Macrocyclic protease inhibitors with reduced peptide character

Article abstract of DOI:10.1002/anie.201404301

There is a real need for simple structures that define a β-strand conformation, a secondary structure that is central to peptide-protein interactions. For example, protease substrates and inhibitors almost universally adopt this geometry on active site binding. A planar pyrrole is used to replace two amino acids of a peptide backbone to generate a simple macrocycle that retains the required geometry for active site binding. The resulting β-strand templates have reduced peptide character and provide potent protease inhibitors with the attachment of an appropriate amino aldehyde to the C-terminus. Picomolar inhibitors of cathepsin L and S are reported and the mode of binding of one example to the model protease chymotrypsin is defined by X-ray crystallography. The incorporation of a pyrrole into a peptide backbone generates simple macrocycles that adopt a β-strand geometry. The attachment of a P1 amino aldehyde to these templates then gives rise to potent protease inhibitors (see example, top, which has K_i values of 440 pM and 920 pM against the cysteine cathepsins L and S, respectively). A crystal structure of a related derivative bound to chymotrypsin (see picture, bottom) confirms the design.

Full text of DOI:10.1002/anie.201404301

Angewandte
Chemie
DOI: 10.1002/anie.201404301
b-Strand Templates
Macrocyclic Protease Inhibitors with Reduced Peptide Character**
Krystle C. H. Chua, Markus Pietsch, Xiaozhou Zhang, Stephanie Hautmann, Hon Y. Chan,
John B. Bruning, Michael Gꢀtschow, and Andrew D. Abell*
Abstract: There is a real need for simple structures that define
a b-strand conformation, a secondary structure that is central to
peptide–protein interactions. For example, protease substrates
and inhibitors almost universally adopt this geometry on active
site binding. A planar pyrrole is used to replace two amino
acids of a peptide backbone to generate a simple macrocycle
that retains the required geometry for active site binding. The
resulting b-strand templates have reduced peptide character
and provide potent protease inhibitors with the attachment of
an appropriate amino aldehyde to the C-terminus. Picomolar
inhibitors of cathepsin L and S are reported and the mode of
binding of one example to the model protease chymotrypsin is
defined by X-ray crystallography.
P
roteases almost uniformly bind their substrates and
inhibitors in a conformation whereby the peptide (or
a peptide-like) backbone adopts a b-strand geometry.[1] This
mode of binding is primarily dictated by the geometry of the
active site subsites (designated S1-Sn and S1’ to Sn’ according
to the nomenclature of Schechter and Berger)[2] that accom-
modate the amino acid side chains of the substrate or
inhibitor. An important approach to inhibitor design is to
then introduce a carefully designed conformational constraint
into the structure to pre-organize its backbone into a b-strand
conformation, thereby reducing entropy loss associated with
ligand-receptor binding, while also enhancing biostability.[3–6]
This typically involves chemically linking either the P1 and P3
or the P1’ and P3’ residues of a peptidomimetic-based
protease inhibitor (see for example macrocycles 1 and 2,
Scheme 1. Macrocyclic protease inhibitors. Cbz=benzyloxycarbonyl,
Boc=tert-butoxycarbonyl.
respectively, in Scheme 1), leading to enhanced potency
against its target protease.[3–6] However the resulting macro-
cycles are somewhat problematic, retaining considerable
peptide-like character owing to the requirement for a tripep-
tide- or modified tripeptide backbone capable of presenting
the necessary side chains for cyclization.
A considerable challenge is to design macrocyclic b-strand
templates of reduced peptide character for incorporation into
protease inhibitors. There would be an additional advantage
in generating such templates in which the P1 or P1’ residues
are not part of the macrocycle. This would then allow the
appendage of a range of groups at these positions (for
example, amino aldehyde or amino dicarbonyl moieties)[3,4,7,8]
to allow the targeting of a particular protease.[9] The approach
reported herein involves replacing two amino acids of the
macrocyclic backbone of first-generation inhibitors of type 2
with a substituted pyrrole, where this group would be
expected to maintain the planar geometry required for an
extended b-strand geometry[10] (see 3–5, Scheme 1). Other
heterocycles have also been used in this context.[11,12] Phen-
ylene groups (0, 1, or 2 as in the P2-P4 linker of 3, 4, and 5,
respectively) were incorporated into the macrocycles to
further constrain the backbone geometry[3–5] and to target
a particular protease through interaction with the respective
active site subsites. In this study, we target ovine m-calpain,
human cathepsins L and S (Cat L and Cat S), bovine a-
chymotrypsin, and human leukocyte elastase (HLE). Cal-
pains are an ubiquitous class of non-lysosomal cysteine
proteases, the over-activation of which is implicated in
[*] Dr. K. C. H. Chua, Dr. M. Pietsch,[+] X. Zhang, Prof. Dr. A. D. Abell
School of Chemistry & Physics
The University of Adelaide
North Terrace, Adelaide, SA 5005 (Australia)
E-mail: andrew.abell@adelaide.edu.au
H. Y. Chan, Dr. J. B. Bruning
School of Molecular and Biomedical Science
The University of Adelaide
North Terrace, Adelaide, SA 5005 (Australia)
S. Hautmann, Prof. Dr. M. Gꢀtschow
Pharmaceutical Institute, Pharmaceutical Chemistry I
University of Bonn
An der Immenburg 4, 53121 Bonn (Germany)
[+] Present address: Department of Pharmacology
University of Cologne
Gleueler Strasse 24, 50931 Cologne (Germany)
[**] We acknowledge the assistance of Dr. J. Morton (Lincoln University)
for the supply of calpain, Dr. S. Heng (University of Adelaide) for
help in optimizing the crystallographic conditions and the Austral-
ian Research Council (ARC) for funding.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201404301.
