Identification of potent and reversible cruzipain inhibitors for the treatment of Chagas disease

Article abstract of DOI:10.1016/j.bmcl.2010.10.015

Identification of potent and reversible cruzipain inhibitors for the treatment of Chagas disease is described. The identified inhibitors bearing an amino nitrile warhead in P1 exhibit low nanomolar in vitro potency against cruzipain. Further SAR in P2 por

Full text of DOI:10.1016/j.bmcl.2010.10.015

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7444–7449
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of potent and reversible cruzipain inhibitors for the treatment
of Chagas disease
Christian Beaulieu a, , Elise Isabel a, Angélique Fortier a, Frédéric Massé a, Christophe Mellon a,
⇑
Nathalie Méthot a, Momar Ndao b, Deborah Nicoll-Griffith a, Doris Lee a, Hyeram Park a, W. Cameron Black a
a Merck Research Laboratories, 16711 Trans Canada Hwy, Kirkland, QC, Canada H9H 3L1
b National Reference Centre for Parasitology, McGill University, Montréal, Québec, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Identification of potent and reversible cruzipain inhibitors for the treatment of Chagas disease is described.
The identified inhibitors bearing an amino nitrile warhead in P1 exhibit low nanomolar in vitro potency
against cruzipain. Further SAR in P2 portion led to the identification of compounds, such as 26, that have
a unique selectivity profile against other cysteine proteases and offering new opportunities for safer treat-
ment of Chagas disease.
Received 8 September 2010
Revised 30 September 2010
Accepted 5 October 2010
Available online 12 October 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Cruzipain
Trypanosoma cruzi
Cysteine protease
Chagas disease
Chagas disease is a parasitic trypanozomial infection endemic
to South America where an estimated 10–12 million individuals
are afflicted and over 15,000 deaths are incurred each year.1 The
causative agent, Trypanosoma cruzi, is transmitted by an insect
vector, blood transfusions, maternal vertical transmission and,
rarely, oral infection. There are currently no FDA-approved drugs
for the treatment of Chagas infections and development of
effective therapies is currently a major focus of organizations such
as the Drugs for Neglected Diseases initiative (DNDi) and
OneWorld Health (iOWH).1–3
While numerous approaches for the treatment of Chagas are
under consideration, one family of enzymes, cysteine proteases
of the cruzipain family, is considered to be a promising target.4–6
The cruzipain enzymes have been identified as critical to the viabil-
ity of the parasite,7 and the ability of the trypanosomal form to
invade tissues8,9 and evade the host immune response.10,11 For
these reasons, cruzipain inhibitors have been proposed as potential
therapies for T. cruzi infection. Elegant studies with the irreversible
inhibitor K-777 (1, Fig. 1) have demonstrated the anti-parasitic
activity of a cruzipain inhibitor.12 K-777 has also shown efficacy
in pre-clinical models of Chagas disease.13,14 Other efforts toward
identifying irreversible covalent inhibitors have also been reported
by several research groups in recent years.15–18 These inhibitors
contain electrophilic groups (e.g., vinyl sulfonate, tetrafluorophen-
oxymethyl ketone, epoxy ketone) that serve as a ‘warhead’ that
binds to cruzipain through a covalent bond to the active site
cysteine. Unfortunately, irreversible inhibitors may also inhibit
host protease enzymes and possibly cause unacceptable side
effects due to off-target activity and/or autoimmune and inflam-
matory phenomena associated with protein covalent binding.7,19
Thus, the identification of reversible and specific inhibitors of
cruzipain would be of particular interest for the development of
a safer therapeutic agent for Chagas disease. However, only rare
examples of reversible covalent inhibitors of cruzipain have been
reported19 and specificity, if reported at all, is poor.
