Design, synthesis, and optimization of novel epoxide incorporating peptidomimetics as selective calpain inhibitors

Article abstract of DOI:10.1021/jm4006719

Hyperactivation of the calcium-dependent cysteine protease calpain 1 (Cal1) is implicated as a primary or secondary pathological event in a wide range of illnesses and in neurodegenerative states, including Alzheimer's disease (AD). E-64 is an epoxide-containing natural product identified as a potent nonselective, calpain inhibitor, with demonstrated efficacy in animal models of AD. By use of E-64 as a lead, three successive generations of calpain inhibitors were developed using computationally assisted design to increase selectivity for Cal1. First generation analogues were potent inhibitors, effecting covalent modification of recombinant Cal1 catalytic domain (Cal1_cat), demonstrated using LC-MS/MS. Refinement yielded second generation inhibitors with improved selectivity. Further library expansion and ligand refinement gave three Cal1 inhibitors, one of which was designed as an activity-based protein profiling probe. These were determined to be irreversible and selective inhibitors by kinetics studies comparing full length Cal1 with the general cysteine protease papain.

Full text of DOI:10.1021/jm4006719

Article
pubs.acs.org/jmc
Design, Synthesis, and Optimization of Novel Epoxide Incorporating
Peptidomimetics as Selective Calpain Inhibitors
Isaac T. Schiefer,† Subhasish Tapadar,† Vladislav Litosh,† Marton Siklos,† Rob Scism,†
Gihani T. Wijewickrama,† Esala P. Chandrasena,† Vaishali Sinha,† Ehsan Tavassoli,† Michael Brunsteiner,†
Mauro Fa’,‡ Ottavio Arancio,‡ Pavel Petukhov,† and Gregory R. J. Thatcher*,†
†Department of Medicinal Chemistry and Pharmacognosy, University of Illinois College of Pharmacy, University of Illinois at
Chicago, 833 S. Wood Street, Chicago, Illinois 60612-7231, United States
‡Department of Pathology and Cell Biology, The Taub Institute for Research on Alzheimer’s Disease and the Aging Brain, Columbia
University, 630W 168th Street, New York, New York 10032, United States
S
* Supporting Information
ABSTRACT: Hyperactivation of the calcium-dependent cysteine protease calpain 1 (Cal1) is implicated as a primary or
secondary pathological event in a wide range of illnesses and in neurodegenerative states, including Alzheimer’s disease (AD). E-
64 is an epoxide-containing natural product identified as a potent nonselective, calpain inhibitor, with demonstrated efficacy in
animal models of AD. By use of E-64 as a lead, three successive generations of calpain inhibitors were developed using
computationally assisted design to increase selectivity for Cal1. First generation analogues were potent inhibitors, effecting
covalent modification of recombinant Cal1 catalytic domain (Cal1cat), demonstrated using LC−MS/MS. Refinement yielded
second generation inhibitors with improved selectivity. Further library expansion and ligand refinement gave three Cal1
inhibitors, one of which was designed as an activity-based protein profiling probe. These were determined to be irreversible and
selective inhibitors by kinetics studies comparing full length Cal1 with the general cysteine protease papain.
substrates and inhibitors is introduced in Figure 1. The majority
of reported calpain inhibitors rely upon the ability of an
electrophilic reactive group, or “warhead”, to either reversibly or
irreversibly modify the active site cysteine of calpain. A natural
product, E-64, L-trans-epoxysuccinylleucylamido(4-guanidino)-
butane (Figure 1), was an early identified cysteine protease
inhibitor, utilizing an epoxide for active site modification.21,22
While being nonreactive toward other protease superfamilies
(i.e., aspartic, serine, etc.), E-64 serves as a benchmark, high
affinity, nonselective, irreversible calpain inhibitor.23,24 In a
transgenic model of AD, E-64 demonstrated excellent in vivo
efficacy.25 Therefore, by use of E-64 as a lead and benchmark, the
object of the present study was to maintain potency while
increasing Cal selectivity and druggability.
CA clan cysteine proteases (i.e., papain, calpains, and
lysosomal cathepsins) have similar P1−P3 substrate binding
pockets, which results in a common preference for hydrophobic
residues at the S2 subsite (i.e., Leu, Ile, Val, Phe, Tyr).26,27 It has
been suggested that inhibitors containing S,S epoxide stereo-
chemistry bind preferentially into the P1−P3 pocket of CA clan
proteases.28,29 Accordingly, E-64 and related S,S epoxides
containing hydrophobic residues at the P2 position have
INTRODUCTION
■
Calpains are a class of ubiquitously expressed calcium-dependent
cysteine proteases that regulate numerous intracellular signaling
cascades and are fundamentally involved in regulating protein
kinases responsible for cytoskeletal dynamics and remodeling.1−4
Under physiological conditions, transient and localized Ca2+
fluxes from extracellular or intracellular calcium stores result in
controlled activation of local calpain populations. While regional
calpain proteolytic activity is essential for native Ca2+ signaling
and cytoskeletal remodeling, pathological conditions may
produce excessive levels of Ca2+, resulting in widespread calpain
activation and unregulated proteolysis.5,6 Calpain proteolytic
activity contributes to secondary degeneration in situations of
acute cellular stress following myocardial ischemia, cerebral
ischemia, and traumatic brain injury.7−10 Enhanced calpain
activity in platelets is a contributor to atherothromobosis and
diabetes pathology.11,12 Calpain hyperactivation associated with
altered Ca2+ homeostasis contributes to pathogenesis of
Huntington’s disease, Parkinson’s disease, cataract formation,
glaucoma, multiple sclerosis, and Alzheimer’s disease
(AD).5,13−18 In this context, an appropriate selective calpain
inhibitor may hold therapeutic potential in selected disease
states.