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a variety of pathophysiological conditions, including cataract
and traumatic brain injury.[8] Cathepsins L and S are lysoso-
mal cysteine proteases that are known to play key roles in
tumor progression and autoimmune disorders, respectively,[13]
while a-chymotrypsin and HLE are typical serine proteases,
with the former being a suitable model enzyme for solving
a ligand/protease X-ray structure to define the mode of
binding.
The key step in the synthesis of the component macro-
cycles of 3–5 involved ring-closing metathesis (RCM)[14] of an
appropriate diene 8 (Scheme 2). These dienes were readily
prepared from 2-ethoxycarbonylpyrrole by Friedel–Crafts
acylation with the respective acid chloride in the presence of
Yb(OTf)3. Ester hydrolysis of 6, followed by HATU- or
EDCI-catalyzed coupling of the resulting pyrrolic acid 7 with
the appropriate olefinic amino acid ester (either (S)-allyl-Gly-
OMe or (S)-allyl-Tyr-OMe), afforded the RCM substrates 8.
Treatment with Grubbs second-generation catalyst then gave
the macrocycles 9, which were not isolated but rather
hydrogenated on treatment with H2 and Pd on C to give the
key macrocycles 10 (see the Supporting Information for
further details). The final steps in the synthesis involved
hydrolysis of the methyl esters 10, coupling the resulting acids
with either (S)-leucinol or (S)-phenylalaninol, in the presence
of HATU, followed by oxidation of the C-terminal primary
alcohol with Dess–Martin periodinane to give the desired
macrocyclic aldehydes 3–5, see Schemes 1 and 2. The acyclic
dienes 13–15 were also prepared as shown in Scheme 2 to
provide suitable acyclic controls for the biological evaluation.
Each aldehyde (3–5 and 13–15) was assayed against three
cysteine proteases (m-calpain, Cat L, and Cat S), and two
serine proteases (a-chymotrypsin and HLE); the data are
shown in Table 1. The assay results for the unsubstituted
macrocycles 3a and 3b against Cat L and Cat S are partic-
ularly significant. Both compounds are highly potent, with
picomolar Ki values against Cat S. The macrocycle of these
compounds is clearly beneficial to activity, with the corre-
sponding acyclic analogues 13a and 13b being clearly less
active (> 6-fold for Cat S and > 100-fold for Cat L). Macro-
cycle 3a, and to a lesser extent 3b, are also highly potent
against m-calpain, exhibiting Ki values of 58 and 140 nm,
respectively. This compares to a Ki of 26 nm (IC50 of 30 nm)3
for the first-generation macrocycle 1 against the same
enzyme.
The alternative macrocycles 4 and 5 that contain one and
two aryl groups respectively, in the constituent macrocycle,
also display good activity against all three cysteine proteases.
Macrocycle 5a is in fact the most potent inhibitor of Cat S (Ki
of 0.74 nm). The Phe containing macrocycle 5b displays an
advantage over its acyclic analogue 15b for the three cysteine
proteases. More generally, the benefit of the macrocycle over
its acyclic analogue is most evident for the inhibition of Cat S
(8-fold).
There is some apparent preference for Leu over Phe at P1
of our macrocyclic inhibitors against the cysteine proteases.
