D-M
N
N
4
Ph
O
N
CO2Et
Ph
O
N
CO2Et
H
H
O
Ph
O
Ph
15
14
syn:anti (7:3)
Scheme 1. Synthetic scheme for the preparation of dipeptidyl epoxyesters 3 and 4.
coupled17 with Cbz-Phe and 4-DMAP furnishing dipep-
tidyl enoate 10. Then compound 10 was deprotected and
epoxidized with TBPLi giving a 7:3 mixture of syn/anti
epoxyalcohols which were separated by chromatogra-
phy. The selectivity of the epoxidation reaction is in
agreement with our previous results related to the nucle-
ophilic epoxidation of c-hydroxy a,b-unsaturated es-
ters.18 Finally Dess–Martin oxidation19 of 11 syn
afforded ketone 3. The isomeric dipeptidyl epoxyester
4 was prepared by an analogous sequence starting from
aldol 7. In this case, the epoxidation of the dipeptidyl
allylic alcohol gave a 7:3 mixture, with the syn isomer
15 predominating.
The stereochemistries of 16 and 17 were assigned on
the basis of coupling constant analysis (J4,5 = 5.5 Hz
for 16 and J4,5 = 7.5 Hz for 17) and by NOE experi-
ments: 16 gave NOE between H-4 and homobenzylic
protons, whilst 17 gave NOE between H-4 and H-5.
The stereochemistries of the epoxides were assigned
through NMR experiments applied to lactones 18 and
19 (Fig. 2). Epoxides 11 and 15 were treated with so-
dium thiophenolate and the resulting diols were further
cyclized in acidic media. In both cases the resulting lac-
tones 18 and 19 gave NOE between H-2 and H-4 denot-
ing the relative syn stereochemistry of the preceding
epoxyalcohols.
The stereochemical assignments of 6 and 7 were verified
by NMR experiments of oxazolidinones 16 and 17,
respectively (Fig. 1). The derivatization of the aldols
into the cyclic compounds was accomplished by thiazoli-
dinethione removal and Curtius degradation sequence.
The dipeptidyl epoxyesters 3 and 4 and their preceding
epoxyalcohols 11 and 15 were screened against cruzain,
rhodesain, and T. brucei cathepsin B20 (Table 1). Alco-
hols 9 and 13 did not inhibit cysteine proteases whilst
O
O
O
O
CO2Et
CO2Et
16
1. H2O2, LiOH
2. DPPA, Et3N
1. H2O2, LiOH
2. DPPA, Et3N
HN
5
HN
H
5
6
7
4
4
H
H
Ph
17
H
NOE
NOE
Ph
Figure 1. Stereochemical assignments of 6 and 7 determined using the oxazolidinones derivatives 16 and 17.

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F. V. Gonzalez et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6697–6700
6699
NOE
O
Ph
O
Ph
O
O
O
OH
O
4
H
H
H
H
1. NaSPh
2
N
N
SPh
Ph
Ph
O
N
H
Ph
Ph
O
N
H
CO2Et
2. CSA, tol., Δ
O
Ph
OH
Ph
18
11
NOE
O
Ph
Ph
O
O
O
OH
O
4
H
H
H
N
H
1. NaSPh
2
N
SPh
O
N
H
O
N
H
CO2Et
2. CSA, tol., Δ
O
Ph
O
OH
Ph
19
15
Figure 2. Stereochemical assignments of epoxides 11 and 15 determined from lactone derivatives 18 and 19.
Table 1. Inhibitory effect and determination of IC50 values for novel dipeptidyl epoxyester inhibitors and previous inhibitors
Inhibition
IC50 (nM)
Compound
Tbb in vitro (%)
Cruzain (%)
Rhodesain (%)
Tbb CathB (%)
Cruzain
Rhodesain
Tbb CathB
11
15
À37
17
5
1
À4
3
6
3
n.d.
n.d.
20
n.d.
n.d.
3.5
30
n.d.
n.d.
3
42
À2
93
55
98
80
51
47
ꢀ1000
400
4
50
>1000
10
1
2
K11777
99
100
72
5
5
2000
Tbb, Trypanosoma brucei brucei.
3 and 4 inhibited cruzain and rhodesain. IC50 determina-
tions21 indicated that 3 and 4 are potent inhibitors of
cruzain and rhodesain, with 3 being more potent than
4, especially against rhodesain. Curiously, compound
3, having stereochemistry opposite to that of E-64c,
was the most active inhibitor. Neither 3 nor 4 inhibited
cathepsin B. In addition, dipeptidyl epoxyester 3 was
much more potent than its analog 1, while 4 was slightly
less potent than the corresponding epoxyketone 2. Com-
pound 3 was also the most active against Trypanosoma
brucei brucei in vitro.22
and both dipeptidyl epoxyesters 3 and 4 showed second
order rate constants that were lower than the values for
epoxyketones 1 and 2.