Attempts to develop calpain inhibitors have been cataloged
Received: February 7, 2013
previously.19,20 The structural nomenclature of cysteine protease
© XXXX American Chemical Society
A
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Figure 1. Protease substrates are designated according to their amino acid residues extending from the scissile bond. “Unprimed” and “primed” substrate
residues are designated P1, P2, etc. and P1′, P2′, etc., respectively. The design rationale is illustrated using E-64 as a lead.
Figure 2. HPLC−UV−vis (λ = 280 nm) chromatogram from incubation of 31 (100 μM) with excess GSH (5 mM) in PBS (50 mM, pH 7.4) at 37 °C for
24 h. Product identity was confirmed using LC−MS/MS and comparison of retention time of authentic sample in the absence of GSH.
shown poor selectivity and good potency for these proteases.
Recent efforts examined an array of P4-P3-P2-epoxysuccinate
peptides for inhibitory activity against CA clan cysteine
proteases, including Cal1, Cal1cat (recombinant calpain 1
catalytic domain), and lysosomal cysteine proteases (cathepsins,
Cath).30 While these peptides do not have optimal druglike
properties, their activity profiles give valuable insight into the
design and development of novel selective calpain inhibitors
using peptidomimetic epoxides. Three successive generations of
inhibitors were synthesized using computationally assisted
design and Cal1 inhibition data. Modification of the active site
cysteine was confirmed using LC−MS/MS and the relative
selectivity assessed against papain using an enzyme kinetics
analysis. The study presents novel, selective Cal1 inhibitors that
because of the presence of the electrophilic epoxide warhead also
provide an ideal activity-based protein profiling (ABPP) probe
for future mechanistic investigation.
RESULTS AND DISCUSSION
■
Design and Synthesis. The epoxysuccinate moiety of E-64
is considered essential for potent cysteine protease inhibition.
Alternatives to the epoxide warhead, such as alkene and aziridine
analogues, possess weak inhibitory activity.21,31 Despite concerns
regarding potential ADMET complications stemming from
B
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a
Scheme 1
a
Reagents: (i) EDCI, HOBT, DIPEA, CH2Cl2, 0 °C; (ii) EDCI, HOBT, DIPEA, DMF, 0 °C; (iii) LiOH, THF/MeOH/H2O, 0 °C.
Table 1. Epoxysuccinate Peptidomimetic Structures and Inhibition Data for Full Length Cal1
a
R1
R2 or R3
epoxide stereochem
Cal1 IC50
E-64
20
L-leucine
R2 = guanidinyl-1-pentyl
S,S
S,S
S,S
R,R
S,S
R,R
S,S
S,S
S,S
S,S
S,S
S,S
R,R
S,S
R,R
S,S
S,S
S,S
S,S
S,S
S,S
S,S
S,S
S,S
S,S
S,S
S,S
S,S
S,S
S,S
100 nM
100 nM
100 nM
>1 μM
50 nM
250 nM
150 nM
100 nM
100 nM
5 μM
L-leucine
R2 = phenyl
21a
21b
22a
22b
23
L-leucine
R2 = 2,6-difluorophenyl
L-leucine
R2 = 2,6-difluorophenyl
L-leucine
R2 = 4-(4-fluorophenyl)thiazol-2-amino
R2 = 4-(4-fluorophenyl)thiazol-2-amino
R2 = 4-F-phenyl-SO2-NH-(CH2)4-NH
R2 = lipoyl-NH-(CH2)4-NH
R2 = D-biotin-NH-(CH2)4-NH
R2 = phenyl
L-leucine
L-leucine
24
L-leucine
25
L-leucine
26
L-histidine
27
L-histidine
R2 = 1,3,5-trimethylanilino
5 μM
28a
28b
29a
29b
30
L/D-histidine
L/D-histidine
L/D-histidine
L/D-histidine
L-Ala(4-thiazyl)
L-Ala(4-thiazyl)
L-Ala(4-thiazyl)
L-His(2-Me)
L-His(4-Me)
L-Ala(4-thiazyl)
L-Ala(4-thiazyl)
L-Ala(4-thiazyl)
L-Ala(4-thiazyl)
L-Ala(4-thiazyl)
L-Ala(4-thiazyl)
L-Ala(4-thiazyl)
L-Ala(4-thiazyl)
L-Ala(4-thiazyl)
L-Ala(4-thiazyl)
R2 = 4-(4-fluorophenyl)thiazol-2-amino
R2 = 4-(4-fluorophenyl)thiazol-2-amino
R2 = 6-fluorobenzo[d]thiazol-2-amino
R2 = 6-fluorobenzo[d]thiazol-2-amino
R2 = phenyl
100 nM
b
>1 μM
530 nM
b
>1 μM
2.5 μM
100 nM
100 nM
5 μM
31
R2 = 4-(4-fluorophenyl)thiazol-2-amino
R2 = 4-(4-ethynylphenyl)thiazol-2-amino
R2 = 4-(4-fluorophenyl)thiazol-2-amino
R2 = 4-(4-fluorophenyl)thiazol-2-amino
R3 = phenyl
32
33
34
225 nM
1 μM
35
36
R3 = 4-fluorophenyl
100 nM
1.5 μM
400 nM
37
R3 = 4-(piperidin-1-ylsulfonyl)phenyl
R3 = benzo[d][1,3]dioxol-5-yl
R3 = 4-sulfamoylphenyl
38
b
39
>1 μM
b
40
R3 = 3,5-bis(trifluoromethyl)phenyl
R3 = 4-bromophenyl
>1 μM
41
40 nM
42
R3 = 4-nitrophenyl
300 nM
b
