Potency and selectivity of P2/P3-modified inhibitors of cysteine proteases from trypanosomes

Article abstract of DOI:10.1016/j.bmcl.2007.11.070

A systematic study of P2 and P3 substitution in a series of vinyl sulfone cysteine protease inhibitors is described. The introduction of a methyl substituent in the P2 phenylalanine aryl ring had a favorable effect on protease inhibition and conferred modest selectivity for rhodesain over cruzain. Rhodesain selectivity could be enhanced further by combining these P2 modifications with certain P3 amide substituents.

Full text of DOI:10.1016/j.bmcl.2007.11.070

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 624–628
Potency and selectivity of P2/P3-modified inhibitors
of cysteine proteases from trypanosomes
Priyadarshini Jaishankar,a Elizabeth Hansell,b,d Dong-Mei Zhao,a
Patricia S. Doyle,b,d James H. McKerrowb,d and Adam R. Rensloa,b,c,
*
aSmall Molecule Discovery Center, University of California, San Francisco, San Francisco, CA 94158, USA
bSandler Center for Basic Research on Parasitic Disease, University of California, San Francisco, San Francisco, CA 94158, USA
cDepartment of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA
dDepartment of Pathology, University of California, San Francisco, San Francisco, CA 94158, USA
Received 16 October 2007; revised 16 November 2007; accepted 19 November 2007
Available online 22 November 2007
Abstract—A systematic study of P2 and P3 substitution in a series of vinyl sulfone cysteine protease inhibitors is described. The
introduction of a methyl substituent in the P2 phenylalanine aryl ring had a favorable effect on protease inhibition and conferred
modest selectivity for rhodesain over cruzain. Rhodesain selectivity could be enhanced further by combining these P2 modifications
with certain P3 amide substituents.
Ó 2007 Elsevier Ltd. All rights reserved.
Chagas’ disease is a trypanosomiasis caused by the proto-
zoan parasite Trypanosoma cruzi. Millions are affected in
Latin America where the disease is associated with signif-
icant morbidity and mortality and has become the leading
cause of cardiomyopathy. Current therapies for Chagas’
disease include the nitroheterocyclics nifurtimox and ben-
znidizole. The use of these drugs is restricted however, due
to significant toxicity (neuropathies, gastrointestinal
symptoms, myelosuppresion) and concerns about the po-
tential mutagenicity of these agents. Furthermore,
although the existing therapies can be effective in treating
acute disease, they are not effective against the chronic
infection that ultimately results in cardiomyopathy.
moiety at P3, and a vinyl sulfone ‘warhead’ that reacts
irreversibly with the thiol function of the active site cys-
teine in cruzain.3 The homophenylalanine-derived moi-
ety at P1 confers stability towards endopeptidases,
leading to superior stability in vivo as compared to ana-
logs bearing a natural amino acid side chain at P1.1 The
vinyl sulfone warhead is modestly electrophilic, reacting
only with active site cysteines, and only when properly
oriented in the protease active site.3 Hence, cysteine pro-
tease inhibitors containing a vinyl sulfone warhead can
exhibit good selectivity and a favorable in vivo safety
profile despite the irreversible nature of inhibition. In-
deed, K-777 has proven well tolerated in preclinical ani-
mal toxicology and efficacy studies aimed at enabling the
clinical evaluation of this compound as a treatment for
Chagas’ disease.4 In an effort to further optimize this
class of inhibitors and possibly identify analogs effective
against other trypanosome proteases, we synthesized and
evaluated a series of analogs closely related to K-777.
In the hope of identifying safer and more selective anti-
trypanosomals, significant interest has been directed at
parasitic proteases as potential drug targets. Along these
lines, inhibitors of cruzain, the major cysteine protease in
T. cruzi, have been shown to kill parasites within infected
macrophages and to cure a T. cruzi infection in ro-
dents.1,2 Among the cruzain inhibitors currently under
investigation is the vinyl sulfone K-777 shown below.
This substrate-like inhibitor bears hydrophobic P1 and
P2 residues, a more hydrophilic N-methylpiperazine
P2
P1'
O
H
N
SO2Ph
Ph
N
N
H
N
O
Keywords: Cysteine protease inhibitors; Trypanosomes; Vinyl sulfones;
Cysteine proteases; Antiparasitics; Antitrypanosomals.
