Design, synthesis and biological evaluation of potent azadipeptide nitrile inhibitors and activity-based probes as promising anti-Trypanosoma brucei agents

Article abstract of DOI:10.1002/chem.201103322

Trypanosoma cruzi and Trypanosoma brucei are parasites that cause Chagas disease and African sleeping sickness, respectively. There is an urgent need for the development of new drugs against both diseases due to the lack of adequate cures and emerging drug resistance. One promising strategy for the discovery of small-molecule therapeutics against parasitic diseases has been to target the major cysteine proteases such as cruzain for T. cruzi, and rhodesain/TbCatB for T. brucei. Azadipeptide nitriles belong to a novel class of extremely potent cysteine protease inhibitors against papain-like proteases. We herein report the design, synthesis, and evaluation of a series of azanitrile-containing compounds, most of which were shown to potently inhibit both recombinant cruzain and rhodesain at low nanomolar/picomolar ranges. A strong correlation between the potency of rhodesain inhibition (i.e., target-based screening) and trypanocidal activity (i.e., whole-organism-based screening) of the compounds was observed. To facilitate detailed studies of this important class of inhibitors, selected hit compounds from our screenings were chemically converted into activity-based probes (ABPs), which were subsequently used for in situ proteome profiling and cellular localization studies to further elucidate potential cellular targets (on and off) in both the disease-relevant bloodstream form (BSF) and the insect-residing procyclic form (PCF) of Trypanosoma brucei. Overall, the inhibitors presented herein show great promise as a new class of anti-trypanosome agents, which possess better activities than existing drugs. The activity-based probes generated from this study could also serve as valuable tools for parasite-based proteome profiling studies, as well as bioimaging agents for studies of cellular uptake and distribution of these drug candidates. Our studies therefore provide a good starting point for further development of these azanitrile-containing compounds as potential anti-parasitic agents. Copyright

Full text of DOI:10.1002/chem.201103322

FULL PAPER
DOI: 10.1002/chem.201103322
Design, Synthesis and Biological Evaluation of Potent Azadipeptide Nitrile
Inhibitors and Activity-Based Probes as Promising Anti-Trypanosoma brucei
Agents
Peng-Yu Yang,[a] Min Wang,[b] Lin Li,[a] Hao Wu,[a] Cynthia Y. He,*[b] and
Shao Q. Yao*[a]
Abstract: Trypanosoma cruzi and Try-
panosoma brucei are parasites that
cause Chagas disease and African
sleeping sickness, respectively. There is
an urgent need for the development of
new drugs against both diseases due to
the lack of adequate cures and emerg-
ing drug resistance. One promising
strategy for the discovery of small-mol-
ecule therapeutics against parasitic dis-
eases has been to target the major cys-
teine proteases such as cruzain for T.
cruzi, and rhodesain/TbCatB for T.
brucei. Azadipeptide nitriles belong to
a novel class of extremely potent cys-
teine protease inhibitors against
papain-like proteases. We herein report
the design, synthesis, and evaluation of
a series of azanitrile-containing com-
pounds, most of which were shown to
potently inhibit both recombinant cru-
zain and rhodesain at low nanomolar/
picomolar ranges. A strong correlation
between the potency of rhodesain in-
hibition (i.e., target-based screening)
and trypanocidal activity (i.e., whole-
organism-based screening) of the com-
pounds was observed. To facilitate de-
tailed studies of this important class of
inhibitors, selected hit compounds from
our screenings were chemically con-
verted into activity-based probes
(ABPs), which were subsequently used
for in situ proteome profiling and cellu-
lar localization studies to further eluci-
date potential cellular targets (on and
off) in both the disease-relevant blood-
stream form (BSF) and the insect-re-
siding procyclic form (PCF) of Trypa-
nosoma brucei. Overall, the inhibitors
presented herein show great promise as
a new class of anti-trypanosome agents,
which possess better activities than ex-
isting drugs. The activity-based probes
generated from this study could also
serve as valuable tools for parasite-
based proteome profiling studies, as
well as bioimaging agents for studies of
cellular uptake and distribution of
these drug candidates. Our studies
therefore provide a good starting point
for further development of these azani-
trile-containing compounds as potential
anti-parasitic agents.
Keywords: azo compounds · click
chemistry · drug discovery · high-
throughput screening · proteomics ·
structure–activity relationships
Introduction
mally does not exist.[2] Human African trypanosomiasis
(HAT, sleeping sickness) is caused by tsetse fly transmitted
parasites of Trypanosoma brucei. Over 60 million people in
sub-Saharan Africa are at risk of this disease, of which there
are already 50,000–70,000 confirmed cases.[3] Existing drugs
used to treat these diseases are generally highly toxic and
often ineffective, and drug resistance has developed in some
cases (see Figure S1 in the Supporting Information).[4] With
no immediate prospect of vaccines, there is an urgent need
to develop new trypanocidal agents with acceptable efficacy
and safety profiles. One encouraging approach to novel anti-
parasitic agents is the development of small-molecule inhibi-
tors targeting parasitic cysteine proteases such as cruzain
(an essential protease for the survival of T. cruzi[5]), rhode-
sain (also known as brucipain, trypanopain) and, more re-
cently, TbCatB (a cathepsin B-like protease) in T. brucei.[6]
Cysteine proteases are fundamental to the metabolism of
many parasites,[7] and their inhibitors have been shown to
kill protozoan parasites in both culture and animal models.
One such inhibitor, N-Mpip-Phe-Hph-VSPh (also known as
K11777; Figure 1A) is currently in late-stage preclinical
trials for treatment of Chagas disease.[8] K11777 was also
Chagas disease (or American trypanosomiasis) is caused by
Trypanosoma cruzi and affects around 12 million people in
Latin America, resulting in approximately 14,000 deaths per
year.[1] More recently, due to immigrant carriers or infec-
tions from contaminated transfusions and organ transplants,
Chagas disease has been reported in areas in which it nor-
[a] P.-Y. Yang, Dr. L. Li, H. Wu, Prof. Dr. S. Q. Yao
Department of Chemistry
National University of Singapore
3 Science Drive 3
Singapore 117543 (Singapore)
Fax : (+65)6779-1691
E-mail: chmyaosq@nus.edu.sg
[b] M. Wang, Dr. C. Y. He
Department of Biological Sciences
National University of Singapore
14 Science Drive 4
Singapore 117543 (Singapore)
E-mail: dbshyc@nus.edu.sg
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201103322.
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Figure 1. A) Representative structures of anti-parasitic cysteine protease inhibitors. B) Proposed inhibitory mechanism of cysteine proteases by azani-
triles (top) and diazomethyl ketones (bottom). C) Overall workflow of chemical screens and characterizations of azanitriles that were screened with
target-based (cruzain and rhodesain) and organism-based assays, and conversion of hit molecules into activity-based probes (ABPs). Putative identifica-
tion of cellular targets (on and off) was achieved by in situ parasite-based proteome profiling. Reaction of the alkyne handle on the probe with rhoda-
mine-azide by using click chemistry allows visualization of target proteins by SDS-PAGE gel or fluorescence microscopy. Alternatively, with biotin-azide,
the probe-labeled parasite proteome could be pulled-down/LC-MS/MS for target identification.
shown to have potent in vitro and in vivo bioactivities
against T. brucei,[9] presumably by targeting rhodesain,
TbCatB, or both (the inhibitor is non-selective).[10] Another
cysteine protease inhibitor, Z-Phe-Ala-CHN2 (6a; Fig-
ure 1A), has also been shown to be lethal to T. brucei in
both in vitro and in vivo studies.[11] Vinyl sulfones, tetrafluor-
ophenoxymethyl ketones, and diazomethyl ketones, all of
which contain an electrophilic “warhead” that can covalent-
ly and irreversibly inactivate cruzain as a result of nucleo-
philic attack by the active-site cysteine (a representative ex-
ample is shown in the bottom Scheme of Figure 1B), are
also known parasiticidal agents.[12,13] Vinyl sulfones are also
well-known proteasome inhibitors.[14] Previous work in de-
veloping antimalarial compounds showed that synthetic pep-
tidyl aldehydes were potent reversible inhibitors of the cys-
teine protease hemoglobinases, falcipain-2 and falcipain-3.[15]
One such compound, Mu-CO-Leu-hPhe-al (Figure 1A),
which exhibits strong inhibitory activity on falcipains and
cultured Plasmodium falciparum parasites, was tested in
a murine malaria model.[15] Recently, Gꢁtschow and co-
workers reported proteolytically stable azadipeptide nitriles
as a novel class of cysteine protease inhibitors that form co-
valent but slow-releasing, reversible isothiosemicarbazide
adducts with the active-site cysteine (Figure 1B; top).[16]
Using cathepsin K as an example, the authors further dem-
onstrated that structural optimizations of azadipeptide ni-
triles could result in the generation of selective cathepsin in-
hibitors.[17] A subsequent study clearly demonstrated that
azadipeptide nitriles also possessed potent antimalarial ac-
tivities.[18] An organelle-specific drug delivery system using
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Azadipeptide Nitrile Inhibitors
FULL PAPER
such compounds was recently reported to directly target
cathepsins in human cells.[19] In the current study, we have
taken efforts to further optimize this class of inhibitors as
potential agents against trypanosome parasites, with the ulti-
mate aim of generating highly potent inhibitors that possess
fewer off-targets and side effects (which are often associated
with irreversible enzyme inhibitors).[20] Herein, we report
the chemical synthesis and biological evaluation of a series
of azanitrile-containing inhibitors, as well as some activity-
based probes derived from selected compounds. By first
screening these compounds against purified recombinant
cruzain and rhodesain (i.e., target-based screening), we
found most of them were indeed highly potent inhibitors,
with IC50 values in the low nanomolar/picomolar ranges. Be-
cause inhibitor sensitivities are often affected by assay con-
ditions such as pH, ionic strength, and the presence of lipids
or other proteins,[21] we subsequently carried out whole-or-
ganism-based screening and quantitatively verified the po-
tencies of some of these compounds. A good correlation be-
tween the potency of rhodesain inhibition and the trypanoci-
dal activity of these compounds was observed. Next, by
taking advantage of the covalent nature of these inhibitors,
we combined the whole-organism-based screening with ac-
tivity-based protein profiling (ABPP) coupled with bioor-
thogonal click chemistry,[22,23] and carried out comprehensive
proteome profiling experiments against selected hit com-
pounds in an effort to identify both on- and off-targets,
which provided invaluable insights into the mode-of-action
and further optimizations.[21] Finally, fluorescence imaging
experiments with the probes provided further information
on the compoundꢂs cellular uptake and sub-cellular distribu-
tion (Figure 1C).
solvent exposure, as seen in the protein structure.[25] In other
words, the urea group at the P3 position in K11777/K11002
did not actively participate in binding interactions with the
enzyme; therefore, this moiety could be replaced by a carba-
mate group, or an amide group.[26] The designed library was
based on the scaffold presented in Scheme 1, in which R3
was either 2,3-dihydro-1,4-benzodioxin-6-yl (resulting in an
amide group at the P3 position) or carbamoyl, that is, tBuO
or BnO, as previously reported.[16] The R2 moiety could be
either phenyl, isopropyl, or isobutyl, so that the amino acid
at the P2 position was either Phe, Val, or Leu, respectively.