43
R3 = 2,6-bis(fluoro)phenyl
>1 μM
b
44
R3 = 2,4,6-tris(methyl)phenyl
>1 μM
a
Inhibition was measured by monitoring fluorescence emission at 480 nm after 20 min incubations with varying concentration of inhibitor in the
presence of the FRET substrate: % inhibition normalized to control incubations in the absence of inhibitor. Data represents the mean SD of
b
triplicate experiments. Inhibition not quantifiable at 1 μM.
incorporation of the epoxysuccinate moiety, E-64 and derivatives
have been approved for clinical studies,32−34 and there are
multiple reports of in vivo efficacy and safety by E-64 and related
epoxysuccinate analogues in mice.35−38 Retention of the
epoxysuccinate group also facilitates the design of ABPP probes
to identify off-target proteins that may contribute to efficacy or
toxicity. Furthermore, we observed that epoxysuccinate contain-
ing peptidomimetics show negligible reactivity after 24 h of
incubation in the presence of excess GSH at physiological pH
and temperature (PBS, 50 mM, pH 7.4, 37 °C; Figure 2). A study
describing the low inherent reactivity of the epoxysuccinate
moiety with thiols has been reported previously.39 Given these
considerations, the epoxysuccinate moiety was retained and
design focused on modifying and evaluating two main portions of
the peptidomimetic scaffold: (1) the P3/P4 cap group and (2)
the P2 site that is occupied by a L-leucine in E-64.
C
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Synthesis was directed at generation of a library of
peptidomimetic epoxides possessing either natural or non-
natural peptidomimetic residues at the P2 position, designated
the R1 substituent, and varying the P3/P4 cap groups, designated
as R2 in Scheme 1. Initially, the R2-amines were coupled to the
commercially available Boc-protected amino acids following
typical peptide coupling procedures either using HOBT and
EDCI or in the presence of CDI to give the corresponding Boc-
protected peptidomimetic scaffolds (9−13). The epoxysuccinate
moiety was synthesized in three steps starting from either L-DET
or D-DET, following published procedures to yield the
unambiguous epoxysuccinate R,R (7) and S,S (8), respectively.40
Following TFA deprotection, the appropriate peptidomimetic
scaffold was coupled to 7 or 8 using EDCI and HOBT in the
presence of DIPEA to give the corresponding epoxide esters
(14−19). Ester saponification using LiOH at 0 °C afforded the
analogous epoxy acids 20−34 (Table 1). In four instances (28a,b
and 29a,b), histidine containing analogues were found to be
diastereomeric mixtures (L/D, S, S) and (L/D, R, R) resulting from
racemization of the peptidyl α-carbon during the initial peptide
coupling using CDI.
Computational guidance was employed, vide infra, along with a
novel click chemistry route to generate a library of triazole
incorporating calpain inhibitors. The key alkynyl intermediate,
19, was functionalized with arylazides using a Cu[II] catalyzed
Huisgen cycloaddition, with the aid of TBTA to give the
corresponding triazole aryl incorporating epoxyester peptidomi-
metics (35E−44E). Ester saponification yielded the desired
epoxyacids (35−44) in good yield over two steps (85−100%,
Scheme 3).
Calpain Inhibition by First Generation Inhibitors: P3/
P4 Cap Group Selection. Calpain activity was measured using
full-length human Cal1 in the presence of a FRET substrate after
20 min. IC50 values were approximated by co-incubation of
varying concentrations (10−1000 nM) in the presence of the
FRET substrate (DABCYL)TPLK-SPPSPR-(EDANS), and %
inhibition was calculated by normalizing to control experiments
containing no inhibitor. A peptide inhibitor, Z-LLY-FMK, was
used as a positive control.
First generation inhibitors 20−25 (Table 1) were designed to
mimic E-64 at the P2 position while varying the P3 cap group to
increase druggability compared to the ionizable guanidino group
of E-64. The majority of first generation inhibitors were found to
be approximately equipotent to E-64, and one inhibitor, 22a,
displayed improved potency with an IC50 value of 50 nM ( 25).