However, the inclusion of Phe at P1, as in 3b, 4b, and 5b,
results in more potent inhibitors of a-chymotrypsin compared
to the corresponding Leu derivatives 3a, 4a, and 5a (4- to 57-
fold). The same is true for the acyclic compounds 13b, 14b,
Scheme 2. Synthesis of aldehydes: a) Yb(OTf)3, CH3NO2, RT, 10-unde-
cenoyl chloride (6, X=(CH2)5, 17%) or p-allyloxyphenylpropionyl
chloride (6, X=C6H4O, 31%); b) KOH, THF, H2O, 40–508C, (7,
X=(CH2)5, 81%; 7, X=C6H4O, 96%); c) HOBt, DIEA, CH2Cl2, RT and
HATU, (S)-allyl-Gly-OMe, (8, X=(CH2)5, Y=CH2, 48%), or EDCI, (S)-
allyl-Gly-OMe, (8, X=C6H4O, Y=CH2, 94%), or HATU, (S)-allyl-Tyr-
OMe, (8, X=C6H4O, Y=CH2C6H4OCH2, 84%); d) Grubbs second-
generation catalyst, CH2Cl2, D, then H2, Pd-C, EtOAc (10, X=(CH2)5,
Y=CH2, 28%; 10, X=C6H4O, Y=CH2, 36%; 10, X=C6H4O,
Y=CH2C6H4OCH2, 56%); e) 2m NaOH, THF, RT; then HATU, HOBt,
DIEA, DMF, RT, and (S)-leucinol, (11a, X=(CH2)5, Y=CH2, 62%;
X=C6H4O, Y=CH2, 81%; X=C6H4O, Y=CH2C6H4OCH2, 54%; 12a,
X=(CH2)5, Y=CH2, 79%; X=C6H4O, Y=CH2, 57%; X=C6H4O,
Y=CH2C6H4OCH2 71%) or (S)-phenylalaninol, (11b, X=(CH2)5,
Y=CH2, 58%; X=C6H4O, Y=CH2, 36%; X=C6H4O,
Y=CH2C6H4OCH2, 55%; 12b, X=(CH2)5, Y=CH2, 92%; X=C6H4O,
Y=CH2, 60%; X=C6H4O, Y=CH2C6H4OCH2, 62%); f) Dess–Martin
periodinane, CH2Cl2, RT (3a, 37%; 3b, 34%; 4a, 37%; 4b, 15%; 5a,
41%; 5b, 65%; 13a, 31%; 13b, 22%; 14a, 21%; 14b, 29%; 15a,
53%; 15b, 48%). DIEA=diisopropylethylamine, DMF=N,N-dimethyl-
formamide, EDCI=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,
HATU=O-(7-azabenzotriazol-1-yl-N,N,N’,N’-tetramethyluronium hexa-
fluoro-phosphate, HOBt=N-hydroxybenzotriazole.
2
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Table 1: In vitro inhibition assay data and Ki values.
Compound
Number of aryl groups
P1
m-Calpain[a,b]
Cat L[a,c]
Cat S[a,c]
a-Chymotrypsin[d]
HLE[a,e]
3a
3b
4a
4b
5a
5b
0
0
1
1
2
2
Leu
Phe
Leu
Phe
Leu
Phe
58
0.44
1.7
0.92
0.91
14
>10000
2500
>10000
430
1900
33
>2500
>2500
n.i.[g]
140
220
130
180
220
6.8
n.d.[f]
3.0
n.d.[f]
0.74
2.3
n.i.[g]
640
600
30
13a
13b
14a
14b
15a
15b
0
0
1
1
2
2
Leu
Phe
Leu
Phe
Leu
Phe
35
63
59
720
6800
1100
130
200
14
250
1.7
53
5.7
95
1.0
17
0.76
19
>10000
2500
940
52
180
300
510
160
180
56
n.i.[g]
690
[a] Ki values [nm] were calculated from IC50 values by applying the Cheng–Prusoff equation for competitive inhibition. [b] Values are the mean of two
experiments in triplicate with seven different inhibitor concentrations. Variation between experiments is less than 10%. The assay was performed with
the fluorogenic substrate BODIPY-FL casein at a final concentration 0.09 mm (0.14Km).[17] [c] Data were calculated from experiments with five different
inhibitor concentrations. IC50 values were obtained by nonlinear regression, with standard errors <25%. Assays were performed with the chromogenic
substrate Cbz-Phe-Arg-pNA at a final concentration of 100 mm (cathepsin L: 5.88Km; cathepsin S: 0.85Km).[18] [d] Ki values are the mean of three
experiments. Variation between experiments is less than 10%. The assays were performed with the chromogenic substrate Suc-Ala-Ala-Pro-Phe-pNA at
final concentrations of 20 mm (0.3Km) and 50 mm (0.75Km). Limits refer to measurements at a substrate concentration of 50 mm and a single inhibitor
concentration (25–125 mm).[19] [e] Data were calculated from experiments with five different inhibitor concentrations. IC50 values were obtained by
nonlinear regression, with standard errors <40% except for 14a (50%). Assays were performed with the chromogenic substrate MeO-Suc-Ala-Ala-
Pro-Val-pNA at a final concentration of 100 mm (1.85Km). Limits refer to measurements at a single inhibitor concentration of 10 mm.[20] [f] n.d.=not
determined. [g] n.i.=no inhibition; refers to less than 50% inhibition at a substrate concentration of 50 mm and an inhibitor concentration of 250 mm
(a-chymotrypsin). For elastase, it refers to less than 20% inhibition at an inhibitor concentration of 10 mm. None of the compounds inhibited bovine
trypsin[21] (less than 20% inhibition at an inhibitor concentration of 10 mm).