The highest variability was introduced at the P1 position
with five different groups (Me, Bn, CH2Bn, CH2CH2Bn, or
n-butyl), thereby enabling a systematic evaluation of the
structure–activity relationship (SAR) between members of
this library and their inhibitory action against cruzain/rhode-
sain. In this context, it is noteworthy that the structural di-
versity of this library can be easily increased by further var-
iations at the P1, P2, and P3 positions using the same chemis-
try developed herein. As shown in Scheme 1, the synthesis
of our library was accomplished by modifications of previ-
ously reported procedures for the preparation of 3b and its
derivatives.[16] Starting from commercially available Boc-pro-
tected amino acids, we prepared the corresponding benzyl
esters and then removed the amine protecting group by
using standard methods, giving 7a–c. The products were
then coupled with 1,4-benzodioxane-6-carboxylic acid to
give 8a–c. Subsequent deprotection was carried out hydro-
genolytically to give 9a–c, which, upon coupling with meth-
ylhydrazine, gave the corresponding hydrazones 10a–c.
These compounds were subjected to reductive amination
using a range of aldehydes (e.g., R2CHO) together with di-
methylamine–borane complex as the reducing regent, giving
the corresponding N1-methyl-N2-alkylhydrazides (12a–o).
Upon treatment with cyanogen bromide under basic condi-
tions, the desired azanitriles 1a–o were obtained. For the
preparation of analogue 2, the key intermediate, N1-methyl-
N2-alkylhydrazide 15, was prepared by using sodium cyano-
borohydride in the presence of acetic acid,[27] instead of di-
methylamine–borane complex. Azanitriles 3a–e were simi-
larly prepared, as shown in Scheme 1, from Cbz-protected
phenylalanine. For the synthesis of aldehyde inhibitor 4 (an
analogue of Mu-CO-Phe-hPhe-al;[15] Figure 1A), initial at-
tempts involving oxidation of the corresponding primary al-
cohol with Dess–Martin periodinane (DMP) resulted in the
formation of a complex mixture of products. Therefore, the
Weinreb amide 22 was prepared and subsequently reduced
with lithium aluminum hydride to furnish 4.
Results and Discussion
Design and synthesis of azadipeptide nitriles: Isoelectronic
replacement of the CaH group by a nitrogen atom to yield
azapeptides is a common structural modification in the
chemistry of peptides and peptidomimetics.[24] This structur-
al modification was first applied to the P1 position of pep-
tide nitriles in 2008.[16] The resulting azadipeptide nitriles
were characterized as highly potent inhibitors of cysteine
cathepsins (i.e., cathepsins L, S, and K). These compounds
À
possess a methylated P2 P1 peptide bond, which accounts
for their stability towards proteolytic cleavage. Such azadi-
peptide nitriles also showed time-dependent inhibition, and
the reversible formation of adducts with the active site cys-
teine was proposed (Figure 1B; bottom). The strategy we
undertook in the current work built on these initial results
to further demonstrate potent inhibition of cruzain and rho-
desain by azanitrile-containing compounds, because both en-
zymes are structurally homologous to cathepsin L. Previous
studies showed that cruzain was effectively inhibited by
vinyl sulfones bearing Leu or Phe residues at the P2 position
(for example, K11777/K11002). However, the P1 and P3
groups were considered to be less important, due to their
Biological screening: With the 21 azanitriles (1a–o, 2 and
3a–e) and aldehyde 4 in hand, we next evaluated the inhibi-
tory activity of the library against cruzain and rhodesain in
a fluorometric microplate assay with the substrate Z-Phe-
Arg-AMC. For comparison, K11002, K11777, and Z-Phe-
Ala-CHN2 (6a) were also assayed under identical condi-
tions.
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S. Q. Yao, C. Y. He et al.
a recent study in which a combi-
nation of optimized P2 and P3
substituents in azadipeptide ni-
triles was used, led to the iden-
tification of a picomolar cathe-
psin K inhibitor with remark-
able selectivity over other cath-
epsins.[17] Compound 2, bearing
a Phe side chain and a Boc
group at the P2 and P3 posi-
tions, respectively, was one of
the most potent inhibitors
against
rhodesain
(IC50 =
60 pM; IC50 cruzain =11.6 nm).
Compound 1a was also a highly
potent inhibitor against rhode-
sain (IC50 =140 pM, IC50
=
cruzain
8.5 nm). Another inhibitor with
excellent inhibitory activity
against rhodesain was 1k
(IC50 =60 pM;
IC50
=
cruzain
340 pM). As
a control, IC50
values of K11002/K11777 were
calculated for rhodesain (IC50 =
0.36 nm and 0.35 nm) and cru-
zain (IC50 =6.6 nm and 4.1 nm).
Interestingly, compound 4, with
an incorporated aldehyde “war-
head”, displayed a potency that
was comparable to K11002/
K11777 against both enzymes
(IC50 values of 0.55 nm and
3.6 nm, respectively, against rho-
desain and cruzain). Although
we did not further evaluate the
inhibitory activity of our com-
pounds against TbCatB due to
the unavailability of this
enzyme, we were encouraged
by the observation that some
compounds show low picomolar
IC50 values against rhodesain,
Scheme 1. Synthesis of inhibitors 1a–o, 2, 3a–e, and 4 used in this study. THF=tetrahydrofuran, EDC=1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide, HOBt=N-hydroxybenzotriazole, DIEA=N,N-diisopropyle-
thylamine, NMM=N-methylmorpholine, ISCF=isobutyl chloroformate, PTS=p-toluenesulfonic acid,
DMAB=dimethylamine borane, LAH=lithium aluminum hydride.
All compounds exhibited greater inhibitory capacity
toward rhodesain than cruzain (Table 1 and Figure S2A in
the Supporting Information). The anti-cruzain and anti-rho-
desain activities were well-correlated. This phenomenon is
not surprising given the structural homology of these two
enzymes. In general, compounds with Phe (1a–e) and Leu
(1k–o) at the P2 position were more active than compounds
with Val (1f–j), whereas compounds with Leu (1k–o) exhib-
ited higher potency against cruzain. For the P1 position,
compounds having a methyl group (1a, 1f, and 1k) yielded
better results. Finally, changes made at the P3 position con-
ferred a subtle effect on enzyme selectivity, that is, 1a, 2
versus 3a, and 1b–d versus 3b–d. This indicates that, unlike
previously assumed,[13] the P3 position may be a recognition
element for certain enzymes in the papain family. Indeed,
and thereby decided to investigate their activities against
live parasites (whole-organism-based screening). In this con-
text, it is noteworthy that TbCatB was previously shown to
be essential for T. brucei survival based on RNA interfer-
ence studies in culture and in mice.[6,28] Whether rhodesain
is essential for the parasite in culture is unclear, but the key
role of rhodesain in T. brucei infection was clearly illustrated
in experiments in which rhodesain-RNAi parasites were
shown to be significantly less efficient in penetrating an in
vitro model of the blood-brain barrier upon blockage by the
cysteine protease inhibitor K11777.[9b] Consequently, these
data validate rhodesain as a viable drug target in T.
brucei.[29]
We next evaluated the trypanocidal activities of these
compounds in cell cultures of both the bloodstream form
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Table 1. In vitro inhibitory effects of the compounds tested against re-
site-killing activity against T. brucei. Similar to data ob-
tained from rhodesain inhibition experiments, compounds
having Phe (1a–e) and Leu (1k–o) at the P2 position inhibit-
ed parasite growth better than compounds having Val (1 f–j)
at the same position. When compounds 1a–e (with Phe at
the P2 position) and 1k–o (with Leu at the P2 position) were
compared, no significant difference in anti-parasitic activity
was observed. Notably, compound 2, which has a Boc group
at the P3 position and exhibited an IC50 value of 60 pM
against rhodesain, was comparatively less potent in its anti-
parasitic activity than compounds 1a and 3a (which bear an
aromatic amide and a Cbz group at the P3 position, respec-
tively). This suggests that the P3 residue is important for the
trypanocidal activity of these azanitriles. For all compounds
tested, there was no clear difference in trypanocidal activity
when the P1 position was changed from a methyl group to
other groups. This series of compounds, however, were able
to block parasite growth much more effectively than
K11002/K11777. These results did not entirely correlate with
inhibition results obtained with recombinant rhodesain
(Table 1), which might be due to differences in their cell
permeability and/or the mechanism of action of these com-
pounds. The exact reason is still under investigation. To
quantitatively verify the above screening results against
some of the compounds tested across a wider range of inhib-
itor concentrations, we subsequently obtained the ED50
values of selected compounds (3b, 3d, 3e, and K11777/
K11002) against both life cycle stages of T. brucei (Table 1
and Figure S2B of the Supporting Information). Interesting-
ly, our results clearly showed that these azanitriles (e.g., 3b,
3d, and 3e) were approximately two-fold more active in
blocking PCF parasite growth (ED50 values of 0.4, 0.7, and
0.7 mm, respectively, after 24 h) than BSF parasite growth
(ED50 values of 1.1, 1.1, and 1.4 mm, respectively). Impor-
tantly, all of them displayed stronger trypanocidal activity
than K11777 and K11002 in both PCF and BSF of T. brucei.
In contrast, both K11777 and K11002 displayed slightly
stronger trypanocidal activity in BSF (ED50 =0.9 and
5.6 mm) than in PCF (ED50 =5.8 and 7.3 mm). In this context,
it is worth noting that the life cycle of T. brucei alternates
between mammalian host stages and insect vector stages,
which are coupled to extensive
combinant cruzain and rhodesain as well as T. brucei.[a]
Compound
IC50 [nm]
Cruzain
ED50 [mm]
PCF
Rhodesain
BSF
1a
1b
1c
1d
1e
1 f
1g
1h
1i
1j
1k
1l
1m
1n
1o
2
3a
3b
3c
3d
3e
4
6a
K11002
K11777
0.14Æ0.06
0.51Æ0.01
1.86Æ0.01
0.24Æ0.004
1.34Æ0.02
5.4Æ0.01
8.5Æ0.002
19.5Æ0.01
101.6Æ0.005
8.1Æ0.008
52.2Æ0.02
18.8Æ0.003
133.6Æ0.02
396.9Æ0.003
410.6Æ0.001
410.6Æ0.01
0.34Æ0.004
4.2Æ0.02
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
15.8Æ0.001
35.1Æ0.002
54.0Æ0.01
54.6Æ0.001
0.06Æ0.001
0.33Æ0.04
0.71Æ0.001
0.14Æ0.02
0.33Æ0.02
0.06Æ0.008
0.4Æ0.01
9.8Æ0.004
2.5Æ0.02
8.4Æ0.002
11.6Æ0.02
3.4Æ0.005
8.4Æ0.01
nd
nd
0.19Æ0.02
0.81Æ0.009
2.0Æ0.002
0.06Æ0.00
0.55Æ0.005
3.1Æ0.4
1.1Æ0.3
0.4Æ0.2
22.7Æ0.003
115.7Æ0.002
3.1Æ0.006
3.6Æ0.002
9.3Æ0.2
nd
nd
1.2Æ0.3
1.4Æ0.4
nd
0.7Æ0.1
0.7Æ0.3
nd
nd
nd
0.36Æ0.02
6.6Æ0.001
5.6Æ0.4
7.3Æ0.3
0.35Æ0.03
4.1Æ0.005
0.9Æ0.3
5.8Æ0.4
[a] Data with standard errors were calculated from duplicate experiments
by using seven different inhibitor concentrations. nd=Not determined.