These results are consistent with the understanding that
hydrophobic amino acid residues at the P2 position deliver
high affinity for Cal1. In the instances of 21a,b and 22a,b the
importance of the S,S epoxide stereochemistry was con-
firmed.28,29
A limitation in the synthesis of epoxide incorporating
inhibitors is the propensity of the epoxide to undergo ring
opening. Hence, convergent synthesis is commonly utilized and
the epoxide functionality is incorporated after derivatization has
taken place, although a divergent synthetic approach is preferred
for library development during lead optimization (Scheme 2).
Scheme 2
Active Site Modification by Novel Calpain Inhibitors.
The recombinant Cal1 catalytic domain (Cal1cat) is composed of
the proteolytic domain of the full-length enzyme and has been
used as a surrogate to study calpain activity.41−44 Cal1cat provides
an advantage because upon Ca2+-induced activation, full length
Cal1 engages in autocatalytic, self-proteolysis complicating
analysis of activity and inhibition. Cal1cat is devoid of the
autocatalytic activity observed for full length Cal1. Recombinant
rat Cal1cat was expressed and purified from E. coli to examine
aspects of inhibition in more detail.
Inhibition kinetics and X-ray crystallography support a
mechanism of calpain inhibition by E-64, resulting from covalent
modification of the active site Cys.45 LC−MS/MS supports a
similar mechanism for 22a. The experimental methodology is
outlined in Figure 3A. The peptide fragment containing the
Copper catalyzed Huisgen cycloadditions, also known as “click
chemistry”, have received much attention because of low cross-
reactivity with other functional groups, making it the ideal tool
for the generation of a library of epoxide incorporating inhibitors.
a
Scheme 3
a
Reagents and conditions: (i) R3-N3, CuSO4, NaAsc, TBTA, H2O/t-BuOH/EtOH (2:1:1), rt, 12 h, 90−100%; (ii) LiOH, THF/MeOH/H2O, 0 °C,
85−100%.
D
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Figure 3. Confirmation of covalent modification of Cal1 active site Cys by epoxysuccinate inhibitors. (A) After in gel digest and capping of free cysteines
with iodoacetamide (IAA), peptide fragments were analyzed by LC−MS/MS. (B) Total ion chromatograms (TIC) for the incubation of recombinant
Cal1cat (5 μM) in the absence or presence of inhibitors (10 μM). A nonactive site and IAA-capped peptide fragment (tR = 41.3 min) was used as an
internal standard for EIC quantitation. (C) Active site Cys modification of Cal1cat was concentration dependent. (D) Co-incubation of Cal1cat (1 μM)
with an inhibitor mixture (5 μM) showed competitive modification of the active site by 22a and E-64.
active site Cys (TDICQGALGDC*WLLAAIASLTNETILHR)
was identified after covalent modification by IAA, 22a, or E-64. A
peptide fragment containing a labeled nonactive site cysteine was
used as an internal standard for semiquantitative analysis of the
degree of active site modification observed (Figure 3B). It is
noteworthy that although Cal1cat was employed in these studies,
analysis of full length Cal1 gave an identical active site peptide
fragment (TIC of control digest from Cal1cat and full length Cal1
are compared in the Supporting Information).
Incubations of Cal1cat (1 μM) with either E-64 or 22a showed
a concentration dependent modification of the active site
cysteine (Figure 3C). Co-incubation of Cal1cat with E-64 and
22a (both 5 μM) demonstrated relatively greater modification by
22a (Figure 3D). This outcome, combined with the potency of
inhibition by 22a, supported retention of the 4-F-phenylthiazole
P3/P4 cap group for subsequent ligand development.
Incorporation of Selectivity in Second Generation
Calpain Inhibitors. CA clan cysteine proteases have similar
“unprimed” substrate binding pockets, with a common
preference for hydrophobic residues at the S2 binding site (i.e.,
Leu, Ile, Val, Phe).26,27,29 Previous work on peptides appended
with an epoxysuccinate ester warhead has indicated that a P2
histidine might confer Cal1 selectivity, the rationale given being
the presence of a stabilizing hydrogen bond occurring within the
S2 pocket with a highly conserved water molecule chelated by
Glu-349 and Thr-210.30 A focused series of L-histidine
incorporating analogues was synthesized and evaluated (26−
29, 33, 34) to probe the S2 pocket.
Facile protonation of the histidine imidazole ring is anticipated
to potentially hinder bioavailability; therefore, thiazolyl ana-
logues were also developed (30−32).46 Screening of the second
generation compounds revealed a general loss of activity on
replacement of L-leucine (Table 1), although the P2 analogues,
28a and 31, were approximately equipotent to E-64 (IC50 ≈ 100
nM). The crystal structures used to rationalize the selectivity and
active site interactions of L-histidine at P2 contained ethyl
epoxysuccinate esters, yet in our hands, simple ethyl esters of first
and second generation epoxysuccinates did not give measurable
inhibition of Cal1 (data not shown).