Further studies on the development of the dipeptidyl
epoxyesters series as cysteine protease inhibitors are
ongoing and the results will be reported.
Acknowledgments
´
This work was financed by Conselleria d’Educacio, Cul-
tura i Ciencia de la Generalitat Valenciana (GV05/071)
and Bancaixa-UJI foundation (P1 1A2005-14). We
thank Prof. William R. Roush for helpful discussions,
and we also acknowledge the National Foundation
Genoma Espan˜a for covering IP related issues.
Kinetic analyses were performed on the most interesting
compounds,23 which confirmed that they are time-
dependent inhibitors of cysteine proteases (Table 2).
The second order rate constant for inactivation of cruz-
ain by 3 is 3-fold greater than that of 4 and 4-fold great-
er in the case of rhodesain. Compound 3 displayed a
second order rate constant higher than that for E-64c,
`
References and notes
Table 2. Second order rate constants, kinact/Ki or kass (sÀ1 MÀ1), of
inhibitors 1, 2, 3, 4 and E-64c against cruzain, rhodesain, and T. brucei
cathepsin B21
1. McKerrow, J. H.; James, M. N. G. In Perspectives in Drug
Discovery and Design; Anderson, P. S., Kenyon, G. L.,
Marshall, G. R., Eds.; ESCOM Science Publishers:
Leiden, 1996; Vol. 6.
Compound Versus cruzain Versus rhodesain Versus Tbb
CathB kass
kinact/Ki
kinact/Ki
2. Otto, H.-H.; Schirmeister, T. Chem. Rev. 1997, 97, 133.
3. Eakin, A. E.; McGrath, M. E.; McKerrow, J. H.;
Fletterick, R. J.; Craik, C. S. J. Biol. Chem. 1993, 268,
6115.
4. McKerrow, J. H.; Sun, E.; Rosenthel, P. J.; Buvier, J.
Annu. Rev. Microbiol. 1993, 47, 821.
3
82,900
25,200
92,090
23,500
—
120
84.5
—
4
1
128,200
330,000
70,600
2
E-64c
—
—
—
—

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F. V. Gonzalez et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6697–6700
6700
5. Godal, T.; Nagera, J. In WHO Division of Control in
Tropical Diseases. World Health Organization: Geneva,
pp 12–13.
6. Molyneux, D. H. In Trypanosomiasis and Leishmaniasis:
Biology and Control; Hide, G., Mottram, J. C., Coombs,
G. H., Holmes, P. H., Eds.; CAB Int.: Oxford, 1997; pp
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multichannel pipettes to an equal volume of enzyme
inhibitor pre-incubated at 25 °C for 5 min. For each assay
set, studies using enzyme with no additions, enzyme with
DMSO vehicle, and enzyme in the presence of the
previously known, highly effective irreversible inhibitor
(N-methyl piperazine-Phe- HPhe-(CH@CHSO2 Ph)) Arris
Pharmaceuticals Inc., South San Francisco, CA) (Palmer,
J. T.; Rasnick, D.; Klaus, J. L.; Bro¨mme, D. J. Med.
Chem. 1995, 38, 3193 and Bro¨mme, D.; Klaus, J. L.;
Okamoto, K.; Rasnick, D.; Palmer, J. T. Biochem. J. 1996,
315, 85.) served as controls. Inhibitors, which had IC50
values of less than 1 lM, were further analyzed. Inhibition
data for all enzymes were determined similarly: rec cruzain
at 4 nM with 5 lM Z FR AMC (Km = 1 lM) in buffer A
(100 mM Na acetate pH 5.5 with 5 mM DTT and 0.001%
Triton X-100 (Sigma)); rec rhodesain at 4 nM enzyme and
5 lM Z-Phe-Arg-AMC (Km = 1 lM) in buffer A; and rec
Tb CatB at 40 nM enzyme and 5 lM Z-Phe-Arg-AMC
(Km = 60 lM) in buffer A. A direct read of the semi log
plot of reaction rate versus inhibitor concentration was
used to determine IC50.
7. WHO. Control and surveillance of African trypanosomi-
asis. World Health Org. Tech. Rep. Ser. 1998, 881.
´
8. Roush, W. R.; Gonzalez, F. V.; Hanssell, E.; McKerrow,
J. H. Bioorg. Med. Chem. Lett. 1998, 8, 2809.
9. Hanada, K.; Tamai, M.; Ohmura, S.; Sawada, J.; Seki, T.;
Tanaka, I. Agric. Biol. Chem. 1978, 42, 529.
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11. Scheidt, K.; Roush, W. R.; McKerrow, J. H.; Selzer, P.