(BSF) and the procyclic form (PCF) of T. brucei by using
a growth assay on a Guava PCA-96 system (Guava Technol-
ogies, USA) following the manufacturerꢂs instructions. As
shown in Figure 2, all compounds induced a potent, dose-de-
pendent inhibition of parasite replication. As expected,
most azanitriles were found to be significantly more potent
than the three positive controls (K11002, K11777 and Z-
Phe-Ala-CHN2 (6a)). Overall, there were similarities in the
structure–activity relationship (SAR) in this series of com-
pounds between the recombinant enzymes (especially rho-
desain) and T. brucei. Compounds 3a–e, with Phe at the P2
position and Cbz at the P3 position, displayed the best para-
alterations in morphology and
metabolism. Although the pre-
cise underlying mechanism for
the above differences is pres-
ently unclear, they appear again
to be related to differences in
the cell permeability and/or cel-
lular targets. Therefore, as de-
scribed in the next paragraphs,
we made an attempt to identify
potential cellular targets (on
Figure 2. Dose-dependent, anti-trypanocidal effects of azanitriles (1a–o, 2, and 3a–e), aldehyde 4, vinyl sul-
fones K11002 and K11777, as well as diazomethylketone Z-Phe-Ala-CHN2 (6a) against the bloodstream form
(BSF) of T. brucei using the Guava ViaCount assay after 24 h. PCF results against selected compounds are
shown in the Supporting Information (Figure S2B). Results represent the average standard deviation from du-
plicate experiments.
and off) of some of the most
potent compounds in T. brucei
by using activity-based protein
profiling (ABPP) coupled to bi-
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S. Q. Yao, C. Y. He et al.
oorthogonal click chemistry.[22,23] To this end, we have suc-
cessfully made cell-permeable activity-based probes (ABPs)
and used them for parasite-based proteome profiling and
bioimaging experiments (see below). It should also be noted
that, due to the high similarity between T. cruzi and T.
brucei, these compounds/probes are possibly also effective
against T. cruzi.
compounds (for example 3b and 6a). Probes 5 and 6b were
conveniently prepared from the key intermediate 23, which
was obtained in four steps from phenylalanine. The subse-
quent transformations leading to 5 were performed in a simi-
lar way to that of 3b. Likewise, compound 23 was coupled
with l-alanine methyl ester hydrochloride (27) to give the
corresponding dipeptide, whereupon subsequent hydrolysis
was carried out under basic conditions, giving 29b. Its acti-
vation with isobutyl chloroformate in the presence of N-
methylmorpholine and subsequent reaction of the mixed an-
hydride with diazomethane gave the diazomethylketone
probe 6b.
Design and synthesis of activity-based probes (ABPs): Our
screening results thus far showed some of the azanitriles
were highly potent inhibitors of cruzain/rhodesain, which
was likely due to the covalent nature of these inhibitors in
binding to the active site of the enzymes. These compounds
possessed excellent trypanocidal activity in T. brucei, likely
through inhibition of endogenous rhodesain/TbCatB activi-
ties, but they fell short in addressing the possibility that
other cellular proteins present in the parasite proteome (for
example, off-targets) might also be targeted. To facilitate
a more detailed understanding of this novel class of inhibi-
tors, we took advantage of the covalent nature of these com-
pounds, and converted two hit compounds identified from
our screening results, 3b, and Z-Phe-Ala-CHN2 (6a), into
activity-based probes (ABPs) that would enable parasite-
based proteome profiling and large-scale pull-down/LC-MS/
MS analysis to identify potential cellular targets (on and off)
based on our previously established protocols.[30] Compoun-
d 6a was chosen as the positive control for our proteomic
studies because this compound is a known covalent inhibitor
that targets parasite cysteine proteases.[11] Its corresponding
probe, 6b, would thus make a suitable ABP for comparative
profiling studies. Briefly, the two probes, 5 and 6b, were de-
signed and chemically synthesized (Scheme 2). In both
cases, a terminal alkyne was introduced at a suitable posi-
tion (for example, near the P3 position) for subsequent click
chemistry,[23] followed by in-gel fluorescence and bioimag-
ing/pull-down experiments. We had previously found, from
an independent study with natural-product-like probes,[30a,b]
that only extremely conservative modifications ensured full
retention of the native biological properties of the parent
In situ proteome profiling: We first confirmed that com-
pounds 5 and 6b retained the full biological activities of
their parent compounds (Figure 3A and Figure 3B); both
the IC50 against recombinant rhodesain and dose-dependent
inhibitory profiles against the growth of BSF/PCF of T.
brucei were comparable to those of 3b and 6a, respectively.
For example, probe 5 and compound 3b displayed nearly
identical IC50 values against rhodesain (0.21 and 0.19 nm, re-
spectively; Figure 3A) and inhibitory profiles against T.
brucei growth. Similar results were obtained for probe 6b
and compound 6a (IC50 =2.9 and 3.1 nm, respectively; for
parasite inhibition profiles, compare Figure 3B and
Figure 2). Taken together, these data showed that the intro-
duction of a terminal alkyne handle in 3b and 6a did not
noticeably affect their trypanocidal activities, and confirmed
5 and 6b were suitable ABPs for subsequent target identifi-
cation/profiling studies. We next compared the in situ pro-
teome reactivity profiles of two probes by using live BSF
and PCF following a previously optimized procedure.[10]
Briefly, probes were directly added to the cell medium in
which parasites were grown. After two hours, the parasites
were washed (to remove excess probes), homogenized, incu-
bated with rhodamine-azide under click chemistry condi-
tions,[30] separated by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE), and analyzed by in-gel
fluorescence scanning (Figure 3C); both probes produced
Scheme 2. Synthesis of activity-based probes (ABPs) 5 and 6b.
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lar, showed noticeable differen-
ces, suggesting the existence of
both common and unique tar-
gets in the two forms of para-
sites.
The covalent labeling of the
two expected cellular targets,
rhodesain and TbCatB (ca. 41
and ca. 31 kDa; Figure 3C),
were unequivocally verified by
affinity pull-down/western blot-
ting with the corresponding an-
tibodies (Figure 3D). Also evi-
dent in Figure 3C and D is that
the two probes labeled only the
mature active enzyme form of
rhodesain (ca. 41 kDa), and not
its proform (ca. 48 kDa).[26a] In
Figure 3. Biological evaluation of two ABPs (5 and 6b) in living T. brucei. A) IC50 curves of probes and their
parent compounds against rhodesain. B) Dose-dependent, anti-trypanocidal effects of the probes against
bloodstream forms of T. brucei after 24 h. Results represent the average standard deviation for duplicated in-
dependent trials. C) In situ proteome-profiling of probes (5, 1 mm; 6b, 10 mm) against BSF and PCF of T.
brucei. Compound 3b (10 mm) or 6a (100 mm) was added to selected lanes in competition experiments. Putative
bands of labeled rhodesain and TbCatB are indicated with arrows. D) Western blotting validation of rhodesain
and TbCatB labeled with 5 (1 mm) or 6b (10 mm), following in situ labeling and affinity pull-down experiments.
E) Venn diagram illustrating the numbers of proteins identified from T. brucei with probe 5 (1 mm) and 6b
(10 mm), after in situ labeling and large-scale pull-down/LC-MS/MS experiments.
addition,
the
fluorescent
TbCatB bands labeled by the
two probes were detected more
prominently in BSF than in
PCF. This is consistent with pre-
vious findings that TbCatB was
up-regulated in BSF.[6,31] Finally,
to identify other proteins that
distinct fluorescently labeled bands, indicating that both
class of compounds were, as expected, covalent inhibitors of
their intended cellular targets. In general, comparable in situ
proteome profiles were obtained for both probes in both
forms of T. brucei, albeit with significant differences in rela-
were also covalently labeled by the two probes, we per-
formed large-scale proteomic analyses using 5 and 6b in
both parasite forms by affinity pull-down followed by LC-
MS/MS experiments (Table 2). All proteins were identified
with a minimum protein score of 30 as well as at least four
tive
reactivity
(compare
Figure 3 lanes 1 and 3 for BSF
and lanes 5 and 7 for PCF). For
example, 1 mm of 5 was able to
give stronger fluorescence-la-
beling profiles than 10 mm of
6b. These results correlated
well with the relative potency
of their trypanocidal activities.
This observation, together with
the fact that the fluorescence
labeling of these two probes
were efficiently inhibited by the
addition of excess 3b and 6a
(Figure 3, lanes 2, 4, 6, and 8),
indicates that all the labeled
proteins were likely true cellu-
lar targets of the probes, and
that both probes had similar
cellular targets in the parasites.
On a related note, as seen in
Figure 3 lanes 1/5 (for probe 5)
and lanes 3/7 (for probe 6b),
the in situ proteome reactivity
profiles between BSF and PCF
Table 2. Representative proteins identified by probe 5 or 6b in T. brucei.[a]
T. brucei gene Protein name
Location Detection
6b
5
Tb927.6.1000
cysteine peptidase precursor (CP), Clan CA, family C1, Cathepsin L-
L
both both
like**
Tb927.6.560
Tb927.4.3950
cysteine peptidase C (CPC), Clan CA, family C1, Cathepsin B-like**
cytoskeleton-associated protein CAP5.5, putative,
cysteine peptidase, Clan CA, family C2, putative (CAP5.5)**
calpain-like cysteine peptidase, cysteine peptidase, Clan CA, family
C2, putative**
L
BSF BSF
n/a
both
BSF
–
–
Tb11.47.0035
n/a
Tb927.3.3410
Tb927.5.1810
Tb11.01.1350
Tb927.6.950
Tb927.8.2540
Tb927.8.1990
aspartyl aminopeptidase
lysosomal/endosomal membrane protein p67 (p67)
S-adenosylhomocysteine hydrolase
cysteinyl-tRNA synthetase
3-ketoacyl-CoA thiolase*
n/a
L
n/a
C
M
M
C
BSF
BSF
PCF
PCF
PCF
PCF
–
–
–
–
–
–
–
BSF
tryparedoxin peroxidase (TRYP2)
Tb10.6k15.2290 protein disulfide isomerase, bloodstream-specific protein 2 precursor
(BS2)*
Tb10.70.5250
metacaspase MCA4, cysteine peptidase, Clan CD, family C13
(MCA4)**
N
–
BSF
Tb927.6.1260
Tb11.02.0100
Tb11.01.3960
Tb927.3.2230
Tb11.02.4080
proteasome beta-1 subunit (TbPSB1)*
carboxypeptidase
flagellar protein essential for flagellar pocket biogenesis (BILBO1)*
succinyl-CoA synthetase alpha subunit*
sterol 14-alpha-demethylase (CYP51)**
n/a
n/a
F
M
n/a
–
–
–
–
–
BSF
PCF
PCF
PCF
PCF
[a] L, C, N, M and F represent lysosome, cytoplasm, nucleus, mitochondrion and flagellum, respectively. n/a=
Not available. Symbols in the protein name column: (*) sensitive to RNA interference; (**) putative drug
of the parasites, although simi- target.