Target selectivity is the primary goal in mechanism-based drug
design; however, for AD pharmacotherapy targeting Cal1, the
selection of an off-target enzyme screen needs consideration.
Many other cysteine proteases, including proapoptotic caspases,
have frequently been proposed as targets for inhibition in AD,
dementia, and neurodegenerative disorders.47,48 Notably, CathB
has variously been proposed as a drug target for inhibition in AD
therapy.49−51 The introduction of P2 histidine into epoxysucci-
nate Cal1 inhibitors by Cuerrier et al. led to selectivity for Cal1
over Cal2, and cathepsins, including CathB. We initially
compared E-64, 22a, and the P2-histidine (28a) and thiazolyl
E
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Figure 4. Plots of secondary and primary kinetic data for inhibition of papain (A) and Cal1 (B) used to derive detailed kinetic parameters reported in
Table 2: E-64 (diamonds, solid green); 22a (closed circles, dashed red); 31 (squares, solid orange); 36 (open circles, dashed blue). The observed rate
constants (kobs) shown in the double reciprocal plots were measured from nonlinear fitting of “progress curves” for product formation from substrate.
Exemplar progress curves are shown for Cal1 inhibition by E-64.
a
Table 2. Detailed Inhibition Constants for Cal1 and Papain and Relative Inhibitor Selectivity
calpain 1
papain
a
a
Ki
ki
ki/Ki ,
Ki
ki
ki/Ki,
102 ×
relative selectivity compared to
b
b
c
(μM) (s−1
)
×10−4 s−1 M−1
rel
1
(μM) (s−1
)
×10−4 s−1 M−1
rel
1
(ki/Ki Cal1)/(ki/Ki papain)
E-64
E-64
22a
31
3.96
6.02
2.59
3.53
2.89
0.12
3.02 0.15
2.74 0.19
2.96 0.14
3.89 0.41
3.31 0.32
5.08
1.62
5.31
9.70
19.2
28.6
563 52
1,050 41
279 47
223 2.4
66.2 7.4
0.54
0.26
1.06
1.74
5.0
1
0.16
0.08
0.14
0.10
0.91
0.98
1.3
17.1
14.8
21.6
12.7
1.9
0.5
2.0
3.3
9.3
0.50
0.40
0.12
32
36
1.1
a
Calculated inhibition rate and apparent equilibrium constants versus full length Cal1 and papain. Inhibition constants were calculated as described
b
in the text, varying both enzyme and inhibitor concentrations. Data represent the mean SD of triplicate experiments. ki/Ki calculated relative to E-
64. Selectivity calculated using ki/Ki relative to E-64 set at 1.0.
c
Figure 5. Selected inhibitors (compounds 22a, 28a, and 31) were docked within the WR-18 X-ray structure of Cal1cat [PDB code 2NQG]: (A)
structural overlay of putative docking poses; (B) magnified S2 pocket illustrating proposed H-bond formed between the conserved H2O molecule and
the P2 moiety. Docking was carried out using the GOLD docking platform. Docking poses were rendered using UCSF Chimera molecular modeling
software.
(31) analogues, using an activity directed profiling assay, to
confirm the predicted selectivity for Cal1 over CathB
(Supporting Information); however, we chose papain for the
quantitative counterscreen, as a promiscuous cysteine protease
generally representative of the family of typically lysosomal or
secreted cysteine proteases, including the human cathepsins (B,
C, F, H, K, L1-2, O, S, W, Z).
For papain, the P1 peptide residue is cationic and P2 is
hydrophobic and includes Leu; therefore, papain represents a
useful counterscreen for Cal1. Several methods have been
reported in the literature for detailed kinetic analysis of the
F
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Figure 6. Crystal structures of epoxide inhibitors bound at the active site of Cal1 show a relaxed, ring-opened structure, whereas a transition state for
epoxide ring opening requires the Cys nucleophile and His general acid to be aligned for SN2 substitution. Triazole-based inhibitors were designed based
upon docking scores biased toward d2 ≈ 2 Å. For comparison, in the crystal structures, 2NQG has d1 = 4.606 and d2 = 1.810 and 1TLO (shown with E-
64, His, and Cys) has d1 = 4.775 and d2 = 1.833.
irreversible inhibition of cysteine proteases, including Cal1. The
FRET assay used for initial screening proved to have insufficient
signal over noise for detailed kinetic analysis; therefore,
conditions were optimized for use of the aminomethylcoumarin
substrate SucLLVYAMC. Furthermore, the truncated rat Cal1cat
is catalytically inferior to the full length enzyme,41,42,44 and
therefore, full length Cal1 was used for kinetics analysis.
changes.52 This is unlikely to be the case for Cal1 and the
epoxide-based ligands. As depicted in Figure 6, reaction at the
active site requires close interaction (d2) of the cysteine
nucleophile with the electrophilic C2 of the epoxide and
probably interaction of the histidine acting as a general acid
toward the ring oxygen of the epoxide. In crystal structures of
Cal1 adducted with ring-opened inhibitors, gross structural
relaxation leads to loss of active site interactions essential for
reaction. In addition to these large structural changes expected
for unbound and bound epoxide-based ligands, conformational
changes to the protein upon ligand binding are expected to be
pronounced based on the comparison of X-ray models of Cal1.