P1
P3
*
Corresponding author. Tel.: +1 415 514 9698; fax: +1 415 514
4507; e-mail: adam.renslo@ucsf.edu
K-777
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.070

P. Jaishankar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 624–628
625
Vinyl sulfones such as K-777 were originally investi-
R
R
gated in the context of human cathepsin inhibition.3
An early study5 of cathepsin specificity using vinyl sulf-
ones showed that the size (surface area) of the P2 side
chain was positively correlated with potency, and that
cathepsin S was able to tolerate greater P2 diversity than
cathepsins K or L. In this study, cathepsin L was most
effectively inhibited by vinyl sulfones bearing Leu or
Phe residues at P2.
X
X
O
a-c
OH
O Ph
BocHN
N
N
H
O
N
O
1a-e
2a-e
R = H, 3-Me, 3-CF3, 4-Me, H (1a-e)
X = CH (1a-d) or N (1e)
d, e
R
H2N
SO2Ph
Ph
Cruzain is considered to be most similar to cathepsin L,
and is distinguished by an S2 pocket that permits the
binding of basic (Arg) as well as hydrophobic (Phe) side
chains. This dual-specificity is conferred by a glutamate
residue (Glu205) at the base of the S2 pocket that is
capable of interacting with Arg-containing substrates,
but swings away from the pocket upon binding more
lipophilic P2 side chains.6 Notably, this glutamate resi-
due is absent in the parasite proteases rhodesain and
cathepsin-B-like protease7 (TbCatB), potential drug tar-
gets in the causative agent of Sleeping Sickness, Trypan-
osoma brucei. These observations, together with the
inspection of published cruzain crystal structures,8,9 sug-
gested that the introduction of small aliphatic substitu-
ents at the meta or para position of the P2 Phe moiety
in K-777 might confer improved potency while impact-
ing selectivity for these three parasitic proteases.
X
O
H
3
N
SO2Ph
Ph
N
N
H
N
O
4a-e
Scheme 1. Reagents: (a) BnOH, DIC, DMAP, DMF; (b) HCl,
dioxane; (c) triphosgene, DIEA, CH2Cl2, then N-methylpiperazine;
(d) H2, Pd/C, EtOH; (e) 3(HCl), EDC, HOBt, DIEA, DMF.
with an amide connection at P3, hydantoin formation
is precluded and hence a shorter three-step sequence
afforded the sixteen P2/P3 modified analogs 6a–9d
(Scheme 2).
For each of the new analogs, second-order rate con-
stants of enzyme deactivation were determined13,14
against three important parasitic proteases: cruzain
from T. cruzi, and both rhodesain and cathepsin-B-like
protease (TbCatB) from T. brucei. The data are pre-
sented in Table 1 as kinact/Ki or kass values (Mꢀ1 sꢀ1).13
The individual Ki (nM) values and rate constants for en-
zyme inactivation kinact (sꢀ1) are provided when the ki-
netic data allowed for the calculation of these values.13
The separate contributions of Ki and kinact to activity
are discussed in those cases where such an analysis is
informative.
In addition to modifications at P2, we sought to remove
or attenuate the basicity of the N-methylpiperazine sub-
stituent at P3 in K-777. Although the piperazine ring is
quite common in drug structures, the presence of this
ring system in a series of cathepsin K inhibitors has been
associated with lysosomotropism—the accumulation of
drug in acidic (e.g., lysosomal) compartments.10 This
can result in an effective loss of enzyme selectivity in cel-
lular contexts, even for compounds that exhibit exquisite
selectivity in biochemical assays. On the other hand, for
an antiparasitic indication the accumulation of drug
within acidic compartments of parasites may in fact be
a favorable property. To better understand these issues,
we examined analogs of K-777 bearing P3 groups of
attenuated basicity.