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S. Q. Yao, C. Y. He et al.
unique peptides; the results are summarized in Figure 3E.
In total, 59 and 95 proteins were identified with probe 5
from BSF and PCF, respectively, of which 35 were detected
in both parasitic forms. For probe 6b, 34 and 92 proteins
were identified in BSF and PCF, respectively, with 24 detect-
ed in both forms. When all proteins identified from both
forms were pooled together, 133 and 105 were uniquely
identified in BSF and PCF, respectively, and 21 were present
in both forms. Among these proteins, some were nonspecific
protein binders as a result of their “sticky” nature as well as
their high endogenous expression level, such as carbohy-
drate-metabolism-related glycosomal proteins, although cor-
rect localization of glycosomal proteins has been shown to
be essential for the survival of the bloodstream form as well
as of the procyclic form of T. brucei. We therefore focused
our attention on other candidate proteins having previously
known nucleophilic cysteine residues in their active sites, be-
cause they are likely to be reactive towards both 5 and 6b,
and therefore may be true cellular targets of the correspond-
ing parent compounds (3b and 6a). As shown in Table 2, in
addition to the expected proteins rhodesain and TbCatB,
other proteins such as CAP5.5, calpain-like cysteine pepti-
dase,[11] BS2, MCA4, and one proteosome subunit were
identified only in BSF with either 5 or 6b.[32] Of note, sterol
14-alpha-demethylase (CYP51),[33] which has recently been
validated as a potential target for anti-trypanosomal therapy,
was identified only with 6b in both forms. Additionally, we
have identified several proteins, including 3-ketoacyl-CoA
thiolase,[34] BILBO1,[35] and succinyl-CoA synthetase
alpha,[36] all of which were previously reported to be essen-
tial for parasite survival from RNAi experiments. Finally, it
should be noted that the LC-MS/MS-based results obtained
above should only be used as preliminary data; owing to the
highly complex cellular environment and the intrinsic limita-
tions of affinity pull-down/mass spectrometric experiments,
false positives and nonspecific protein binding could be
minimized but not eliminated. Consequently, proper follow-
up studies and validation experiments will be needed before
any biological conclusions can be made for some of these
protein hits.
Figure 4. Fluorescence microscopy of A) cellular uptake and B) sub-cellu-
lar localization of the probes in BSF and PCF of T. brucei. Live parasites
were treated with 5 (0.1 mm) or 6b (10 mm) for 2 h, reacted with rhoda-
mine-azide (10 mm) in situ under click chemistry conditions, and then
imaged. A) Panels a, c, e and g: merged images of bright-field with
stained nuclei (DAPI; in blue); Panels b, d, f and h: 554 nm channel
(pseudocolored in red) detecting cellular uptake of the probe. B) Panels
a, e, i and m: bright-field images; Panels b, f, j and n: 554 nm channel
(pseudocolored in red) detecting cellular localization of the probe;
Panels c, g, k and o: Immunofluorescence (IF) staining at 488 nm channel
(pseudocolored in green) to detect cellular localization of rhodesain.
Anti-rhodesain primary antibody and FITC-conjugated anti-rabbit IgG
secondary antibody were used; Panels d, h, l and p: merged images of
Panels b/c, f/g, j/k, and n/o together with stained nuclei (DAPI; in blue).
All images were acquired under the same settings. Scale bar=10 mm.
Cellular imaging using activity-based probes: Having accom-
plished putative target identification using the correspond-
ing ABPs 5 and 6b, we next investigated whether these
probes could be used for drug uptake studies, as well as vis-
ualization of their sub-cellular distribution and potential cel-
lular targets in live parasites (both BSF and PCF). To do
this, live parasites were treated with probe 5 or 6b, fixed,
and reacted in situ with rhodamine-azide (Figure 4). Consis-
tent with in situ labeling profiles, as low as 0.1 mm of probe 5
was sufficient to stain the parasites strongly, rendering them
visible under a confocal fluorescence microscope (Fig-
ure 4A, Panels b and f ), whereas 10 mm of 6b was required
to produce fluorescent images with comparable probe label-
ing intensity (Figure 4A, Panels d and h). There was no
other noticeable difference in the cellular uptake of the two
probes between BSF and PCF parasites. The sub-cellular
distribution of the two probes, however, were notably differ-
ent (Figure 4B); probe 6b stained mostly lysosomal com-
partments of the BSF parasites, which was expected for
a probe designed to target the endocytic cysteine proteases,
rhodesain/TbCatB (Figure 4, Panel j). This result was further
confirmed by immunofluorescence (IF) experiments with
the anti-rhodesain antibody (Figure 4, Panel k).[10] Interest-
ingly, the same probe showed mostly cytosolic distribution
in PCF parasites (Figure 4, Panel n). Probe 5, on the other
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Azadipeptide Nitrile Inhibitors
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hand, did not appear to entirely co-localize with rhodesain
(Figure 4, Panels d and h), and showed both lysosomal and
cytosolic distribution in both BSF and PCF parasites
(Figure 4, Panels b and f). Surprisingly, staining of HepG2
cells (Human hepatocellular liver carcinoma) with the
probes revealed that 5 (at lmm) resided mostly in lysosomal
compartments and co-localized with cathepsin L (see Fig-
ure S6 in the Supporting Information). On the other hand,
even as high as 50 mm of 6b only weakly stained HepG2
cells under similar conditions. In this context, it is notewor-
thy that cathepsin L is a lysosomal protease that is involved
in all stages of cancer progression, including growth, antian-
giogenesis, invasion, and metastasis.[37] Recent research on
cathepsin L also shows that proteolysis by this enzyme is re-
quired for entry and replication of the SARS and Ebola vi-
ruses in human cells.[38] Collectively, these results demon-
strate that our newly synthesized ABPs could be used as po-
tential cell-permeable bioimaging agents to study drug
uptake and sub-cellular distribution.
ble in infected human cells due to parasite localization, for
example, parasites reside in the cytoplasm of host cells,
whereas the cathepsins are located in the less accessible ly-
sosomes.[12c] In fact, K11777—currently the most advanced
inhibitor in pre-clinical trials against Chagas disease—also
potently inhibits a variety of recombinant human cathepsin-
s.[10,12a] Nevertheless, Gutschow et al. have recently shown
that it is possible to achieve selective cathepsin inhibition by
fine-tuning the structure of azadipeptide nitriles.[17b]
Experimental Section
Chemistry: Unless otherwise stated, all reactions were carried out under
an atmosphere of dry argon or nitrogen in dried glassware. Indicated re-
action temperatures refer to those of the reaction bath; room tempera-
ture (RT) is noted as 258C. Commercially available starting materials
and regents were purchased from Aldrich, Alfa Aesar, or Fluka and used
as received. Tetrahydrofuran (THF) was distilled over sodium benzophe-
none and used immediately. Reaction progress was monitored by analyti-
cal thin layer chromatography (TLC) on pre-coated silica plates (Merck
60 F254, 250 mm thickness) and spots were visualized by irradiation
under a 254 nm UV lamp, and by treatment with ceric ammonium mo-
Conclusion
1
lybdate, basic KMnO4, or iodine. H and 13C NMR spectra were recorded
with a Bruker Avance 300 MHz, DPX-300 MHz, or DPX-500 MHz NMR
spectrometer. Chemical shifts are reported in ppm relative to internal
standard tetramethylsilane (TMS) or residual solvent peaks (CDCl3 d=
7.26, 77.00 ppm; [D6]DMSO d=2.50, 39.50 ppm for 1H and 13C, respec-
tively). Data are reported as follows: chemical shift, multiplicity (s=sin-
glet, d=doublet, t=triplet, q=quartet, br=broad, m=multiplet), cou-
pling constants, and number of protons. Mass spectra were obtained with
a Shimadzu IT-TOF-MS system. The syntheses and characterizations of
16, 17b, 17c, 18e, 18a–c, 18e, 3a–c, 3e, 19, 21, and 23 were previously re-
ported.[10,16]
We have synthesized a focused library of azanitrile-contain-
ing compounds and screened them for inhibitory activities
against recombinant cruzain and rhodesain, as well as for
their trypanocidal activities in both BSF and PCF of T.
brucei. All compounds exhibited excellent activities and
showed promise as potential anti-parasitic agents. By com-
paring the structure–activity relationship (SAR) of these
compounds, inhibition of rhodesain protease activity and
trypanocidal activities were found to be well-correlated. The
rational approach taken to design this novel class of com-
pounds may provide a starting point for future development
of compounds that are suitable for therapeutic applications,
and be used to address selectivity profiles of different
classes of cysteine protease inhibitors (i.e., azanitriles vs. di-
azomethylketones).[39] Furthermore, based on the most
potent hits from our screening results, two activity-based
probes, 5 and 6b, were subsequently developed. These cell-
permeable probes enabled parasite-based proteome profil-
ing/identification of potential cellular targets (on and off) of
the parent compounds, and unequivocally confirmed that
both rhodesain and TbCatB were targets of our compounds.
The probes could be used in cell imaging experiments for
further assessment of drug uptake and sub-cellular distribu-
tion. Clearly, additional studies will be required for further
target validation, as well as to determine drug efficacy and
toxicity in animal models. Furthermore, we expect the azadi-
peptide nitrile inhibitors reported in this study will likely be
potent against human cathepsins (see Figure S3 in the Sup-
porting Information and references [16] and [19]) due to the
high sequence homology of these enzymes with rhodesain/
cruzain. This, however, should not deter further investiga-
tions into their use as potential anti-parasitic agents. Previ-
ous studies clearly indicated that preferential inhibition of
parasite cysteine proteases over human cathepsins is possi-
(S)-N-[1-(2-Cyano-1,2-dimethylhydrazinyl)-1-oxo-3-phenylpropan-2-yl]-
2,3-dihydrobenzo[b]ACTHNUTRGNEU[GN 1,4]dioxine-6-carboxamide (1a): This general proce-
dure was adapted from a prior publication.[16] Anhydrous sodium acetate
(164 mg, 2.0 mmol) and cyanogen bromide (106 mg, 1.0 mmol) were
added to a stirred solution of 12a (185 mg, 0.5 mmol) in anhydrous
MeOH (10 mL). The mixture was stirred at RT for 12 h, then two addi-
tional equivalents of cyanogen bromide (106 mg, 1.0 mmol) were added
and stirring was continued for 24 h. The solvent was removed under re-
duced pressure, the oily residue was suspended in H2O (10 mL), and the
solution was adjusted to pH 1–2 (10% KHSO4), extracted with EtOAc
(5ꢃ20 mL), and the combined organic layers were washed with H2O, sat.