Cruzipain is a cysteine protease enzyme, closely related to the
cathepsin family of enzymes.20 Recently, the discovery of revers-
ible cathepsin K (Cat K) inhibitors has been reported. Odanacatib
(2) (Fig. 2), a cathepsin K inhibitor having an amino nitrile
warhead,21 is currently in Phase III clinical studies.22 This nitrile
O
O
S
O
H
N
N
N
H
N
O
1
⇑
Corresponding author.
E-mail address: beaulieu.ch@videotron.ca (C. Beaulieu).
Figure 1. Structure of K-777 (1).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.015

C. Beaulieu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7444–7449
7445
F
CF3
CF3
H
N
H
N
CN
CN
N
H
N
H
O
O
N
MeO2S
HN
2
3
Figure 2. Structure of odonacatib (2) and of basic cruzipain inhibitor 3.
moiety forms a reversible thioimidate21 with the active site
cysteine of cathepsin K. Given the structural similarities between
cruzipain and the cathepsins, a drug discovery effort was mounted
to identify an amino nitrile-containing cruzipain inhibitor.
Focused screening of our cysteine protease inhibitors collection
identified compound 323 (Fig. 2) as a potent cruzipain inhibitor
with an IC50 of 1.8 nM. In vitro studies with T. cruzi and a prelimin-
ary in vivo proof-of-concept study in mice demonstrated that this
basic cruzipain inhibitor significantly reduces the blood parasite-
mia in mice infected with the Brazilian strain of T. cruzi without
any evidence of toxicity.24 However the basic nature of 3 is likely
associated with lysosomotropism in which the accumulation of
the compound in lysosomes could lead to unexpected activity
P2
O
F
CF3
H
N
CN
N
H
MeO2S
P3
Figure 3. Structure of non-basic cruzipain inhibitor 4.
P1
4
Table 1
P1 SAR: IC50 values for 4–11
F
CF3
H
N
CN
N
H
R1 R2
O
O
S
O
R1
R2
H
Cruz. IC50 (nM)
Cat L IC50 (nM)
966
Cat B IC50 (nM)
Cat F IC50 (nM)
a
a
a
a
Compds
4
8
122
67
5
6
H
H
H
183
130
3819
2628
312
668
547
654
CF3
7
8
H
H
510
>10,000
>10,000
397
3242
>10,000
>10,000
>10,000
9
H
H
6
1
219
49
79
9
12
4
N
F
10
CN
F
11
H
17
711
260
76
CN
IC50 values represent an average of at least three titrations. Standard deviations for these assays were typically within 35% of the IC50 values. See Refs. 28,29 for assay
a
conditions.

7446
C. Beaulieu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7444–7449
against human cathepsins such as Cat B, L, and F.25 This prompted
us to develop a non-basic inhibitor of cruzipain for safer treatment
of Chagas disease. Fortunately, among the compounds tested in our
screening campaign we also identified the non-basic inhibitor 4
(Fig. 3) as a potent inhibitor of cruzipain. However, the potency,
selectivity profile against human cathepsins and pharmacokinetic
profile in mice (T1/2 <30 min) of 4 were sub-optimal and optimiza-
tion of these parameters became the main objective of the SAR
studies conducted with this new series of compounds.
The scaffold of this series was divided into three portions that
we identified as P1, P2, and P3 (Fig. 3). SAR effort around the P1
portion highlighted the importance of this substituent for potency
against cruzipain (Table 1). Presence of the phenylalanine-like
motif in P1, was clearly important. Indeed, the absence of P1 group
or its replacement by a group smaller than phenylalanine, as in 5
and 6, resulted in a >10-fold loss of potency against cruzipain.
Homologation of the phenylalanine moiety in compound 7 was
also found to be detrimental for potency, and attempts to further
functionalize the phenylanine’s methylene (8) were also unsuc-
cessful. Interestingly, heterocycles were found to be tolerated in
P1, but did not contribute to additional potency or selectivity as
observed with compound 9. Further exploration of the substitution
pattern on the phenylalanine moiety led to the discovery of the
4-cyano-2-fluoro-phenylalanine 10 which exhibited an eightfold
increase in potency over the unsubstituted phenylalanine 4. In
addition, the synthesis of the R isomer (11) demonstrated the
importance of having the S stereochemistry in P1 for potency.