and 15b compared to 13a, 14a, and 15a. These data clearly
reflect the known primary substrate specificity of a-chymo-
trypsin for aromatic amino acids at the P1 position. Macro-
cycle 5b, with its two aryl groups, is particularly potent
towards a-chymotrypsin and has a Ki of 33 nm. This com-
pound also shows a significant gain in inhibitory potency with
macrocyclization (36-fold, 5b vs. 15b). Interestingly, the
macrocycles are not potent inhibitors of HLE to provide
some selectivity within the serine protease family. It should be
noted that Leu is preferred over Phe, in accordance with the
specificity of HLE for aliphatic residues at the P1 position.
The most potent HLE inhibitor of the series is compound 13a
Figure 1. Macrocycle 5b bound to a-chymotrypsin (left) and 5b
(Ki of 52 nm) that lacks aromatic groups in its side chains.
A crystal structure of the macrocyclic aldehyde 5b
covalently bound to serine 195 of bovine a-chymotrypsin
was solved to define the mode of protease binding of our new
macrocycles, and particularly the extended backbone with its
constituent pyrrole (see Figure 1 and the Supporting Infor-
mation for further details). An overlay of this structure with
that of an extended peptide-based inhibitor (PMP-C, PDB:
1L1)[15] of bovine a-chymotrypsin is also shown in Figure 1.
PMP-C binds in an extended b-strand geometry that extends
from P5 to P4’. As expected, the P1 Phe side chain of 5b binds
in the S1 pocket as defined by a cavity formed by the main
chain atoms of Cys191, Gly216, and the disulfide bond formed
between Cys191 and Cys220. Importantly and exactly as per
our design, the backbone of 5b superimposes nicely with that
of b-strand bound PMP-C, with the pyrrole of 5b clearly
defining the required geometry while occupying the P3 and
P4 amino acid positions. An overlay of the structure of 5b
with a simple peptidic aldehyde (N-acetyl-l-Leu-l-phenyl-
(yellow) overlayed with truncated PMP-C (P1Leu-P2Thr-P3Cys-P4Ala,
gray; right).
alanal)[16] bound to a-chymotrypsin (see the Supporting
Information for further details) also shows that 5b forms
a b-strand conformation nearly identical to that of N-acetyl-l-
Leu-l-phenylalanal bound to chymotrypsin,[16] with both
phenyl groups positioned similarly in the S1 cavity (Support-
ing Information, Figure S2). Compound 5b forms the desired
b-strand geometry with two peptide torsion angles f= 1478,
y = À988 (calculated between C’33 and C’37, N31 and N35)
and f= À1658, y = À888 (calculated between C’29 and C’33,
N25 and N31).
In conclusion, we present a new class of macrocyclic
protease inhibitors in which a backbone amino acid and the
associated peptide bond are replaced with a planar pyrrole.
This results in reduced peptide character, while retaining
a backbone b-strand geometry to facilitate active site binding.
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The macrocycle is introduced between the P2 amino acid and
N-terminus to leave the P1 position free for incorporation of
an appropriate functionality for targeting a particular pro-
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Received: April 14, 2014
Published online: && &&, &&&&
Keywords: inhibitors · macrocycles · peptidomimetics ·
.
proteases · b-strands
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Angew. Chem. Int. Ed. 2014, 53, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Communications
b-Strand Templates
The incorporation of a pyrrole into a pep-
tide backbone generates simple macro-
cycles that adopt a b-strand geometry.
The attachment of a P1 amino aldehyde to
these templates then gives rise to potent
protease inhibitors (see example, top,
which has Ki values of 440 pm and 920 pm
against the cysteine cathepsins L and S,
respectively). A crystal structure of
a related derivative bound to chymotryp-
sin (see picture, bottom) confirms the
design.
K. C. H. Chua, M. Pietsch, X. Zhang,
S. Hautmann, H. Y. Chan, J. B. Bruning,
M. Gꢁtschow,
A. D. Abell*
&&&& — &&&&
Macrocyclic Protease Inhibitors with
Reduced Peptide Character
Angew. Chem. Int. Ed. 2014, 53, 1 – 5
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!