A comparative study of warheads for design of cysteine protease inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.10.002

The effects on potency of cruzain inhibition of replacing a nitrile group with alternative warheads were explored. The oxime was almost an order of magnitude more potent than the corresponding nitrile and has the potential to provide access to the prime side of the catalytic site. Dipeptide aldehydes and azadipeptide nitriles were found to be two orders of magnitude more potent cruzain inhibitors than the corresponding dipeptide nitriles although potency differences were modulated by substitution at P1 and P3. Replacement of the α methylene of a dipeptide aldehyde with cyclopropane led to a loss of potency of almost three orders of magnitude. The vinyl esters and amides that were characterized as reversible inhibitors were less potent than the corresponding nitrile by between one and two orders of magnitude.

Full text of DOI:10.1016/j.bmcl.2017.10.002

Accepted Manuscript
A comparative study of warheads for design of cysteine protease inhibitors
Daniel G. Silva, Jean F.R. Ribeiro, Daniela De Vita, Lorenzo Cianni, Caio
Haddad Franco, Lucio H. Freitas-Junior, Carolina Borsoi Moraes, Josmar R.
Rocha, Antonio C.B. Burtoloso, Peter W. Kenny, Andrei Leitão, Carlos A.
Montanari
PII:
S0960-894X(17)30976-9
https://doi.org/10.1016/j.bmcl.2017.10.002
BMCL 25331
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 August 2017
28 September 2017
1 October 2017
Please cite this article as: Silva, D.G., Ribeiro, J.F.R., De Vita, D., Cianni, L., Haddad Franco, C., Freitas-Junior,
L.H., Moraes, C.B., Rocha, J.R., Burtoloso, A.C.B., Kenny, P.W., Leitão, A., Montanari, C.A., A comparative study
of warheads for design of cysteine protease inhibitors, Bioorganic & Medicinal Chemistry Letters (2017), doi:
https://doi.org/10.1016/j.bmcl.2017.10.002
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A comparative study of warheads for design of cysteine protease inhibitors
Daniel G. Silvaa, Jean F. R. Ribeiroa, Daniela De Vitaa, Lorenzo Ciannia, Caio Haddad Francob, Lucio
H. Freitas-Juniorb, Carolina Borsoi Moraesb, Josmar R. Rochaa, Antonio C. B. Burtolosoc, Peter W.
Kenny*a, Andrei Leitão*a, Carlos A. Montanari*a
a Grupo de Química Medicinal do IQSC/USP, Instituto de Química de São Carlos, Universidade de São
Paulo, São Carlos, São Paulo, Brazil
b Laboratório Nacional de Biociências (LNBio), Centro Nacional de Pesquisa em Energia e Materiais
(CNPEM), Campinas, São Paulo, Brazil
c Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, São Paulo, Brazil
* Corresponding author. E-mail: pwk.pub.2008@gmail.com (PWK); andleitao@iqsc.usp.br (AL);
carlos.montanari@usp.br (CAM)
Keywords
Aldehyde, covalent inhibitor, cysteine protease, nitrile, oxime, warhead
Abstract
The effects on potency of cruzain inhibition of replacing a nitrile group with alternative warheads were
explored. The oxime was almost an order of magnitude more potent than the corresponding nitrile and
has the potential to provide access to the prime side of the catalytic site. Dipeptide aldehydes and
azadipeptide nitriles were found to be two orders of magnitude more potent cruzain inhibitors than the
corresponding dipeptide nitriles although potency differences were modulated by substitution at P1 and
P3. Replacement of the α methylene of a dipeptide aldehyde with cyclopropane led to a loss of potency
of almost three orders of magnitude. The vinyl esters and amides that were characterized as reversible
inhibitors were less potent than the corresponding nitrile by between one and two orders of magnitude
1