The conformations of the gating loops43 in domain I and
especially in domain II, which are both part of the binding site
and directly interact with the ligands, vary quite dramatically
depending on the presence and the structure of the ligand. For
example, the gating loop in domain II could be considered
“open” in the 2ARY (no ligand), 2G8J, 2NQG, 2NQI, 2R9C,
2R9F (with ligand) and “closed” in 1KXR (no ligand), 1TL9,
1TLO, 1ZCM, 2G8E (with ligand) X-ray structures found in the
PDB.
To make use of the structural information, we hypothesized
that IC50 observed for inhibition of calpain by a covalently bound
ligand is proportional to the probability of reaction that in turn is
proportional to the number of protein−ligand conformations
where such reaction can occur. A conceptually similar approach
was successfully used for fast computational prediction of P450
drug metabolism.53−55 First, we docked a 1,2-substituted
ethylene oxide portion general for all the ligands in Table 1 to
2ARY and 1KXR (both are ligand free). The “substrate” was then
positioned manually with the assumption that the epoxide should
adopt a conformation optimal for nucleophile attack of thiol and
its likely interaction with the oxyanion hole formed by the side
chain of Gln109. The placement of the non-hydrogen atoms of
the oxirane ring was then used to generate rmsd with the
corresponding atoms in the docked ligands. To evaluate the
probability of reaction using docking, we decided to approximate
it as the number of docking poses (NSig) within the rmsd
threshold at or below 2 Å. We hypothesized that the higher the
number of docking poses complying with this criterion, the
better would be the IC50. To test this computational approach
and to explore P3/P4 modifications further, a click chemistry
route was used to generate a virtual library of analogues of
compound 31, using a divergent synthetic strategy (Scheme 2).
Thirty-two synthetically accessible analogues of compound 31
were screened in silico to select 10 for synthesis and assay
(Scheme 3). We found that compounds with low NSig values,
and therefore predicted to be poor inhibitors (39, 40, 43, and
By use of SucLLVYAMC as substrate, kinetics parameters
were derived using the method of Davies and co-workers,
whereby “progress curves” were initially obtained measuring
product formation as a function of time and fitted to y = P∞(1 −
e(−kx)) (Figure 4).30 Plots of P∞ versus 1/[I] gave excellent linear
correlations supporting the validity of the approach (not shown).
Kinetics parameters (ki, Ki, ki/Ki) were obtained from double
reciprocal plots (Table 2). By use of ki/Ki to assess relative
selectivity for inhibition of Cal1 over papain, the data presented
in Table 2 show that 22a is less selective for Cal1 than is the lead
compound E-64, whereas 31, 32, and 36 all show Cal1 selectivity.
Reference to Table 2 and Figure 4 clearly shows that the cause of
this selectivity is not enhanced inhibition of Cal1 but attenuated
inhibition of papain caused by the P2-thiazole. Thus,
introduction of the thiazolyl side chain leads to diminished
inhibitory activity against general cysteine proteases as
represented by papain while maintaining efficiency for inhibition
of Cal1. The P3 cap group of 36 also contributes to the higher
selectivity of this derivative by reducing binding affinity to papain
but not Cal1.
Structural overlay of docking results for 22a, 28a, and 31
predicts a common binding motif, with the P3 cap group
extended into the solvent exposed S4−S3 region and the P2
peptidomimetic moiety situated within the S2 pocket (Figure 5).
Docking poses are compatible with the potential of 28a and 31 to
interact with the conserved water molecule at the S2 site. It is
important to note that incorporation of imidazolyl or thiazolyl
groups did not increase affinity for the Cal1 binding site relative to
the leucine-based inhibitors, as indicated by IC50 values.
In Silico Guidance toward P3/P4 Refinement. Computer
aided molecular design, based upon crystal structures of cysteine
proteases modified by epoxide inhibitors, is expected to be
problematic, since nucleophilic attack by the active site Cys thiol
leads to ring opening, bond rotations, and relaxation of the
resulting ligand−protein adduct. We attempted a variety of
docking approaches and programs and found that docking to the
X-ray models of the Cal1 that contained a cocrystallized ligand
resulted in no correlation between the docking scores and the
IC50 values. A common assumption for accurate prediction of
binding energies of covalently linked ligands is that the binding
site and the ligands should undergo minimal conformational
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experiments from Figure 2D were only examined at 5 μM E-64 and 22a.
The reaction was quenched with EDTA (10 μM), and the reaction
mixture ran on a SDS−PAGE gel. The Cal1cat containing band was cut
from the gel and submitted to in-gel alkylation with IAA (100 mM) for
60 min, followed by trypsin digestion. LC−MS/MS was carried out on
an Agilent 6300 ion-trap LC−MS instrument. HPLC separation
employed a Phenomenex Jupiter reverse phase HPLC column (5 μm,
150 mm, 2.00 mm); mobile phase ACN (0.1% formic acid)/water (0.1%
formic acid). The resulting TIC and EIC were analyzed for anticipated
m/z modified peptide fragments. Chromatograms are shown in Figure
3, and m/z peak assignments are included in the Supporting
Information.