The effects of P2 modification alone are revealed by con-
sidering the data for analogs 4a–e in Table 1. K-777 (4a)
itself exhibits little selectivity for cruzain over rhodesain
and is much less effective (ca. 500-fold) against the
cathepsin-B-like protease (TbCatB) in T. brucei. The
introduction of a methyl substituent at the meta or para
The synthesis of substituted phenylalanine analogs was
accomplished using a slight modification of the proce-
dure reported previously for the preparation of K-777
and derivatives.11,12 Starting from commercially avail-
able Boc-phenylalanines (1a–e), we prepared the corre-
sponding benzyl esters and then removed the amine
protecting group using standard methods. Next, the
urea function was introduced via reaction of an interme-
diate isocyanate with N-methylpiperazine, affording
analogs 2a–e (Scheme 1). Hydrogenolysis of the benzyl
esters 2a–e and EDC-mediated coupling of the resulting
acids with the vinyl sulfone amine 311 afforded the de-
sired P2-modified analogs 4a–e with no detectable
epimerization.
R
R
H
N
a
SO2Ph
Ph
OH
BocHN
BocHN
O
O
1a-d
R = H, 3-Me, 3-CF3, 4-Me
5a-d
b, c
Ar =
F
R
6a-d
8a-d
7a-d
9a-d
F3C
F
O
H
N
SO2Ph
Ph
Ar
N
O
O
H
O
N
Although installation of the vinyl sulfone prior to urea
formation could in principle save two steps, a facile for-
mation of hydantoin product upon isocyanate forma-
tion precluded this approach. In the case of analogs
6-9
Scheme 2. Reagents: (a) 3(HCl), EDC, HOBt, DIEA, CH2Cl2; (b)
HCl, dioxane; (c) ArCOOH, EDC, HOBt, DIEA, DMF.

626
P. Jaishankar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 624–628
Table 1. Inhibition kinetics of P2/P3 modified vinyl sulfones against three trypanosome proteases
R
X
O
H
N
SO2Ph
Ph
N
H
Ar
O
Compound
R
H
X
Ar
Cruzain
Rhodesain
TbCatB
kinact/Ki
(Mꢀ1 sꢀ1
Ki (nM)
kinact (sꢀ1
)
kinact/Ki
(Mꢀ1 sꢀ1
Ki (nM)
kinact (sꢀ1
)
kinact/Ki
(Mꢀ1 sꢀ1
)
)
)
K-777 (4a)
CH
N-MePip
510,000
220
0.11
432,000
270
0.11
1000a
4b
4c
4d
4e
3-Me
3-CF3
4-Me
H
CH
CH
CH
N
N-MePip
N-MePip
N-MePip
N-MePip
257,000
53,000
440,000a
45,000
3300
170
—
0.86
0.009
—
517,000
81,000a
852,000
55,000a
170
—
0.088
—
19,000
400a
120
—
0.11
—
10,000
800a
3600
0.16
6a
7a
8a
9a
H
CH
CH
CH
CH
3,5-DiFPh
4-CF3Ph
2-Pyridyl
DHBD
31,000
100,000
120,000
70,000
980
97
0.31
317,000
104,000
150,000
223,000
66
87
0.021
0.0090
0.14
<10b
<5a
0.0097
0.030
0.0056
250
80
910
85
70a
0.019
540a
6b
7b
8b
9b
3-Me
3-CF3
4-Me
CH
CH
CH
CH
3,5-DiFPh
4-CF3Ph
2-Pyridyl
DHBD
105,000
38,000
36,000
14,000
50
92
0.0052
0.0035
0.010
456,000
116,000
173,000
252,000
50
43
0.023
16,000
50b
0.0050
0.040
0.0093
280
71
230
37
100a
650a
0.0010
6c
7c
8c
9c
CH
CH
CH
CH
3,5-DiFPh
4-CF3Ph
2-Pyridyl
DHBD
2400b
55b
—
—
—
—
52,000
900b
6000
2350b
33
—
0.0017
—
<10b
<10b
500a
35b
3100
3300b
150
—
0.00047
—
280
—
0.0017
—
6d
7d
8d
9d
CH
CH
CH
CH
3,5-DiFPh
4-CF3Ph
2-Pyridyl
DHBD
34,000
109,000
16,000
350
45
0.012
366,000
420,000
327,000
30
33
0.011
0.014
0.290
0.031
<10b
<10b
60a
0.0049
0.027
0.0060
1700
19
880
20
311,000
1,570,000
600a
TbCatB, cathepsin-B-like protease in T. brucei; N-MePip, N-methyl piperazine; DHBD, 2,3-dihydro-1,4-benzodioxin-6-yl.
a Value is kass (Mꢀ1 sꢀ1).