___________________________________________________________________________________
Cysteine proteases have been linked to a range of human diseases.1-4 In particular, members of this
target class are seen as potential targets for therapeutic intervention in the treatment of parasitic diseases
such as Chagas disease,5 African sleeping sickness6 and leishmaniasis.7 A commonly employed tactic in
design of cysteine protease inhibitors is to incorporate an electrophilic moiety that can form a covalent
bond between the catalytic cysteine and the inhibitor.3,4,6,7-13 These functional groups are commonly
termed ‘warheads’ and the electrophilic center is typically an unsaturated carbon atom. Formation of a
covalent bond between inhibitor and enzyme allows the cysteine thiol to be exploited as a molecular
recognition element even when the associated part of the protein molecular surface is suboptimal for
formation of non-covalent interactions.10 For example, the molecular surface associated with the
catalytic thiol of a cysteine protease is typically saddle-shaped (i.e. concave in one direction but convex
in another).11 The nitrile group3,11-14 is considered to be a particularly useful warhead for cysteine
protease inhibition on account of its metabolic stability, polarity and small contribution to molecular
size.
Although covalent binding to proteins is often believed to be irreversible, this does not have to
be the case. While reversibility of binding may reduce the risk of adverse effects, irreversible binding
can result in prolonged duration of action.3,9,10 Selectivity can be achieved15 with irreversible inhibitors
but irreversibility to the extent that a drug remains covalently bound to peptide fragments after protein
degradation is generally undesirable because it can lead to immunogenicity.11 The functional behavior
of warheads is sometimes assumed to be determined entirely by electrophilicity although atoms bonded
to the electrophilic center can form non-covalent interactions with target proteins that also modulate
affinity. Examples of these non-covalent interactions are shown in Figure 1 for inhibitors based on
nitrile16 and α-ketoamide17 warheads. Figure 1 also illustrates how a 1,2-dicarbonyl warhead provides
easier access than the nitrile to the prime side of the catalytic site.
2

Figure 1. (a) Binding mode of nitrile inhibitor to cathepsin L (PDB refcode: 3HHA). (b) Binding mode
of a ketoamide inhibitor to cathepsin K (PDB refcode: 1TU6). (c) Schematic view of a dipeptidyl nitrile
bound to a papain-like cysteine protease.
Warhead exchange can be seen as analogous to bioisosteric substitution18 and knowledge of
potency differences between warheads is potentially valuable in situations where activity has been
observed for a cysteine protease inhibitor in a cell-based assay but the target is unknown.19 The
exchange of one warhead with another can be seen as a structural transformation and these can be
written20 as [X→Y] where the labels X and Y may be structure numbers, warhead names (e.g. nitrile) or
substructures encoded in line notation (e.g. C#N). The potency difference, ∆pKi, corresponding to the
[X→Y] transformation can be written as:
∆pKi[X→Y] = pKi[Y] − pKi[X]
3

This study has three objectives. First, to investigate the transferability of published effects of warhead
replacement, for example [nitrile→aldehyde], to cruzain which is currently of interest as an antichagasic
target. Second, to evaluate warheads such as the oxime that are based on a carbon-nitrogen double bond.
Third, to explore the potential of substitution for mitigating potential liabilities (e.g. instability,
irreversible binding) associated with warheads.
Synthesis of compounds is summarized in Schemes 1-3. Compounds 1-4, 6, 7 were synthetized
from commercially available amines and the N-Cbz-protected (L)-phenylalanine activated by EDC
(Scheme 1), followed by a mild oxidation of the alcohol to the aldehyde with IBX for compounds 3 and
4. Reaction of aldehyde 3 with hydroxylamine gave the oxime 5, as a mixture of E and Z isomers
(scheme 1). Vinyl esters 8-10 and amides 11, 12 were prepared from aldehydes 3 or 4 and commercial
or freshly prepared phosphonates by the Horner–Wadsworth–Emmons reaction (Scheme 2). Finally, 1,2-
dimethylhydrazides D, E, G, H (Scheme 3) were reacted with cyanogen bromide to give the
azadipeptide nitriles 13-16. Due to the sensitivity of the azanitrile group to the acidic environment
required for Boc removal, P3 modification to obtain the final compounds 15 and 16 was necessary
before the introduction of the nitrile group. Compounds were assayed fluorimetrically for their
inhibitory activity against cruzain monitoring the hydrolysis rate of the fluorogenic substrate Z-Phe-
Arg-7-amido-4-methylcoumarin and the results are presented in Tables 1-2. Cell-based assay results are
given in Table 3 for some of the compounds. Full experimental details and a spreadsheet with structures
(as SMILES strings) and assay results are provided as supporting information. Compounds 1, 2, 3, 8, 13
have been reported in the literature and additional information is provided for these compounds in the
supporting information spreadsheet.
4