NaHCO3, and brine. The solvent was dried (Na2SO4) and evaporated. Pu-
rification by flash column chromatography (silica gel) using 20 to 50%
EtOAc in hexanes gave 1a as a white solid (146 mg, 74%). 1H NMR
(500 MHz, CDCl3): d=3.00–3.23 (m, 2H), 3.24 (s, 3H), 3.33 (s, 3H), 4.26
(dd, J=5.0, 15.0 Hz, 4H), 5.41–5.46 (m, 1H), 6.71 (d, J=5.0 Hz, 1H),
6.84 (d, J=10.0 Hz, 1H), 7.18–7.34 ppm (m, 7H); 13C NMR (126 MHz,
CDCl3): d=29.7, 30.5, 37.9, 41.1, 50.7, 64.1, 64.5, 113.5, 116.6, 117.2,
120.6, 126.7, 127.3, 128.8, 129.2, 129.6, 135.6, 143.3, 146.8, 166.7,
173.4 ppm; LC-IT-TOF/MS: m/z calcd for C21H22N4O4: 395.1641 [M+H]+
; found: 395.1654.
(S)-N-[1-(2-Benzyl-2-cyano-1-methylhydrazinyl)-1-oxo-3-phenylpropan-2-
yl]-2,3-dihydrobenzo[b]ACTHNUTRGNEUG[N 1,4]dioxine-6-carboxamide (1b): Prepared ac-
cording to the general procedure using 12b (102 mg, 0.23 mmol), sodium
acetate (75 mg, 0.9 mmol), and cyanogen bromide (96 mg, 0.9 mmol) in
anhydrous methanol (10 mL) to afford 1b (84 mg, 79%) as a white solid.
1H NMR (500 MHz, CDCl3): d=2.97–3.14 (m, 2H), 3.16 (s, 3H), 4.23–
4.27 (m, 4H), 4.64 (s, 2H), 5.52–5.56 (m, 1H), 6.84–6.87 (m, 2H), 7.17–
7.53 ppm (m, 13H); 13C NMR (126 MHz, CDCl3): d=32.3, 37.8, 51.3,
58.9, 64.1, 64.5, 112.3, 115.9, 116.6, 117.2, 120.6, 126.8, 127.2, 127.4, 128.2,
128.7, 128.8, 129.1, 129.3, 129.4, 129.5, 129.7, 130.3, 131.5, 135.8, 143.3,
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S. Q. Yao, C. Y. He et al.
146.7, 166.8, 173.8 ppm; LC-IT-TOF/MS: m/z calcd for C27H26N4O4:
471.1954 [M+H]+; found: 471.1968.
sodium acetate (108 mg, 1.3 mmol), and cyanogen bromide (141 mg,
1.3 mmol) in anhydrous methanol (10 mL) to afford 1h (97 mg, 67%) as
a white solid. 1H NMR (500 MHz, CDCl3): d=0.89 (t, J=10.0 Hz, 6H),
1.94–2.04 (m, 1H), 3.06–3.13 (m, 2H), 3.19 (s, 3H), 3.60–3.73 (m, 1H),
3.84–3.89 (m, 1H), 4.27–4.30 (m, 4H), 4.72 (t, J=10.0, 1H), 6.36 (d, J=
10.0 Hz, 1H), 6.89 (d, J=10 Hz, 1H), 7.23–7.38 ppm (m, 7H); 13C NMR
(126 MHz, CDCl3): d=17.9, 19.5, 31.0, 31.4, 32.9, 54.1, 54.8, 64.2, 64.6,
112.6, 116.6, 117.3, 120.5, 127.1, 127.2, 128.8, 129.2, 136.3, 143.4, 146.7,
166.9, 174.0 ppm; LC-IT-TOF/MS: m/z calcd for C24H28N4O4: 437.2111
[M+H]+; found: 437.2121.
(S)-N-[1-(2-Cyano-1-methyl-2-phenethylhydrazinyl)-1-oxo-3-phenylpro-
pan-2-yl]-2,3-dihydrobenzo[b]ACTHUNRTGNEUNG[1,4]dioxine-6-carboxamide (1c): Prepared
according to the general procedure using 12c (138 mg, 0.3 mmol), sodium
acetate (100 mg, 1.2 mmol), and cyanogen bromide (128 mg, 1.2 mmol) in
anhydrous methanol (10 mL) to afford 1c (118 mg, 81%) as a white
solid. 1H NMR (500 MHz, CDCl3): d=2.85–2.89 (m, 2H), 3.06–3.16 (m,
2H), 3.18 (s, 3H), 3.71–3.76 (m, 1H), 3.83–3.89 (m, 1H), 4.26 (dd, J=5.0,
10.0 Hz, 4H), 5.11–5.16 (m, 1H), 6.38 (d, J=10.0 Hz, 1H), 6.85 (d, J=
10.0 Hz, 1H), 7.03 (d, J=10.0 Hz, 1H), 7.15–7.36 ppm (m, 11H);
13C NMR (126 MHz, CDCl3): d=31.6, 32.9, 37.7, 50.6, 54.8, 64.2, 64.5,
112.3, 116.6, 117.3, 120.5, 126.9, 127.2, 127.3, 128.8, 128.9, 129.0, 129.2,
129.5, 135.4, 136.2, 143.4, 146.8, 166.6, 173.4 ppm; LC-IT-TOF/MS: m/z
calcd for C28H28N4O4: 485.2111 [M+H]+; found: 485.2102.
(S)-N-{1-[2-Cyano-1-methyl-2-(3-phenylpropyl)hydrazinyl]-3-methyl-1-
oxobutan-2-yl}-2,3-dihydrobenzo[b]ACTHNUTRGENUGN[1,4]dioxine-6-carboxamide (1i): Pre-
pared according to the general procedure using 12i (127 mg, 0.3 mmol),
sodium acetate (98 mg, 1.2 mmol), and cyanogen bromide (127 mg,
1.2 mmol) in anhydrous methanol (10 mL) to afford 1i (101 mg, 75%) as
a white solid. 1H NMR (500 MHz, CDCl3): d=1.02 (d, J=5.0 Hz, 3H),
1.08 (d, J=5.0 Hz, 3H), 2.09–2.18 (m, 3H), 2.78–2.85 (m, 2H), 3.21 (s,
3H), 3.43–3.51 (m, 2H), 4.26 (dd, J=5.0, 10.0 Hz, 4H), 5.18 (dd, J=5.0,
10.0 Hz, 1H), 6.54 (d, J=10.0 Hz, 1H), 6.89 (d, J=10 Hz, 1H), 7.19–
7.34 ppm (m, 7H); 13C NMR (126 MHz, CDCl3): d=17.8, 19.7, 28.1,
31.38, 31.42, 32.6, 53.6, 53.9, 64.2, 64.5, 113.7, 116.6, 117.3, 120.5, 126.3,
127.2, 128.3, 128.6, 140.1, 143.4, 146.7, 166.8, 174.0 ppm; LC-IT-TOF/MS:
m/z calcd for C25H30N4O4: 451.2267 [M+H]+; found: 451.2284.
(S)-N-{1-[2-Cyano-1-methyl-2-(3-phenylpropyl)hydrazinyl]-1-oxo-3-phe-
nylpropan-2-yl}-2,3-dihydrobenzo[b]
G
(1d):
Prepared according to the general procedure using 12d (160 mg,
0.34 mmol), sodium acetate (112 mg, 1.4 mmol), and cyanogen bromide
(143 mg, 1.4 mmol) in anhydrous methanol (10 mL) to afford 1d (123 mg,
73%) as a white solid. 1H NMR (500 MHz, CDCl3): d=2.10–2.16 (m,
2H), 2.74–2.86 (m, 2H), 3.00–3.04 (m, 2H), 3.22 (s, 3H), 3.45–3.55 (m,
2H), 4.26 (dd, J=5.0, 10.0 Hz, 4H), 5.44–5.48 (m, 1H), 6.63 (d, J=
10.0 Hz, 1H), 6.84 (d, J=10.0 Hz, 1H), 7.13–7.33 ppm (m, 11H);
13C NMR (126 MHz, CDCl3): d=28.2, 31.6, 32.6, 38.0, 50.8, 53.6, 64.2,
64.6, 112.5, 116.6, 117.3, 120.6, 126.4, 126.9, 127.4, 128.4, 128.7, 128.8,
129.2, 129.6, 135.7, 140.0, 143.4, 146.8, 166.6, 168.4, 173.5 ppm; LC-IT-
TOF/MS: m/z calcd for C29H30N4O4: 499.2267 [M+H]+; found: 499.2329.
(S)-N-[1-(2-Butyl-2-cyano-1-methylhydrazinyl)-3-methyl-1-oxobutan-2-
yl]-2,3-dihydrobenzo[b]ACTHNUTRGNEUG[N 1,4]dioxine-6-carboxamide (1j): Prepared ac-
cording to the general procedure using 12j (124 mg, 0.34 mmol), sodium
acetate (112 mg, 1.4 mmol), and cyanogen bromide (144 mg, 1.4 mmol) in
anhydrous methanol (10 mL) to afford 1j (94 mg, 71%) as a white solid.
1H NMR (500 MHz, CDCl3): d=1.02 (d, J=5.0 Hz, 3H), 1.08 (d, J=
5.0 Hz, 3H), 1.49–1.58 (m, 2H), 1.73–1.81 (m, 2H), 2.13–2.19 (m, 1H),
3.24 (s, 3H), 3.37–3.50 (m, 2H), 4.26–4.30 (m, 4H), 5.14 (dd, J=5.0,
10.0 Hz, 1H), 6.54 (d, J=5.0 Hz, 1H), 6.89 (d, J=10.0 Hz, 1H), 7.28–
7.34 ppm (m, 2H); 13C NMR (126 MHz, CDCl3): d=13.7, 17.6, 19.7, 19.8,
53.8, 54.0, 64.2, 64.5, 112.7, 116.6, 117.3, 120.5, 127.2, 143.4, 146.7, 166.8,
173.9 ppm; LC-IT-TOF/MS: m/z calcd for C20H28N4O4: 389.2111 [M+H]+
; found: 389.2134.
(S)-N-[1-(2-Butyl-2-cyano-1-methylhydrazinyl)-1-oxo-3-phenylpropan-2-
yl]-2,3-dihydrobenzo[b]ACHTUNGTRENNUNG[1,4]dioxine-6-carboxamide (1e): Prepared ac-
cording to the general procedure using 12e (155 mg, 0.38 mmol), sodium
acetate (123 mg, 1.5 mmol), and cyanogen bromide (160 mg, 1.5 mmol) in
anhydrous methanol (10 mL) to afford 1e (113 mg, 69%) as a white
1
solid. H NMR (500 MHz, CDCl3): d=0.99 (t, J=10.0 Hz, 3H), 1.47–1.56
(m, 2H), 1.74–1.83 (m, 2H), 2.96–3.21 (m, 2H), 3.26ACTHUNRTGNEUNG(s, 3H), 3.42- 3.54
(m, 2H), 4.25 (d, J=10.0 Hz, 4H), 5.39–5.43 (m, 1H), 6.79–6.87 (m, 2H),
7.18–7.33 ppm (m, 6H); 13C NMR (126 MHz, CDCl3): d=13.7, 19.6, 19.8,
28.6, 31.5, 37.7, 50.9, 53.8, 64.1, 64.5, 112.5, 116.6, 117.2, 120.6, 126.9,
127.2, 128.7, 129.2, 129.5, 135.8, 143.3, 146.7, 166.6, 173.5 ppm; LC-IT-
TOF/MS: m/z calcd for C24H28N4O4: 437.2111 [M+H]+; found: 437.2102.