Given the minimal effect that P1 modifications had on the selec-
tivity of cruzipain inhibitors over human cathepsins, we evaluated
the SAR in P3 to explore the potential to improve the selectivity
profile of our compounds. The ease of the chemistry in this region
allowed the evaluation of a wide range of moieties in P3. The most
potent analogs were found to share a common para-substituted
biphenyl motif in P3. As shown in Table 2, most of the P3 groups
introduced were well tolerated (12–16). However, none of these
changes contributed to a significant gain of potency or selectivity
compared to the 4-methanesulfonyl biphenyl moiety starting
point. Nonetheless, the variety of functional groups being accepted
in P3 could be very useful to modulate the physicochemical
properties and the metabolic stability of inhibitors.
At this point, we knew very little about the SAR around the P2
portion of the series. However, previous studies in the Cat K
program had indicated that this region was instrumental in achiev-
ing selectivity amongst the cathepsins.21 Indeed, this synthetically
more demanding portion represented our last option to improve
the selectivity of this series. Replacement of the fluoroleucine in
4 by a simple leucine (17) resulted in a 16-fold increase in potency
(Table 3). However, inhibitor 17 was found to be almost equipotent
on Cat F and was metabolically unstable due to methine oxidation
of the leucine.21 The cyclopropylalanine analog 18 and phenylala-
nine analog 20 were found to be equipotent to 4, but did not pro-
vide additional selectivity. To improve the metabolic stability of
leucine analog 17, the dichloro alanine analog 19 was prepared.
This halogenated derivative exhibited sub-nanomolar potency
Table 2
P3 SAR: IC50 values for 12–16
F
CF3
H
N
CN
N
H
O
R
a
a
a
a
Compds
R
Cruz. IC50 (nM)
Cat L IC50 (nM)
Cat B IC50 (nM)
Cat F IC50 (nM)
4
8
966
122
27
67
37
MeO2S
O
12
13
3
3
426
H2N
O
1084
64
48
22
64
N
O
F
F
14
8
1086
OH
15
16
8
436
379
54
59
51
24
N
33
N
HN
a
IC50 values represent an average of at least three titrations. Standard deviations for these assays were typically within 35% of the IC50 values. See Refs. 28,29 for assay
conditions.

C. Beaulieu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7444–7449
7447
Table 3
P2 SAR: IC50 values for 17–23
CF3
R
H
N
CN
N
H
O
O
S
O
a
a
a
a
Compds
R
Cruz. IC50 (nM)
Cat L IC50 (nM)
Cat B IC50 (nM)
Cat F IC50 (nM)
F
4
8
966
122
67
17
18
19
0.2
16
117
5246
42
163
231
383
0.3
147
4
Cl
0.1
Cl
20
7
458
577
769
21
22
2
7
631
751
433
68
174
80
23
380
>10,000
>10,000
>10,000
a
IC50 values represent an average of at least three titrations. Standard deviations for these assays were typically within 35% of the IC50 values. See Refs. 28,29 for assay
conditions.
Table 4
Best P1, P2, and P3 matches: IC50 values for 24–26
a
a
a
a
a
a
a
Compds
Cruz. IC50
(nM)
Cat L IC50
(nM)
Cat B IC50
(nM)
Cat F IC50
(nM)
Cat S IC50
(nM)
Cat K IC50
(nM)
Cat V IC50
(nM)
CF3
H
N
CN
N
H
0.2
0.2
121
143
23
16
169
408
0.9
28
79
O
24
CF3
MeO2S
F
CN
H
N
CN
N
H
98
41
3
O
O
25
CF3
H2N
F
CN
H
N
CN
N
H
O
0.4
1060
87
56
322
2
69
F
F
26
F
CN
OH
a
IC50 values represent an average of at least three titrations. Standard deviations for these assays were typically within 35% of the IC50 values. See Refs. 28,29 for assay
conditions.