b Value is kobs/[I] (Mꢀ1 sꢀ1) at a single inhibitor concentration.
position (4b and 4d) confers a subtle effect on enzyme
selectivity. Thus, the substituted analogs are marginally
less potent than K-777 against cruzain while exhibiting
slightly improved potency against rhodesain and a 10-
to 20-fold improvement against TbCatB. The improved
rhodesain activity of 4b and 4d derives from an approx-
imately twofold improvement in Ki, presumably a result
of the additional binding affinity contributed by the
additional methyl substituent. That no comparable
gains in potency are realized versus cruzain possibly re-
flects an unfavorable interaction between the introduced
methyl substituent and the side chain of Glu205. In sup-
port of this notion, we note that the 3-methyl analog 4b
exhibits a 20-fold weaker Ki for cruzain than for rhodes-
ain while K-777 shows similar Ki values for the two en-
zymes. Perhaps surprisingly then, the isosteric 3-
trifluoromethyl analog 4c exhibits a cruzain Ki much
closer to that of K777 than 4b. In the case of 4c, the rate
constant of enzyme inactivation kinact is notably
affected.
showing only comparable activity against cruzain. In
the case of P2/P3 modified analogs 6–9, substitution at
the 4 position is clearly favored over 3-substitution, with
3-methyl analogs 6b–9b no more potent than the des-
methyl analogs 6a–9a. As was the case with 4c, the
introduction of an electron-withdrawing trifluoromethyl
substituent results in significant losses in potency (ana-
logs 6c–9c). Overall, the SAR at P2 suggests good toler-
ance for small alkyl substituents and augurs that
substitution with larger groups may be a fruitful strategy
for engineering improved activity against rhodesain and
TbCatB.
To explore SAR at P3, we replaced the N-methylpiper-
azine ring of analogs 4a–d with one of four aromatic
or heteroaromatic ring systems (Scheme 2). As measured
by the kinact/Ki ratio, these new analogs (6–9) display
activities generally inferior to the parent piperazine ana-
logs, but with an enhanced selectivity profile that favors
rhodesain over cruzain—in some cases by a significant
margin (e.g., 8d). Looking solely at kinact/Ki, it is difficult
to discern a consistent SAR trend with respect to P3
substitution. Examined separately however, the kinact
and Ki values reveal a more interesting story. Thus,
compared to the parent piperazine analogs 4, the non-
basic benzoyl P3 congeners 6, 7, and 9 exhibit superior
The P2 effects noted above are to some extent recapitu-
lated in the case of analogs bearing non-piperazine P3
substituents (Table 1). Hence, compared to the parent
P2 Phe analogs 6a–9a, the corresponding 4-methyl ana-
logs 6d–9d exhibit improved rhodesain activity while

P. Jaishankar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 624–628
627
binding affinities (Ki as low as 19 nM) but significantly
reduced rate constants of inactivation (kinact). Appar-
ently, while a lipophilic P3 substituent can confer stron-
ger binding through enhanced interactions with S3, the
vinyl sulfone moiety may, as a result, be nudged into a
less favorable orientation for efficient reaction. Interest-
ingly, analogs 8a–d bearing a more hydrophilic P3 group
(pyridyl) display Ki and kinact values that are more com-
parable to the parent piperazine analogs.
non-piperazine analogs examined, only the P3 pyridyl
analog 8a was as effective as K-777. While it is tempting
to attribute this effect to the presence of a basic nitrogen
in 8a, the 2-pyridyl ring in 8a is unlikely to be signifi-
cantly protonated, even at lysosomal pH. Instead, the
activity of 8a may derive from improved permeability
(or active transport) into the parasite and/or sequestra-
tion to high concentration within particular compart-
ments. Certainly, the superior efficacy of 8a as
compared to 6a–c and 7a and 7b cannot be explained so-
lely on the basis of relative enzymatic activities.
In some cases the combination of P2 and P3 modifica-
tion confers a noteworthy synergistic effect. For exam-
ple, the combination of a 3-Me Phe at P2 with either
3,5-difluorophenylamide or benzodioxane moieties at
P3 produces analogs (6b and 9b) with noteworthy selec-
tivity for rhodesain over cruzain (up to eighteenfold).