(S)-N-[1-(2-Cyano-1,2-dimethylhydrazinyl)-4-methyl-1-oxopentan-2-yl]-
2,3-dihydrobenzo[b]ACTHNUTRGNEU[GN 1,4]dioxine-6-carboxamide (1k): Prepared according
to the general procedure using 12k (118 mg, 0.35 mmol), sodium acetate
(115 mg, 1.4 mmol), and cyanogen bromide (149 mg, 1.4 mmol) in anhy-
drous methanol (10 mL) to afford 1k (97 mg, 77%) as a white solid.
1H NMR (500 MHz, CDCl3): d=1.00 (d, J=5.0 Hz, 3H), 1.05 (d, J=
5.0 Hz, 3H), 1.56–1.81 (m, 3H), 3.23 (s, 3H), 3.34 (s, 3H), 4.26–4.30 (m,
4H), 5.23–5.28 (m, 1H), 6.58 (d, J=10.0 Hz, 1H), 6.88 (d, J=10.0 Hz,
1H), 7.27–7.32 ppm (m, 2H); 13C NMR (126 MHz, CDCl3): d=21.5, 32.3,
25.1, 29.7, 30.5, 41.0, 41.3, 48.2, 64.2, 64.5, 113.6, 116.6, 117.3, 120.6, 126.8,
143.4, 146.8, 167.0, 174.7 ppm; LC-IT-TOF/MS: m/z calcd for
C18H24N4O4: 361.1798 [M+H]+; found: 361.1808.
(S)-N-[1-(2-Cyano-1,2-dimethylhydrazinyl)-3-methyl-1-oxobutan-2-yl]-
2,3-dihydrobenzo[b]ACHTUNGTRENNUNG[1,4]dioxine-6-carboxamide (1 f): Prepared according
to the general procedure using 12 f (106 mg, 0.33 mmol), sodium acetate
(107 mg, 1.3 mmol), and cyanogen bromide (138 mg, 1.3 mmol) in anhy-
drous methanol (10 mL) to afford 1 f (80 mg, 70%) as a white solid.
1H NMR (500 MHz, CDCl3): d=1.04 (d, J=10.0 Hz, 3H), 1.07 (d, J=
10.0 Hz, 3H), 2.09–2.17 (m, 1H), 3.24 (s, 3H), 3.32 (s, 3H), 4.26–4.30 (m,
4H), 5.10–5.13 (m, 1H), 6.57 (d, J=10.0 Hz, 1H), 6.88 (d, J=5.0 Hz,
1H), 7.29–7.35 ppm (m, 2H); 13C NMR (126 MHz, CDCl3): d=18.1, 19.6,
30.2, 31.4, 41.2, 54.0, 64.2, 64.5, 113.9, 116.6, 117.3, 120.6, 127.0, 143.4,
146.8, 166.9, 173.0 ppm; LC-IT-TOF/MS: m/z calcd for C17H22N4O4:
347.1641 [M+H]+; found: 347.347.1648.
(S)-N-[1-(2-Benzyl-2-cyano-1-methylhydrazinyl)-4-methyl-1-oxopentan-2-
yl]-2,3-dihydrobenzo[b]ACTHNUTRGNEUG[N 1,4]dioxine-6-carboxamide (1l): Prepared ac-
cording to the general procedure using 12l (132 mg, 0.32 mmol), sodium
acetate (106 mg, 1.3 mmol), and cyanogen bromide (138 mg, 1.3 mmol) in
anhydrous methanol (10 mL) to afford 1l (131 mg, 81%) as a white solid.
1H NMR (500 MHz, CDCl3): d=0.96 (t, J=5.0 Hz, 6H), 1.50–1.81 (m,
3H), 3.14 (s, 3H), 4.28 (dd, J=5.0, 15.0 Hz, 4H), 4.63 (s, 2H), 5.41–5.45
(m, 1H), 6.67 (d, J=10.0 Hz, 1H), 6.89 (d, J=10.0 Hz, 1H), 7.31–
7.57 ppm (m, 7H); 13C NMR (126 MHz, CDCl3): d=21.4, 23.3, 25.0, 32.3,
41.3, 48.4, 58.9, 64.2, 64.6, 112.4, 116.7, 117.3, 120.7, 127.0, 127.4, 128.8,
129.3, 129.8, 130.4, 131.5, 143.4, 146.8, 167.1, 175.1 ppm; LC-IT-TOF/MS:
m/z calcd for C24H28N4O4: 437.2111 [M+H]+; found: 437.2124.
(S)-N-[1-(2-Benzyl-2-cyano-1-methylhydrazinyl)-3-methyl-1-oxobutan-2-
yl]-2,3-dihydrobenzo[b]ACHTUNGTRENNUNG[1,4]dioxine-6-carboxamide (1g): Prepared ac-
cording to the general procedure using 12g (140 mg, 0.35 mmol), sodium
acetate (115 mg, 1.4 mmol), and cyanogen bromide (149 mg, 1.4 mmol) in
anhydrous methanol (10 mL) to afford 1g (115 mg, 78%) as a white
solid. 1H NMR (500 MHz, CDCl3): d=1.03 (d, J=10.0 Hz, 3H), 1.05 (d,
J=10.0 Hz, 3H), 2.10–2.17 (m, 1H), 3.13 (s, 3H), 4.29 (dd, J=5.0,
10.0 Hz, 4H), 4.62 (dd, J=15.0, 20.0 Hz, 2H), 5.26–5.30 (m, 1H), 6.54 (d,
J=10.0 Hz, 1H), 6.91 (d, J=5.0 Hz, 1H), 7.30–7.44 (m, 6H), 7.55 ppm
(d, J=10.0 Hz, 1H); 13C NMR (126 MHz, CDCl3): d=18.0, 19.7, 31.5,
32.2, 54.2, 59.0, 64.2, 64.6, 112.7, 116.7, 117.3, 120.6, 127.2, 129.3, 129.7,
130.3, 131.6, 143.4, 146.8, 167.0, 174.1 ppm; LC-IT-TOF/MS: m/z calcd
for C23H26N4O4: 423.1594 [M+H]+; found: 423.1606.
(S)-N-[1-(2-Cyano-1-methyl-2-phenethylhydrazinyl)-4-methyl-1-oxopen-
tan-2-yl]-2,3-dihydrobenzo[b]ACTHUNRTGNEUNG[1,4]dioxine-6-carboxamide (1 m): Prepared
according to the general procedure using 12m (132 mg, 0.31 mg), sodium
acetate (98 mg, 1.2 mmol), and cyanogen bromide (127 mg, 1.2 mmol) in
anhydrous methanol (10 mL) to afford 1m (110 mg, 79%) as a white
solid. 1H NMR (500 MHz, CDCl3): d=0.91 (d, J=5.0 Hz, 3H), 0.93 (d,
J=5.0 Hz, 3H), 1.48–1.69 (m, 3H), 3.11–3.14 (m, 2H), 3.16 (s, 3H), 3.69–
3.74 (m, 1H), 3.81–3.87 (m, 1H), 4.25–4.29 (m, 4H), 5.11–5.15 (m, 1H),
(S)-N-[1-(2-Cyano-1-methyl-2-phenethylhydrazinyl)-3-methyl-1-oxobu-
tan-2-yl]-2,3-dihydrobenzo[b]
ACHTUNGTRENUN[NG 1,4]dioxine-6-carboxamide (1 h): Prepared
according to the general procedure using 12 h (136 mg, 0.33 mmol),
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Azadipeptide Nitrile Inhibitors
FULL PAPER
6.58 (d, J=5.0 Hz, 1H), 6.87 (d, J=10.0 Hz, 1H), 7.23–7.35 ppm (m,
7H); 13C NMR (126 MHz, CDCl3): d=21.5, 23.2, 25.1, 31.6, 33.0, 41.1,
48.3, 54.9, 64.2, 64.5, 112.4, 116.6, 117.2, 120.6, 127.0, 127.2, 128.8, 129.0,
136.1, 143.3, 146.7, 167.0, 175.0 ppm; LC-IT-TOF/MS: m/z calcd for
C25H30N4O4: 451.2267 [M+H]+; found: 451.2260.
172.9, 199.1 ppm; LC-IT-TOF/MS: m/z calcd for C24H29N3O4: 424.2158;
[M+H]+; found: 424.2164.
(S)-4-Ethynylbenzyl
1-(2-Benzyl-2-cyano-1-methylhydrazinyl)-1-oxo-3-
phenylpropan-2-ylcarbamate (5): Prepared according to the general pro-
cedure using 26 (88 mg, 0.2 mmol), anhydrous sodium acetate (66 mg,
0.8 mmol), and cyanogen bromide (85 mg, 0.8 mmol) in anhydrous
MeOH (5 mL) to afford 5 (81 mg, 87%) as a white solid. 1H NMR
(500 MHz, CDCl3): d=2.83–2.87 (m, 2H), 3.09 (s, 1H), 3.12 (s, 3H), 4.52
(dd, J=10.0, 15.0 Hz, 2H), 5.02–5.08 (m, 4H), 5.38 (d, J=10.0 Hz, 1H),
7.13–7.48 ppm (m, 14H); 13C NMR (126 MHz, CDCl3): d=32.3, 38.4,
53.5, 59.0, 66.4, 77.6, 83.2, 112.2, 127.3, 127.7, 128.7, 129.2, 129.3, 129.5,
129.7, 129.8, 130.2, 131.2, 132.2, 135.4, 136.8, 155.8, 173.4 ppm; LC-IT-
TOF/MS: m/z calcd for C28H26N4O3: 467.2005 [M+H]+; found: 467.2104.
(S)-N-{1-[2-Cyano-1-methyl-2-(3-phenylpropyl)hydrazinyl]-4-methyl-1-
oxopentan-2-yl}-2,3-dihydrobenzo[b]
G
(1n):
The general procedure was followed using 12n (123 mg, 0.28 mmol),
sodium acetate (92 mg, 1.1 mmol), and cyanogen bromide (117,
1.1 mmol) in anhydrous methanol (10 mL) to afford 1n (95 mg, 73%) as
a white solid. 1H NMR (500 MHz, CDCl3): d=0.98 (d, J=5.0 Hz, 3H),
1.03 (d, J=5.0 Hz, 3H), 1.57–1.67 (m, 2H), 1.80–1.81 (m, 1H), 2.11–2.17
(m, 2H), 2.77–2.86 (m, 2H), 3.21 (s, 3H), 3.47–3.56 (m, 2H), 4.25–4.29
(m, 4H), 5.28–5.33 (m, 1H), 6.68 (d, J=10.0 Hz, 1H), 6.88 (d, J=5.0 Hz,
1H), 7.21–7.32 ppm (m, 7H); 13C NMR (126 MHz, CDCl3): d=21.5, 23.3,
25.1, 28.2, 31.5, 32.6, 41.2, 48.3, 53.4, 61.1, 64.5, 112.5, 116.6, 117.2, 120.6,
126.4, 127.0, 128.3, 128.6, 140.1, 143.3, 146.7, 166.9, 175.0 ppm; LC-IT-
TOF/MS: m/z calcd for C26H32N4O4: 465.2424 [M+H]+; found: 465.2638.