7448
C. Beaulieu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7444–7449
CF3 R1
O
R1
a,b,c
OH
OMe
CF3
N
H
H2N
O
O
Br
Br
Br
27
28
29
R2
R2
CN
d
N
N
CN
30
31
32
H2N
CN
e,f
R2
33
H2N
CN
CF3 R1
CF3 R1
g
H
N
OH
R2
CN
N
H
N
H
R2
O
O
Br
Br
29
34
33
CF3 R1
H
N
CN
N
H
h
R2
O
R3
35
Scheme 1. General synthesis of compounds. Reagents and conditions: (a) K2CO3, MeOH, 50 °C; (b) Zn(BH4)2, MeCN, À40 °C; (c) 1 N HCl; (d) NaH, DMF, 0 °C; (e) AcOH, H2O,
THF, 40 °C; (f) HPLC chiral resolution; (g) HATU, 2,6-lutidine, DMF, 0 °C; (h) ArB(OH)2, Pd(dppf)Cl2, Na2CO3, DMF, 90 °C
against cruzipain (17), but exhibited poor selectivity against Cat F.
This unnatural amino acid also represented a synthetic challenge,
especially for large scale synthesis. Fortunately, the replacement
of the fluoroleucine in P2 by a valine afforded the potent and selec-
tive compound 21. With a potency of 2 nM and an encouraging
selectivity profile of >80-fold against human cathepsins L, B, and
F, the identification of 21 confirmed the potential of P2 SAR to
modulate the selectivity against human cathepsins. However, the
SAR around the valine in P2 was found to be very tight: replacing
the isopropyl with a cyclopropyl (22) or a t-butyl group (23) re-
sulted in compounds significantly less potent and/or less selective
than 21.
Due to its unique potency and selectivity profile, valine was
judged the optimal P2 substituent and the compounds combining
the best P1 and P3 portions were prepared. From this exercise,
three compounds (24–26, Table 4) were found to show an
excellent profile with sub-nanomolar potency on cruzipain and
selectivity >80-fold against Cat L, B, F, S, and V. The low selectivity
of these compounds against Cat K was not judged to be a signifi-
cant development issue, as a selective Cat K inhibitor in clinical
trials has shown an excellent safety profile.22 The pharmacokinetic
profile of compounds 24–26 was evaluated and 26 was determined
to have the best profile with moderate bioavailability in rats and
dogs (F = 20% and 30%, respectively) and good half-lives in both
species (5.2 and 4.6 h, respectively). In addition, the difluorometh-
ylcarbinol moiety enclosed within the P3 portion of compound 26
has demonstrated its potential to deliver compounds with suitable
profile for drug development.26 The overall profile of 26 is superior
to any other known inhibitor of cruzipain and represents a great
opportunity to evaluate a selective and reversible cruzipain inhib-
itor for the treatment of Chagas disease.
The general synthetic route to prepare compounds is outlined in
Scheme 1. The P2 portion was first prepared through a stereoselec-
tive reductive amination27 of the amino ester (28) with
1-(4-bromophenyl)-2,2,2-trifluoroethanone (27) to yield acid 29
with diastereomeric ratio >10:1 in favor of the desired diastereo-
mer. The P1 aminonitrile was prepared by the alkylation of (diph-
enylmethylidene)amino acetonitrile with the substituted benzylic
bromide (30). Hydrolysis of the resulting imine 32 yielded the
racemic amino nitrile which was then resolved by chiral HPLC to
yield the desired pure S isomer (33). Both P1 and P2 portions were
then coupled to afford the bromophenyl intermediate 34. The use
of a Suzuki reaction with an appropriate aryl boronic acid allowed
introduction of the P3 group to provide the final compounds.