M.; Hansell, E.; Rosenthal, P. Bioorg. Med. Chem. 1998,
6, 2477.
12. Roush, W. R.; Gwaltney, S. L., II; Cheng, J.; Scheidt, K.;
McKerrow, J. H.; Hanssell, E. J. Am. Chem. Soc. 1998,
120, 10994.
13. Roush, W. R.; Cheng, J.; Knapp-Reed, B.; Alvarez-
Hernandez, A.; McKerrow, J. H.; Hansell, E.; Engel, J. C.
Bioorg. Med. Chem. Lett. 2001, 11, 2759.
22. Mackey, Z. B.; Baca, A. M.; Mallari, J. P.; Apsel, B.;
Shelat, A.; Hansell, E. J.; Chiang, P. K.; Wolff, B.; Guy,
K. R.; Williams, J.; McKerrow, J. H. Chem. Biol. Drug.
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14. Evans, D. A.; Downey, C. W.; Shaw, J. T.; Tedrow, J. S.
Org. Lett. 2002, 7, 1127.
23. Kinetic assays of irreversible inhibitors. Kinetic analyses
of the irreversible cysteine protease inhibitors were
performed as follows.20 (Tian, W.-X.; Tsou, C.-L. Bio-
chemistry 1982, 21, 1028.) Enzyme in 100 lL of assay
buffer was added to inhibitor dilutions in 100 lL of 10 lM
Z-Phe-Arg-AMC in buffer A (Ref. 20) using the FLEX
Station in FLEX mode. Enzyme was added by the
instrument while fluorescent emission was being followed,
allowing the first seconds of inhibition to be recorded.
Progress curves were recorded over 5 min at 25 °C (less
than 5% of substrate consumed) over a 10-fold range of
dilutions of inhibitor, starting at 10 lM, and 5 lM (10, 5,
1, 0.5, 0.1, 0.05, 0.01, and 0 lM). Inhibitor dilutions which
gave simple exponential progress curves over a wide range
of kobs were used to determine kinetic parameters
(excluding those values where inhibitor concentration
´
´
15. Rodrıguez, S.; Vidal, A.; Monroig, J. J.; Gonzalez, F. V.
Tetrahedron Lett. 2004, 45, 5359.
16. Ninomiya, K.; Shiori, T.; Yamada, S. Tetrahedron Lett.
1974, 30, 2151.
17. Schuemacher, A. C.; Hoffmann, R. W. Synthesis 2001, 2,
243.
´
´ ´
18. Rodrıguez, S.; Izquierdo, F.; Lopez, I.; Gonzalez, F. V.
Tetrahedron 2006, 62, 11112.
19. Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155.
20. Inhibition and kinetic assays were performed using puri-
fied recombinant enzymes, see Ref. 3 and Caffrey, C. C.;
Hansell, E.; Lucas, K. D.; Brinen, L. S.; Alvarez Hernan-
dez, A.; Cheng, J.; Gwaltney, S. L., II; Roush, W. R.;
Stierhof, Y.-D.; Bogyo, M.; Steverding, D.; McKerrow, J.
H. Mol. Biochem. Parasitol. 2001, 118, 61. For the used
methods, see: Beith, J. G. Methods Enzymol. 1995, 248, 59.
21. IC50 determination. The inhibition of the cysteine prote-
ases cruzain, rhodesain, and Tb CatB was evaluated by
quantitation of the fluorescence emission resulting from
proteolytic cleavage of the synthetic substrate Z-Phe-Arg-
AMC (Bachem). Inhibitors were used at concentrations
ranging from 10 nM to 10,000 nM (500–10,000 nM for Tb
CatB), with the minimum inhibitor concentration used for
calculations, at least 10-fold greater than enzyme; serial
dilutions were prepared in DMSO.20 These assays were
performed in a 96-well plate, scanning fluorescence reader
(Flex Station, Molecular Devices) at ex 355 and em
460 nm at 25 °C, with delivery of 100 lL of substrate by
was not 10-fold greater than enzyme). The value of kobs
,
the rate constant for loss of enzyme activity, was deter-
mined from an equation for pseudo first order dynamics
using Prism3.16 (GraphPad). When kobs varied linearly
with inhibitor concentration, kass was determined by linear
regression analysis.20 If the variation was hyperbolic,
indicating saturation inhibition kinetics, kinact and Ki were
determined from an equation describing a two-step
irreversible inhibitor mechanism (kobs = kinact [I]o/
([I]o + Ki (1 + [S]o/Km))) and nonlinear regression anal-
*
ysis using Prism.2 The value of kobs/[I] is given at a single
concentration of inhibitor only when the kobs was signif-
icantly different from the kobs for solvent, at the highest
concentration tested.