Scheme 1. Synthesis of nitrile, aldehyde, oxime and heteroaromatic warheads
The nitrile 1 (pKi = 6.3) is used as the reference compound for this study and its cyclopropane
analogue 2 (pKi = 5.2) is an order of magnitude less potent (Table 1). Substitution of the P1 methylene
group in dipeptide nitriles with cyclopropane typically leads11 to a 0.3 to 0.4 increase in the potency of
cruzain inhibition so the result for 2 indicates a degree of non-additivity in the structure-activity
relationship (SAR). The aldehyde 3 (pKi = 8.1) is almost two orders of magnitude more potent than 1.
Aldehydes would generally be expected to be less stable than the corresponding nitriles under
physiological conditions since they are more readily oxidized and can also enolize. Replacement of a P1
methylene linker in dipeptidyl nitriles with cyclopropane appears to lead to improved physicochemical
properties11 and would also prevent enolization of an aldehyde. However, this replacement results in a
reduction in potency to the extent that 4 (pKi = 5.4) is a weaker inhibitor than the reference nitrile 1,
probably reflecting the different torsional characteristics of trigonal and tetrahedral carbon when bonded
to tetrahedral carbon. The differing effects of the [nitrile→aldehyde] replacement for 1 and 2 illustrate
5

the importance of substructural context and can be interpreted as non-additivity in the structure activity
relationship (SAR).
Table 1. Cruzain inhibition results
Cmpd pKi SE(pKi)
102 × kinact
102 × SE(kinact
)
a
b
(s-1) c
(s-1) d
1 e
2 f
3 e
4 f
5 e
6 f
7 f
8 g
6.3 0.02
5.2 0.03
8.1 0.04
5.4 0.03
7.0 0.04
5.0 0.03
4.6 0.02
5.8 0.05
1.7
0.27
9 e
5.2 0.04
3.9 0.05
3.7 0.03
3.4 0.06
8.7 0.02
10 e
11 e
12 e
13 e
14 e
15 f
16 f
8.0 0.04
8.8 0.03
8.8 0.04
a –log10(Ki/M)
b Standard error in pKi
c Inactivation rate constant
d Standard error in inactivation rate constant
e Reversible inhibitor
f Not assessed in reversibility assay
g Irreversible inhibitor
The [nitrile→aldehyde] transformation of N-(tert-butoxycarbonyl)-phenylalanyl-glycine-nitrile
results in ∆pKi values13 of +1.3 (papain), +1.4 (cathepsin L), +1.4 (cathepsin S) and +1.7 (cathepsin K)
which are comparable with the ∆pKi value of +1.8 observed for [1→3] in the current study. The ∆pKi
values21 corresponding to N-acetyl-phenylalanyl-glycine-nitrile (+1.6; papain), N-acetyl-glycine-nitrile
6