4-Ethynylbenzyl
[(S)-1-{[(S)-4-diazo-3-oxobutan-2-yl]amino}-1-oxo-3-
phenylpropan-2-yl]carbamate (6b): Prepared according to the general
procedure using 29b (0.39 g, 1.0 mmol), NMM (0.15 mL, 1.2 mmol),
ISCF (0.17 mL, 1.2 mmol), and the diazomethane-ether solution. Purifi-
cation by flash column chromatography (silica gel) using 25 to 50%
EtOAc in hexanes gave 6b (0.37 g, 89%) as a white solid. 1H NMR
(500 MHz, [D6]DMSO): d=1.22 (d, J=10.0 Hz, 3H), 2.78 (dd, J=5.0,
105.0 Hz, 1H), 3.03 (dd, J=5.0, 10.0 Hz, 1H), 4.18 (s, 1H), 4.26–4.32 (m,
2H), 4.96 (s, 2H), 5.80 (s, 1H), 7.16–7.32 (m, 6H), 7.44 (d, J=5.0 Hz,
2H), 7.62 (d, J=10.0 Hz, 1H), 8.45 ppm (d, J=5.0 Hz, 1H); 13C NMR
(126 MHz, [D6]DMSO): d=16.9, 37.4, 52.2, 55.9, 64.7, 80.8, 83.2, 120.9,
126.2, 127.5, 128.0, 129.3, 131.6, 137.9, 138.0, 155.7, 171.3, 195.1 ppm; LC-
IT-TOF/MS: m/z calcd for C23H22N4O4: 419.1641 [M+H]+; found:
419.1760.
(S)-N-[1-(2-Butyl-2-cyano-1-methylhydrazinyl)-4-methyl-1-oxopentan-2-
yl]-2,3-dihydrobenzo[b]ACHTUNGTRENNUNG[1,4]dioxine-6-carboxamide (1o): Prepared ac-
cording to the general procedure using 12o (132 mg, 0.35 mmol), sodium
acetate (115 mg, 1.4 mmol), and cyanogen bromide (148 mg, 1.4 mmol) in
anhydrous methanol (10 mL) to afford 1o (94 mg, 67%) as a white solid.
1H NMR (500 MHz, CDCl3): d=0.98–1.05 (m, 9H), 1.50–1.82 (m, 7H),
3.23 (s, 3H), 3.41–3.53 (m, 2H), 4.26–4.30 (m, 4H), 5.24–5.29 (m, 1H),
6.58 (d, J=10.0 Hz, 1H), 6.88 (d, J=5.0 Hz, 1H), 7.28–7.32 ppm (m,
2H); 13C NMR (126 MHz, CDCl3): d=13.7, 19.8, 21.4, 23.3, 25.1, 28.7,
31.5, 41.3, 48.3, 53.7, 64.2, 64.5, 112.6, 116.6, 117.2, 120.6, 127.0, 143.4,
146.7, 166.9, 174.9 ppm; LC-IT-TOF/MS: m/z calcd for C21H30N4O4:
403.2267 [M+H]+; found: 403.2314.
Enzyme assays: Cruzain and rhodesain were assayed in freshly prepared
100 mm acetate buffer (pH 5.5), containing 5 mm dithiothreitol (DTT)
and 0.001% Triton X-100. Reagents (25 mL; cruzain, or rhodesain at 4 nm
final concentration) were added to a 384-well black plate (Greiner, Ger-
many) that contained the test compound (1 mL; concentration ranging
from 10 mm to 10 pM). The enzyme–compound mixture was incubated for
5 min at RT, then substrate Z-FR-AMC (25 mL; Bachem, 10 mm final con-
centration, in the same buffer solution as above) was added to initiate
the reaction. The rate of increase in fluorescence (resulting from the pro-
teolytic cleavage of the substrate leading to the release of fluorogenic
AMC) was monitored with an automated microtiter plate spectrofluorim-
eter (BioTek Synergy 4 Fluorescence Plate Reader) with fluorescence
readout setting of excitation at 355 nm and emission at 460 nm. Control
experiments were performed by using enzyme alone, enzyme with
DMSO vehicle, and enzyme in the presence of the previously known,
highly effective irreversible inhibitors K11002, K11777, and Z-Phe-Ala-
CHN2 (6a). IC50 values were determined by sigmoid dose-response curve
fitting with GraphPad Prism software using inhibitor concentrations in
the linear portion of a plot of inhibition versus log[I] (seven concentra-
tions tested with at least two in the linear range). Data are given as the
mean values of at least two independent assays.
(S)-tert-Butyl
[1-(2-Cyano-1,2-dimethylhydrazinyl)-1-oxo-3-phenylpro-
pan-2-yl]carbamate (2): Prepared according to the general procedure
using 15 (307 mg, 1.0 mmol), anhydrous sodium acetate (230 mg,
3 mmol), and cyanogen bromide (350 mg, 3.3 mmol) in anhydrous MeOH
(25 mL) to afford 2 (246 mg, 74%) as a pale-yellow solid. 1H NMR
(500 MHz, CDCl3): d=1.37 (s, 9H), 2.84–2.31 (m, 2H), 3.20 (s, 3H), 3.24
(s, 3H), 4.99–5.08 (m, 2H), 7.20–7.33 ppm (m, 5H); 13C NMR (126 MHz,
CDCl3): d=28.2, 30.4, 38.5, 41.2, 51.4, 80.0, 113.4, 127.1, 128.6, 129.2,
129.5, 135.6, 155.3, 173.6 ppm; LC-IT-TOF/MS: m/z calcd for
C17H24N4O3: 333.1848 [M+H]+; found: 333.1896.
(S)-Benzyl {1-[2-Cyano-1-methyl-2-(3-phenylpropyl)hydrazinyl]-1-oxo-3-
phenylpropan-2-yl}carbamate (3d): Prepared according to the general
procedure using 18d (112 mg, 0.25 mmol), sodium acetate (82 mg,
1.0 mmol), and cyanogen bromide (106 mg, 1.0 mmol) in anhydrous
methanol (10 mL) to afford 3d (82 mg, 72%) as a white solid. 1H NMR
(500 MHz, CDCl3): d=2.09–2.11 (m, 2H), 2.65–2.90 (m, 4H), 3.08–3.16
(m, 2H), 3.19 (s, 3H), 3.32–3.37 (m, 1H), 3.44–3.49 (m, 1H), 4.98–5.15
(m, 2H), 5.32 (br s, 1H), 7.14–7.34 ppm (m, 15H); 13C NMR (126 MHz,
CDCl3): d=28.1, 31.5, 32.5, 38.4, 52.2, 53.6, 67.0, 112.3, 126.4, 127.3,
127.9, 128.2, 128.3, 128.5, 128.7, 128.8, 129.2, 129.5, 135.4, 139.9, 155.8,
173.4 ppm; LC-IT-TOF/MS: m/z calcd for C28H30N4O3: 471.2318 [M+H]+
; found: 471.2365.
Trypanosoma brucei assay: The bloodstream forms of T. brucei were
grown at 378C and 5% CO2 in HMI-9 medium supplemented with 20%
heat-inactivated fetal bovine serum (FBS). Procyclic forms YTAT 1.1
were grown at 288C and 5% CO2 in Cunninghamꢂs medium supplement-
ed with 15% heat-inactivated fetal bovine serum (FBS). Trypanosomes
number and percentage viability were determined in 96-well plate format
using the Guava ViaCount assay with a Guava PCA-96 system (Guava
Technologies, USA) following the manufacturerꢂs instructions. Briefly,
trypanosomes were harvested in the exponential growth phase and dilut-
ed to a concentration of 1ꢃ105 cells/mL in complete growth medium. For
larger screens, the diluted trypanosomes were aliquoted (200 mL) in ster-
ile 96-well flat white opaque culture plates (Greiner, Germany) using
a Sciclone ALH 3000 Liquid Handling Workstation (Caliper Life Scien-
ces, USA). For small-scale screens, diluted trypanosomes were dispensed
manually using a multichannel pipette (BrandTech, USA). Positive and
negative control experiments were performed by using K11002, K11777,
Z-Phe-Ala-CHN2 (6a) and 1.25% DMSO, respectively. After 24 h incu-
bation with compounds, the cell density and viability were evaluated by
using the ViaCount assay on a Guava PCA-96 system. ED50 values were
Mu-CO-Phe-hPh-al (4): LiAlH4 (57 mg, 1.5 mmol) was added with vigo-
rous stirring to a solution of 22 (480 mg, 1 mmol) in anhydrous THF
(25 mL) at 08C. The mixture was stirred for an additional 20 min at 08C,
whereupon cold water was carefully added until effervescence ceased.
Cold HCl (1m) was added to break up the gelatinous emulsion until
pH 6–7 was reached. The mixture was diluted with H2O (50 mL) and ex-
tracted with EtOAc (3ꢃ25 mL). The combined organic extracts were
washed with saturated aqueous NaHCO3 and brine, dried over Na2SO4,
filtered, and concentrated in vacuo. Purification by flash column chroma-
tography (silica gel) using 50 to 75% EtOAc in hexanes gave 4 (350 mg,
73%) as a white solid. 1H NMR (500 MHz, CDCl3): d=1.79–1.85 (m,
1H), 2.10–2.13 (m, 1H), 2.50–2.60 (m, 2H), 3.11–3.13 (m, 2H), 3.25–3.36
(m, 4H), 3.57–3.62 (m, 4H), 4.25–4.31 (m, 1H), 4.76 (dd, J=10.0,
15.0 Hz, 1H), 5.60 (t, J=10.0 Hz, 1H), 7.11–7.42 (m, 10H), 9.31 ppm (s,
1H); 13C NMR (126 MHz, CDCl3): d=30.1, 31.2, 38.7, 43.9, 55.7, 58.3,
66.2, 126.3, 127.0, 128.3, 128.48, 128.52, 129.25, 129.31, 136.7, 140.3, 157.1,
Chem. Eur. J. 2012, 00, 0 – 0
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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S. Q. Yao, C. Y. He et al.
calculated by sigmoid curve fitting with GraphPad Prism 5.0 software
(San Diego, USA). All data were collected in duplicate.
2.2.07, Matrix Science, UK) with MS tolerance of 10 ppm and MS/MS
tolerance of 0.8 Da. Two missed cleavage sites of trypsin were allowed.
Carbamidomethylation (C) was set as a fixed modification, and oxidation
(M) and phosphorylation (S, T and Y) were set as variable modifications.