The combination of a benzodioxane P3 substituent with
4-Me Phe at P2 affords 9d, a rhodesain-selective analog
that is fourfold more potent than K-777. Finally, the
combination of a pyridyl P3 group with a 4-Me Phe
P2 substituent affords 8d, the most highly rhodesain-
selective (twentyfold) analog described in this study.
Interestingly, in each of these cases (6b, 9b, 9d, 8d) rho-
desain selectivity derives not from increased binding
affinity (Ki) relative to cruzain but from faster rate con-
stants of inactivation (kinact).
In summary, we have conducted a systematic explora-
tion of P2 and P3 substitution in a series of vinyl sul-
fone-based cysteine protease inhibitors. Our results
suggest that the introduction of small substituents on
the phenyl ring of the P2 side chain represents a viable
strategy for generating more rhodesain-selective inhibi-
tors. We continue to explore P2/P3 modification in this
context and plan a more extensive biological evaluation
of these analogs, particularly with respect to activity
against T. brucei parasites.
Acknowledgments
Research at the SCBRPD was supported by the Sandler
Family Supporting Foundation and NIAID Grant
AI35707. The SMDC has received generous research
support from the Sandler Family Supporting Founda-
tion, the UCSF School of Medicine, the Cardiovascular
Research Institute at UCSF, and the California Institute
for Quantitative Biology (QB3). We thank Drs. James
Palmer and Mark Burlingame for helpful discussions
and Ymain Shew for technical assistance with the T. cru-
zi-infected macrophage assay.
Next, we examined selected analogs for their ability to
prolong survival of T. cruzi-infected J774 macrophages
using an established protocol1 (Table 2). This assay pro-
vides information about antiparasitic efficacy and also
provides a qualitative measure of compound toxicity
to the macrophage itself. The experiment was carried
out over 42 days and a number of the new P2/P3-mod-
ified analogs were found to exert an antitrypanosomal
effect (Table 2). The 3-methyl and 3-trifluoromethyl ana-
logs 4b and 4c were as effective as K-777, although the
latter showed some toxicity. A number of P3 benzoyl
amide analogs were also examined in this assay and
showed varying degrees of efficacy but were generally
less effective than the P3 piperazine analogs. Of the
References and notes
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macrophage assay (10 lM compound)
a T. cruzi-infected J774
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R
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O
H
N
SO2Ph
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H
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O
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Compound
R
Ar
J774 macrophage Macrophage
survival (days)
toxicity
No drug
K-777 (4a)
4b
—
H
—
N-MePip 42
5
—
No
No
Yes
3-Me N-MePip 42
3-CF3 N-MePip 42
4c
6a
6b
6c
7a
7b
8a
H
3,5-DiFPh 12
Yes
Yes
No
No
Yes
No
3-Me 3,5-DiFPh 12
3-CF3 3,5-DiFPh 23
H
3-Me 4-CF3Ph
4-CF3Ph
8
8
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H
2-Pyridyl 42

628
P. Jaishankar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 624–628
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used to determine kinetic parameters. The value of kobs,
the rate constant for loss of enzymatic activity, was
determined from an equation for pseudo first-order
dynamics using Prism 3.16 (GraphPad). When kobs
varied linearly with inhibitor concentration, kass was
determined by linear regression analysis. If the variation
was hyperbolic, indicating saturation inhibition kinetics,
kinact and Ki were determined from an equation
describing two-step irreversible inhibitor mechanism
½kobs ¼ kinact½Iꢁo=ð½Iꢁo þ Kꢂi ð1þ ½Sꢁo=KmÞÞꢁ and non-linear
regression analysis using Prism.
13. Kinetic analyses were performed as follows: Cruzain
(2 nM), rhodesain (2 nM), or TbCatB (40 nM) in 100 lL
assay buffer was added to inhibitor dilutions in 100 lL of
10 lM Z-Phe-Arg-AMC in buffer A. Progress curves were
determined for 150 s (cruzain) or 300 s (rhodesain and
TbCatB) at room temperature for inhibitor concentra-
tions ranging from 10 to 0.01 lM (10–0.1 lM for
TbCatB). Inhibitor dilutions which gave simple expo-
nential progress curves over a wide range of kobs were
14. Tian, W. X.; Tsou, C. L. Biochemistry 1982, 21, 1028.