(+2.1; papain) and N-benzoyl-glycine-nitrile (+2.2; papain) are consistent with a view that the presence
of the phenylalanine side chain stabilizes the covalently-bound nitrile to a greater extent than the
covalently-bound aldehyde. Note that Ki values reported21 by Bendall et al for aldehydes were scaled to
account for proportion of hydrate and the ∆pKi values derived from them are not directly comparable
with what we report in this study.
Warheads based on a carbon-nitrogen double bond have the potential to provide better access to
the prime side of the catalytic site than either nitriles or aldehydes. Imines and Schiff bases are generally
thought to be insufficiently stable under physiological conditions to be useful warheads but dipeptide
hydrazones have been reported as cysteine protease inhibitors. For example, a benzoyl hydrazone has
been shown22 to be equipotent with the corresponding aldehyde against both cathepsins B and S
although some caution is needed when interpreting these results, given the size of the benzoyl group.
Hydrazone-based warheads are typically substituted with electron-withdrawing groups which increases
both the steric footprint and molecular complexity23 of inhibitors. In contrast, unsubstituted oxime-
based warheads can be used and, if necessary, these may be synthetically elaborated by linking oxygen
to saturated carbon. Compound 5 (pKi = 7.0) was synthesized to evaluate the oxime group as a warhead
and it was found to be more potent than the reference nitrile 1 (pKi = 6.3). It was not possible to
separate the geometric isomers and 5 was isolated as a 54/46 E/Z mixture.
The isoxazole ring can be thought of as having an oxime embedded in it. Compound 6 (pKi =
5.0) is over an order of magnitude less potent than the reference nitrile 1 (pKi = 6.3). The ∆pKi value of
−1.3 for the [1→6] transformation is sufficiently small to be consistent with covalent bond formation,
although in the absence of structural data for cruzain complexed with 6, it is not possible to say
definitely whether or not this is the case. The isoxazole ring would be expected to be less electrophilic
than an oxime and the ring is also likely to introduce geometric and steric constraints. Unsurprisingly,
the oxazole 7 (pKi = 4.6) is even less potent than 6.
7

Scheme 2. Synthesis of vinyl esters and amides
Vinyl sulfones are of interest as antichagasic agents and these compounds are typically
irreversible inhibitors of cruzain and other cysteine proteases.15 A number of vinyl amides and esters
were synthesized to test the hypothesis that replacement of sulfonyl with a less strongly electron
withdrawing group would lead to reversible inhibition. The vinyl ester 8 (kinact/Ki = 9.8 ×103 s-1 M-1) is
an irreversible inhibitor of cruzain although less active than the vinyl sulfone K-777 (kinact/Ki = 5.1 × 105
s-1 M-1).15 Compound 9 (pKi = 5.2), the Z-isomer of 8, was isolated as minor byproduct and its
reversibility was not characterized. Compounds 10 (pKi = 3.9), 11 (pKi = 3.7) and 12 (pKi = 3.4) are all
between one and two orders of magnitude less potent than 2. Vinyl esters and amides do not appear to
offer any advantage over the more potent nitriles since they are of higher molecular weight and would
also be associated with increased risk of poor pharmacokinetics on account of the hydrolytically
unstable ester and amide functional groups.
Azadipeptide nitriles have been shown to be potent and proteolytically stable inhibitors of
papain-like cysteine proteases.24 Azadipeptide nitriles have also been reported to exhibit
antitrypanosomal activity against the bloodstream form of T. Brucei and appear useful as activity-based
probes.25 The azadipeptide nitrile 13 (pKi = 8.7) is two orders of magnitude more potent against cruzain
8

than the corresponding dipeptide nitrile 1 (pKi = 6.3). Previously reported11 pKi values for dipeptide
nitriles can be used to derive ∆pKi values for the [NHCH2CN→ N(Me)N(Me)CN] transformation
corresponding to the azadipeptide nitriles 14 (+2.1), 15 (+1.6) and 16 (+1.6). Analogous values of ∆pKi
have been reported for 13 against other cysteine proteases ranging from +2.0 (papain) to +4.1 (cathepsin
K).24 The pyrazole-based P3 substituent of 15 and 16 was shown to be advantageous for inhibition of
both cruzain11 and cathepsin L16 when presented to these targets on a dipeptide nitrile scaffold. Our
results suggest that the binding modes of 15 and 16 to cruzain prevent the full benefit of this P3
substituent from being realized.
Scheme 3. Synthesis of azadipeptide nitriles
The potency of the azadipeptide nitriles is especially significant given that these compounds lack
the P1 hydrogen bond donor that is exploited by dipeptide nitriles to interact with cysteine protease
9