Calpain and Papain Inhibition Kinetics Studies. Full length
porcine calpain (156 nM) or papain (236 pM) was added to a solution of
100 mM NaCl, 50 mM HEPES, pH 7.6, 1 mM TCEP, 30 μM Suc-
LLVY-AMC substrate, and inhibitor (0.5−50 μM). Calpain reactions
also contained CaCl2 (1 and 100 mM for porcine and rat, respectively).
Both substrate and inhibitors were dissolved in acetonitrile/DMSO
(1:1) with the exception of E-64, dissolved in water. Organic solvent
remained <2% in all reactions and most often <1%. Reactions were
carried out in microtiter 96-well plates, with 150 μL per well, 30 °C, and
product formation was monitored over time by fluorescence (excitation
and emission of 346 and 444 nm, respectively, with 420 nm cutoff filter).
Kinetics values of kobs were determined via nonlinear regression using
one-phase association analysis, and linear plots of 1/kobs vs 1/[I]
provided kinetics constants ki and Ki. Representative primary data are
supplied in Supporting Information.
44), yielded relatively poor IC50 values, whereas compounds with
high NSig, and therefore predicted to be effective inhibitors,
displayed higher activity against calpain (36, 38, 41, and 42).
These promising results require further validation with a larger
set of ligands and suggest that this approach originally developed
for phase I metabolic enzymes may have broader application.
CONCLUSION
■
Calpain inhibition has been proposed as a promising therapeutic
intervention in many disease states associated with disrupted Ca
signaling and hyperactivated Cal1, including neuronal stress
leading to neurodegeneration. E-64 and simple analogues have
been reported to display in vivo efficacy in murine models of
muscular dystrophy and AD; however, the nonselective nature of
these compounds is seen as unfavorable for clinical use.
Exploiting E-64 as a lead compound, novel peptidomimetic
epoxides were designed and generated as Cal1 inhibitors. Active
site modification was confirmed by LC−MS/MS. The negligible
reactivity toward GSH of epoxysuccinate containing peptidomi-
metics contrasted with the observed covalent modification of the
active site cysteine of Cal1. Potency was maintained via
incorporation of the correct epoxide stereochemistry and a 4-
F-phenylthiazole P3 cap group (22a), allowing selectivity to be
achieved by incorporating a P2 thiazolyl group (31). Diversity
was achieved by developing a divergent synthetic route leading to
36. Increased selectivity for Cal1 was an objective of this study;
however, several cysteine proteases, including caspases and Cath
B, have been proposed as targets for AD, dementia, and
neurodegenerative disorders47−51 and would not be appropriate
counterscreens predictive of adverse effects. Therefore, papain
was used as a counterscreen for selectivity. Retention of the
electrophilic epoxide in the present work was deemed as
important for the preparation of a Cal1 inhibitor (32) that could
be used in future studies as an ABPP chemical probe for
identification of on-target and off-target proteins to aid in further
refinement of both drug targets and drug optimization. Several
inhibitors reported herein have shown activity in AD mouse
models, and these results in addition to toxicity data will be
presented in subsequent manuscripts.
Synthesis. General Methods. Unless stated otherwise, all reactions
were carried out under an atmosphere of dry argon in oven-dried
glassware. Indicated reaction temperatures refer to those of the reaction
bath, while room temperature (rt) is noted as 25 °C. Dichloromethane
(CH2Cl2) was distilled over CaH2, and THF was distilled over Na(s). All
other solvents were of anhydrous quality purchased from Aldrich
Chemical Co. and used as received. Pure reaction products were
typically dried under high vacuum in the presence of phosphorus
pentoxide. Commercially available starting materials and reagents were
purchased from Aldrich, TCI, and Fisher Scientific and were used as
received unless specified otherwise. Analytical thin layer chromatog-
raphy (TLC) was performed (column dimensions of 5 cm × 20 cm, 60
Å, 250 μm). Visualization was accomplished using a 254 nm UV lamp.
1H and 13C NMR spectra were recorded on either a Bruker Avance 400
MHz spectrometer or Bruker DPX 400 MHz spectrophotometer.
Chemical shifts are reported in ppm with the solvent resonance as
internal standard (CDCl3 at 7.27 and 77.23 ppm, DMSO-d6 at 2.5 and
39.51 ppm, and MeOD-d4 at 4.78 and 49.0 for 1H and 13C, respectively).
Data are reported as follows: chemical shift, multiplicity (s = singlet, d =
doublet, dd = doublet of doublet, t = triplet, q = quartet, br = broad, m =
multiplet, abq = ab quartet), number of protons, and coupling constants.