In situ proteomic profiling: T. brucei parasites were plated into 6-well
plates (PCF, 2 mL at ca. 1ꢃ107 cells/mL) or 25 mL cell culture flasks
(BSF, 10 mL at ca. 2ꢃ106 cells/mL), and incubated with the probe for 2 h
at culture temperature with or without competing inhibitors, 3b or Z-
Phe-Ala-CHN2 (6a). All compounds were solubilized in DMSO. To
avoid adverse effects on parasite growth, the final DMSO concentration
in the assay never exceeded 1.25% in cultivation medium. After incuba-
tion, the parasite cells were pelleted at 2000 rpm for 10 min, washed
twice with PBS and resuspended in PBS (100 mL). Cells were homogen-
ized by sonication and diluted to approximately 1 mgmLÀ1 with PBS. To
initiate the click chemistry reaction, 20 mL of freshly premixed solution
containing rhodamine-azide[30] (100 mm final concentration), TCEP (1 mm
final concentration), TBTA (100 mm final concentration), and CuSO4
(1 mm final concentration) was added. The reaction was incubated at
108C for 4 h with gentle mixing. The reaction was terminated by addition
of prechilled acetone (0.5 mL). The resulting solution was then placed at
À208C for 30 min, followed by centrifugation (13000 rpm ꢃ 10 min) at
48C. The supernatant was discarded and the precipitated protein pellets
were washed with prechilled methanol (2ꢃ200 mL), air-dried for 10 min,
resuspended in 1 ꢃ standard reducing SDS-loading buffer (25 mL) then
heated for 10 min at 958C. Finally, the protein sample (ca. 20 mg/lane)
was loaded onto 12% SDS-PAGE gel, separated, and analyzed by in-gel
fluorescence scanning with a Typhoon 9410 Variable Mode Imager scan-
ner (GE Amersham).
Pull-down and western blotting analysis: Pull-down samples from in situ
labeling with 5 (1 mm) or 6b (10 mm) were separated on 12% SDS-PAGE
gels together with a pull-down sample from DMSO-treatment (negative
control). After SDS-PAGE gel separation, proteins were then transferred
to a PVDF membrane and subsequently blocked with 3% (w/v) BSA/
PBST overnight at 48C. Membranes were incubated for 1 h at RT with
the respective antibodies (anti-cathepsin L for HepG2; anti-rhodesain or
anti-TbcatB for T. brucei), and washed with PBST (3ꢃ15 min with gentle
agitation), then incubated with an anti-mouse-IgG conjugated secondary
antibody in the blocking buffer mentioned above. After washing with
PBST (3ꢃ15 min with gentle agitation), the SuperSignal West Pico kit
(Pierce) was used to develop the blot.
Fluorescence microscopy: For drug uptake analysis, trypanosomes (1ꢃ
105 cells/mL for both forms) were incubated in growth medium contain-
ing different concentrations of 5 or 6b at culture temperature and 5%
CO2 for 2 h. Medium containing 1% DMSO was used as a negative con-
trol. The parasites were then washed twice with PBS, fixed with 4% par-
aformaldehyde in PBS for 15 min at RT, washed with PBS (2ꢃ5 min with
gentle agitation), and then sedimented to poly-l-lysine-coated coverslips.
Fixed cells were permeabilized with 0.25% Triton-X 100 in PBS for
15 min at RT, and washed with PBS (2ꢃ5 min with gentle agitation). The
cells were blocked with 3% BSA in PBS for 30 min at RT, and washed
with PBS (2ꢃ5 min with gentle agitation). The cells were then treated
with a freshly pre-mixed click chemistry reaction solution (rhodamine-
azide (10 mm final concentration from a 10 mm stock solution in DMSO),
TCEP (1 mm final concentration from a 50 mm freshly prepared stock so-
lution in deionized water), TBTA (100 mm final concentration from
a 10 mm stock solution in DMSO), and CuSO4 (1 mm final concentration
from a 100 mm freshly prepared stock solution in deionized water)) in
PBS for 1 h at RT. The cells were washed with PBS (5 min with gentle
agitation), and cold methanol (5 min with gentle agitation), followed by
1% Tween-20 and 0.5 mm of EDTA in PBS (3ꢃ2 min with gentle agita-
tion), and with PBS (2ꢃ5 min with gentle agitation). The cells were then
incubated in PBS containing 2 mgmLÀ1 DAPI for 15 min at RT to stain
the kinetoplast and nuclear DNA, and washed with PBS (2ꢃ5 min with
gentle agitation) and finally washed with deionized water (1ꢃ5 min with
gentle agitation) before mounting onto the Fluoromount G (Emsdiasum,
USA). For immunofluorescence (IF) analysis, cells were then incubated
for lh in PBS with rabbit anti-rhodesain (1:1000) and washed with PBS
(3ꢃ5 min with gentle agitation), then labeled with FITC-conjugated goat
anti-rabbit IgG antibody (1:500) and finally washed with PBS (3ꢃ5 min
with gentle agitation) before mounting. Confocal images were taken with
a Leica TCS SP5X Confocal Microscope System equipped with Leica
HCX PL APO 100ꢃ/1.40 oil objective, 405 nm Diode laser, White laser
(470 to 670 nm, with 1 nm increments, with 8 channels AOTF for simulta-
neous control of eight laser lines, each excitation wavelength provides
1.5 mV, PMT detector range from 420 to 700 nm) was used to measure
the steady-state fluorescence. DAPI, FITC, and rhodamine were excited
with a krypton/argon laser at 405, 488, and 554 nm, respectively, and the
emission was collected through 420–470, 500–550, and 565–650 nm filters,
respectively. Images were processed with Leica Application Suite Ad-
vanced Fluorescence (LAS AF).
Affinity pull-down and LC/MS-MS experiments: For proteomic experi-
ments, BSF and PCF trypanosomes (ca. 2ꢃ109 cells, ca. 5 mg each), la-
beled in Cunninghamꢂs media (1ꢃ107 cells/mL) with 5 (1 mm), 6b (10 mm)
or DMSO (negative control), were harvested, washed, and homogenized
in PBS. CuAAC reagents were added at the same concentrations as de-
scribed above, except that biotin-azide[30] was substituted for rhodamine-
azide. Acetone-precipitated, methanol-washed protein pellets were solu-
bilized in PBS containing 0.1% (w/v) SDS by brief sonication. Insoluble
materials were precipitated by centrifugation (13,000 g ꢃ 10 min) at 48C.
The supernatants were then incubated with gentle shaking at 48C over-
night with Neutravidin agarose beads (50 mLmgÀ1 protein, Prod #29204,
Thermo Scientific, USA), which had been prewashed twice with PBS.
After centrifugation, the bead/complexes were washed extensively eight
times with 1% (w/v) SDS in PBS, three times with PBS, and twice with
250 mm ammonium bicarbonate (ABC). Elution of bound proteins from
beads was then performed twice using boiling buffer (200 mm Tris,
pH 6.8, 400 mm DTT, 8% (w/v) SDS), then pooled. Protein samples were
concentrated using an YM-10 Centricon spin column (Millipore, USA).
Following SDS-PAGE separation, protein bands were visualized by Coo-
massie blue staining. Gel lanes corresponding to both DMSO- and
probe-treated samples were then each cut into several slices. Subsequent
trypsin digestion (using In-Gel Trypsin Digestion Kit, Pierce Co., USA)
and peptide extraction (with 50% acetonitrile and 1% formic acid) gen-
erated the corresponding LCMS samples for each pull-down experiment.
All samples were dried in vacuo and stored at À208C prior to LCMS
analysis. Each LCMS sample was resuspended in 0.1% formic acid for
mass spectrometry analysis as previously described.[40] Briefly, peptides
were separated and analyzed with a Shimadzu UFLC system (Shimadzu,
Kyoto, Japan) coupled to an LTQ-FT Ultra (Thermo Electron, Germa-
ny). Mobile phase A (0.1% formic acid in H2O) and mobile phase B
(0.1% formic acid in acetonitrile) were used to establish the 60 min gra-
dient, which was comprised of 45 min of 5–35% B, 8 min of 35–50% B,
and 2 min of 80% B followed by re-equilibrating at 5% B for 5 min.
Peptides were then analyzed with an LTQ-FT with an ADVANCE Capti-
veSpray Source (Michrom BioResources, USA) at an electrospray poten-
tial of 1.5 kV. A gas flow of 2 LminÀ1, ion transfer tube temperature of
1808C and collision gas pressure of 0.85 mTorr were used. The LTQ-FT
was set to perform data acquisition in the positive ion mode as previously
described, except that an m/z range of 350–1600 was used in the full MS
scan.[41] The raw data were converted into mgf format as previously de-
scribed.[41] The database (76708 sequences, 33362815 residues) used for
Mascot search was a concatenated T. brucei protein database. The data-
base search was performed by using an in-house Mascot server (version
In situ proteome profiling and fluorescence microscopy in HepG2 mam-
malian cells: Our previous published procedures were followed.[30,42]
Briefly, cells were grown to 80–90% confluence in 24-well plates, and
medium was removed, washed twice with cold PBS, then treated with
0.5 mL of DMEM-containing probe for 2 h (the final DMSO concentra-
tion in the assay never exceeded 1% in cultivation medium) as men-
tioned above. After incubation, the growth medium was aspirated and
cells were washed twice with PBS to remove the excess probe, trypsined,
and pelleted at 1,000 rpm for 10 min, washed twice with PBS, and resus-
pended in PBS (100 mL). Cells were homogenized by sonication, diluted
to approximately 1 mgmLÀ1 with PBS, then submitted to click chemistry,
SDS-PAGE gel analysis, and in-gel fluorescence scanning (Figure S3 in
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Azadipeptide Nitrile Inhibitors
FULL PAPER
the Supporting Information). For cellular imaging, cells were grown to
approximately 50% confluence in 24-well plates containing sterile glass
coverslips, and medium was removed, washed twice with cold PBS, then
treated with 0.5 mL DMEM-containing probe for 2 h. After incubation,
the growth medium was aspirated and cells were washed twice with PBS.
Cells were fixed, permeabilized, and blocked, then submitted to click
chemistry, washing, staining (for IF, using mouse anti-cathepsin L; 1:100),
mounted, and imaged (Figure S6 in the Supporting Information).
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zain and rhodesain) and antibodies (anti-rhodesain and anti-TbCatB)
from Dr. Conor R. Caffrey and Professor James H. McKerrow (Universi-
ty of California, San Francisco). We thank Professor Siu Kwan Sze (Na-
nyang Technological University, Singapore) for his continuous support
for LC-MS/MS experiments. This work was supported by the Ministry of
Education (R-143–000–394–112), the Agency for Science, Technology
and Research (A*Star) (R143–000–391–305), and the Competitive Re-
search Program (NRF-G-CRP 2007–04) of the National Research Foun-
dation (NRF). This work was also supported in part by funding from the
Singapore National Research Foundation, awarded to C. Y. H as a re-
search fellow.
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Received: October 21, 2011
Published online: && &&, 2012
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Azadipeptide Nitrile Inhibitors
FULL PAPER
Drug Discovery
P.-Y. Yang, M. Wang, L. Li, H. Wu,
C. Y. He,* S. Q. Yao* ........ &&&& — &&&&
Design, Synthesis and Biological Eval-
uation of Potent Azadipeptide Nitrile
Inhibitors and Activity-Based Probes
as Promising Anti-Trypanosoma
brucei Agents
Killer instinct: We report the design,
synthesis, and evaluation of a series of
azanitriles as highly potent inhibitors
against recombinant cruzain and rho-
desain and the growth of T. brucei par-
asites (see figure). Selected hit com-
pounds were then converted into activ-
ity-based probes and used for parasite-
based proteome profiling to further
elucidate potential cellular targets.
Bioimaging experiments with these
probes were carried out to study cellu-
lar uptake and sub-cellular distribution
of the compounds in T. brucei.
Chem. Eur. J. 2012, 00, 0 – 0
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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