targets. The observation that methylation of the P1 amide nitrogen leads to a 200-fold reduction in
potency of 1 against cathepsin L24 gives an indication of the importance of this molecular recognition
element. In agreement with the literature,24 slow binding (Table 2) was observed for the azadipeptide
nitriles and this probably reflects the presence of the electron-releasing methylimino group that would
be expected to render the nitrile carbon less electrophilic. The presence of this group would also be
expected to render the imine nitrogen of the covalently bound adduct more basic and the pKa values of
thiazoline (5.2)26 and 2-aminothiazoline (9.1)27 provide an indication of the nature of the effect although
the difference between these pKa values is likely to be greater than the corresponding difference for the
enzyme-bound inhibitors. Protonation of the imine nitrogen after the rate-determining step may explain
the striking differences in potency between dipeptide nitriles and their aza analogs.
Table 2. Binding kinetics of azadipeptide nitriles
Compound
10-6 × kon 10-6
(M-1s-1) a SE(kon)
(M-1s-1) b
× 103 × koff 103 × SE(koff)
d
(s-1) c
5.0
0.47
7.7
11
0.2
0.04
0.3
0.7
11
5.3
12
15
0.8
0.6
0.8
1.8
13
14
15
16
a On-rate
b Standard error in on-rate
c Off-rate
d Standard error in off-rate
High-content screening was performed for the amastigote form of the T. cruzi Y strain infecting
U2OS host cells (Table 3).28,29 The pCC50 values measured for the azadipeptide nitriles 13, 14 and 16
were very similar to the corresponding pEC50 values suggesting that the azadipeptide nitriles may be
engaging one or more cysteine proteases of the host cell. Although not definitive, the effects on the
10

U2OS host cells observed for 13, 14 and 16 suggest that the possibility of cytotoxicity should be
anticipated when working with azapeptide nitriles. In general, warhead replacement is not a particularly
useful design tactic for increasing selectivity. It is unlikely that the weak potency of 7 (pEC50 = 4.4) in
the cell-based assay is due to cruzipain inhibition.
Table 3. Cell-based assay results
a
c
Cmpd
1
pEC50
< 4
< 4
SE(pEC50) b pCC50
SE(pCC50) d
< 4
< 4
< 4
2
< 4
6
4.4
5.3
0.09
0.22
< 4
5.0
7
0.06
0.02
0.6
13
4.3
4.8
4.4
4.9
0.14
0.6
14
16
a
–log10(EC50/M)
b Standard error in pEC50
c –log10(CC50/M)
d Standard error in pCC50
We have introduced the oxime group as a warhead and shown that it contributes more than the
nitrile group to potency while providing better access to prime region of the catalytic site. We have
demonstrated that dipeptide aldehydes and azadipeptide nitriles are significantly more potent inhibitors
of cruzain than the corresponding dipeptide nitriles. We have also presented examples of the effects of
warhead exchange being modulated by molecular recognition elements, in particular the P1 and P3
substituents, which are remote from the warheads.
11

Supporting Information
A PDF file with full experimental details for synthesis and assays a comma-separated text file of
structures (as SMILES) and assay results are made available as supporting information.
Funding Sources
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) through the granting award
numbers 2013/18009-4, 2013/01128-0, 2016/10362-5, 2015/14304-7, 2016/0946-5. Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq) through granting award number 400658-2014-3.
Acknowledgement
We are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for project
funding through grant number 2013/18009-4 and Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) through granting award number 400658-2014-3. We are also grateful to FAPESP
for scholarships to DGS (2013/01128-0; 2016/10362-5), DDV (2015/14304-7) and LC (2016/0946-5).
We thank Juliana Gomes and Cristian Camilo for helpful discussions and Jonatan Catai for obtaining
mass spectra.
Abbreviations
Boc, tert-butyloxycarbonyl; Cbz, carboxybenzyl; CC50, half maximal cytoxicity concentration; EC50,
half maximal effective concentration; EDC, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride; IBX, 2-iodoxybenzoic acid; Ki, inhibitory constant; pCC50, −log10(CC50/M); pEC50,
−log10(EC50/M); pKi, −log10(Ki/M); SAR, structure activity relationship; ∆pKi, change in pKi resulting
from a structural transformation.
12

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Graphical abstract
17