In conclusion, a new and unique series of potent, selective, and
reversible cruzipain inhibitors has been identified. Compound 26
has a good selectivity profile (>100-fold) against human cathepsins
L, B, S, and F and its pharmacokinetic profile is suitable for further
evaluation in the search for a safe and effective treatment of
Chagas disease.
References and notes
1. Nature Outlook, June 24th, 2010.
2. Ribeiro, I.; Sevcsik, A.-M.; Alves, F.; Diap, G.; Don, R.; Harhay, M. O.; Chang, S.;
Pecoul, B. PLoS Neglected Trop. Dis. 2009, 3, e484.
3. Cavalli, A.; Bolognesi, M. L. J. Med. Chem. 2009, 52, 7339.
4. Urbina, J. A. Curr. Pharm. Des. 2002, 8, 287.
5. Cazzulo, J. J.; Stoka, V.; Turk, V. Curr. Pharm. Des. 2001, 7, 1143.
6. Cazzulo, J. J. Curr. Top. Med. Chem. 2002, 2, 1261.
7. McKerrow, J. H.; Engel, J. C.; Caffrey, C. R. Bioorg. Med. Chem. 1999, 7, 639.
8. Meirelles, M. N. L.; Juliano, L.; Carmona, E.; Silva, S. G.; Costa, E. M.; Murtha, A.
C. M.; Scharfstein, J. Mol. Biochem. Parasitol. 1992, 52, 175.
9. Aparicio, I. M.; Scharfstein, J.; Lima, P. C. A. Infect. Immun. 2004, 72, 5892.

C. Beaulieu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7444–7449
7449
10. Berasain, P.; Carmona, C.; Frangione, B.; Cazzulo, J. J.; Goni, F. Mol. Biochem.
Parasitol. 2003, 130, 23.
11. Engel, J. C.; Doyle, P. S.; Palmer, J.; Hsieh, I.; Bainton, D. F.; McKerrow, J. H. J. Cell
Sci. 1998, 111, 597.
12. Engel, J. C.; Doyle, P. S.; Hsieh, I.; McKerrow, J. H. J. Exp. Med. 1998, 188, 725.
13. Barr, S. C.; Warner, K. L.; Kornreic, B. G.; Piscitelli, J.; Wolfe, A.; Benet, L.;
McKerrow, J. H. Antimicrob. Agents Chemother. 2005, 49, 5160.
14. McKerrow, J. H.; Doyle, P. S.; Engel, J. C.; Podust, L. M.; Robertson, S. A.; Ferreira,
R.; Saxton, T.; Arkin, M.; Kerr, I. D.; Brinen, L. S.; Craik, C. S. Mem. Inst. Oswaldo
Cruz 2009, 104, 263.
Massé, F.; McKay, D. J.; Nicoll-Griffith, D. A.; Oballa, R. M.; Palmer, J. T.; Percival,
M. D.; Riendeau, D.; Robichaud, J.; Rodan, G. A.; Rodan, S. B.; Seto, C.; Thérien,
M.; Truong, V. L.; Venuti, M. C.; Wesolowski, G.; Young, R. N.; Zamboni, R.;
Black, W. C. Bioorg. Med. Chem. Lett. 2008, 18, 923; (b) Oballa, R. M.; Truchon, J.-
F.; Baily, C. I.; Chauret, N.; Day, S.; Crane, S.; Berthelette, C. Bioorg. Med. Chem.
Lett. 2007, 17, 998.
22. Stoch, S. A.; Zajic, S.; Stone, J.; Miller, D. L.; Van Dyck, K.; Gutierrez, M. J.; De
Decker, M.; Liu, Q.; Liu, L.; Scott, B. B.; Panebianco, D.; Jin, B.; Duong, L. T.;
Gottesdiener, K.; Wagner, J. A. Clin. Pharm. Ther. 2009, 86, 175.
23. Black, W. C.; Bayly, C. I.; Davis, D. E.; Desmarais, S.; Falgueyret, J.-P.; Léger, S.; Li,
C. S.; Massé, F.; McKay, D. J.; Palmer, J. T.; Percival, M. D.; Robichaud, J.; Tsou,
N.; Zamboni, R. Bioorg. Med. Chem. Lett. 2005, 15, 4741.
15. Moreira, D. R. M.; Leite, A. C. L.; Dos Santos, R. R.; Soares, M. B. P. Curr. Drug
Targets 2009, 10, 212.
16. Choe, Y.; Brinen, L. S.; Price, M. S.; Engel, J. C.; Lange, M.; Grisostomi, C.;
Weston, S. G.; Pallai, P. V.; Cheng, H.; Hardy, L. W.; Hartsough, D. S.; McMakin,
M.; Tilton, R. F.; Baldino, C. M.; Craik, C. S. Bioorg. Med. Chem. 2005, 13, 2141.
17. Brak, K.; Doyle, P. S.; McKerrow, J. H.; Ellman, J. A. JACS 2008, 130, 6404.
18. Chen, Y. T.; Lira, R.; Hansell, E.; McKerrow, J. H.; Roush, W. R. Bioorg. Med. Chem.
Lett. 2008, 18, 5860.
19. Mott, B. T.; Ferreira, R. S.; Simeonov, A.; Jadhav, A.; Ang, K K.-H.; Leister, W.;
Shen, M.; Silveira, J. T.; Doyle, P. S.; Arkin, M. R.; McKerrow, J. H.; Inglese, J.;
Austin, C. P.; Thomas, C. J.; Shoichet, B. K.; Maloney, D. J. J. Med. Chem. 2010, 53,
52.
24. Ndao, M., manuscript in preparation.
25. Black, W. C.; Percival, M. D. ChemBioChem 2006, 7, 1525.
26. Isabel, I.; Bateman, K. P.; Chauret, N.; Cromlish, W.; Desmarais, S.; Duong, L. T.;
Falgueyret, J. P.; Gauthier, J. Y.; Lamontagne, S.; Lau, C. K.; Léger, S.; LeRiche, T.;
Lévesque, J.-F.; Li, C. S.; Massé, F.; McKay, D. J.; Mellon, C.; Nicoll-Griffith, D. A.;
Oballa, R. M.; Percival, M. D.; Riendeau, D.; Robichaud, J.; Rodan, G. A.; Rodan, S.
B.; Seto, C.; Thérien, M.; Truong, V. L.; Wesolowski, G.; Young, R. N.; Zamboni,
R.; Black, W. C. Bioorg. Med. Chem. Lett. 2010, 20, 887.
27. Hughes, G.; Devine, P. N.; Naber, J. R.; O’Shea, P. D.; Foster, B. S.; McKay, D. J.;
Volante, R. P. Angew. Chem., Int. Ed. 2007, 46, 1839.
20. Murta, A. C. M.; Persechini, P. M.; Padron, T. S.; Souza, W.; Guimaraes, J. A.;
Scharfstein, J. Mol. Biochem. Parasitol. 1990, 43, 27.
21. (a) Gauthier, J. Y.; Chauret, N.; Cromlish, W.; Desmarais, S.; Duong, L. T.;
Falgueyret, J. P.; Kimmel, D. B.; Lamontagne, S.; Léger, S.; LeRiche, T.; Li, C. S.;
28. Isabel, E.; Mellon, C.; Beaulieu, C. WO 2009/067797 A1, 2009.
29. Falgueyret, J. P.; Black, W. C.; Cromlish, W.; Desmarais, S.; Lamontagne, S.;
Mellon, C.; Riendeau, D.; Rodan, S.; Tawa, P.; Wesolowski, G.; Bass, K. E.;
Venkatraman, S.; Percival, M. D. Anal. Biochem. 2004, 335, 218.