Low-resolution (LR) mass spectra were acquired on an Agilent 6300
ion-trap LC−MS instrument. High resolution mass spectral data were
collected in-house using a Shimadzu QTOF 6500. All reported
EXPERIMENTAL SECTION
■
Calpain Inhibition FRET Assay. The Calbiochem InnoZyme
activity kit was used for measuring the inhibition effect inhibitors on
human erythrocyte calpain 1 activity. A calpain FRET substrate,
(DABCYL)-TPLKSPPPSPR-(EDANS), was used to detect the activity
of Cal1. 20 μM FRET substrate, 10 nM of native Cal1, and 20 μM TCEP
(reducing agent) were added to the reaction mixture containing assay
buffer (Tris [10 mM], NaCl [100 mM], pH 7.4). Cal1 was activated by
the addition of 10 μM calcium. Cleavage of the scissile bond amide
bond, K−S, releases the fluorophore (EDANS) from the internal
quenching molecule (DABCYL), resulting in an increase in fluorescence
measured at 320 nm excitation and 480 nm emission wavelengths for 20
min. Each inhibitor was added to the reaction mixture at varying
concentrations (10 nM, 100 nM, 1 μM, and 10 μM) to detect inhibition
of Cal1, and reduction in fluorescence was measured by 96-well plate
reader. Approximate IC50 values of each compound were generated
using linear regression within Graphpad Prism software.
1
compounds were fully characterized by H NMR and 13C NMR. The
structures of all novel final compounds were supported by HRMS. All
compounds submitted for biological testing were confirmed to be >95%
pure by analytical HPLC. Synthetic procedures, tabulated spectral data,
HRMS, and purity analysis for final compounds and novel intermediates
are described below. Consult the Supporting Information for extended
synthetic procedures and characterization data.
HPLC Analysis of Stability/Reactivity. 31 (100 μM) was
incubated with reduced glutathione (5 mM) in PBS (50 mM, pH 7.4,
[2% DMSO v/v]) in a temperature controlled HPLC autosampler (37
°C) and reaction progress monitored via 20 μL injections every hour for
24 h. Product identity was confirmed using LC−MS/MS and
comparison of retention time of authentic sample in the absence of
GSH.
LC−MS/MS Examination of Inhibitor Modified Cal1cat Active
Site. Expression from E. coli and purification protocols can be found in
Supporting Information. Method A refers to the data shown in Figure
3A,B, and method B describes the semiquantitative approach shown in
Figure 3C,D. The recombinant Cal1cat (method A, 5 μM; method B, 1
μM) was activated via addition of CaCl2 (10 mM) and incubated with
either method: for method A, E-64 or 22a (10 μM) for 30 min; for
method B, E-64 and/or 22a (1, 5, 10 μM) for 20 min. The competition
Optimized Procedure for the Coupling of Arylamines to N-
Protected Peptidomimetics. A round-bottom flask was charged with
the appropriate Boc-protected carboxylic acid (1.0 equiv) dissolved in
minimal DMF (∼3 mL/mmol) under argon and maintained at 0 °C.
EDCI (1.2 equiv) was then added in one portion and the suspension
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stirred until homogeneous (typically <5 min), followed by the addition
of HOBt (1.5 equiv). After the mixture was stirred for an additional 15
min, the amine (1.0 equiv) in DMF (∼2 mL/mmol) was added
dropwise, and the mixture was allowed to warm to room temperature,
monitored by TLC. In instances when the amine remained on TLC after
8 h, the mixture was again brought to 0 °C, and additional EDCI (0.5
equiv) was added, followed by stirring for an additional 4 h. The mixture
was acidified to pH ≈ 4 with 1 N HCl and extracted with CH2Cl2 (3×).
Combined organic extracts were washed with 1 N HCl (2×) and brine
(1×), dried over Na2SO4, concentrated in vacuo, and purified by column
chromatography to give the desired peptidomimetic scaffolds.
General Procedure for Coupling of the Epoxide Monoester
with the Peptidomimetic Amine. TFA (10 equiv) was added to a
suspension of the Boc protected peptidomimetic (1 equiv) in CH2Cl2
(20 mL/1 mmol peptidomimetic) at 0 °C and the reaction mixture
stirred at the same temperature for 2 h. Excess TFA and CH2Cl2 were
removed under vacuum and the residual TFA salt was either dried under
high vacuum and subjected to the next reaction without further
purification or dissolved in MeOH/H2O and brought to a pH of ∼7
using a saturated NaHCO3 solution, followed by extraction with CH2Cl2
(3 × 50 mL) and removal of solvent in vacuo to afford the pure free
amine, which was used without further purification. The intermediate
deprotected amine (1 equiv), the epoxide monoester (1 equiv), and
HOBt (1.1 equiv) were dissolved in minimal DMF, and then DIPEA (1
mL, 5.6 mmol) and EDCI (326 mg, 1.7 mmol) were added. After the
mixture was stirred at ambient temperature for 12 h, H2O was added to
the reaction mixture and the mixture was diluted with 200 mL of
CH2Cl2. The organic layer was separated and washed with 1 N HCl (20
mL), saturated NaHCO3 solution (10 mL), water (30 mL), brine (10
mL), dried over anhydrous Na2SO4, and concentrated in vacuo.
Chromatographic purification of the crude mixture gave the title
compounds.
Optimized Procedure for Coupling of the Epoxide Monoester
with the Peptidomimetic Amine Boc Deprotection: TFA (10
equiv) was added to a suspension of the appropriate Boc-protected
peptidomimetic (1 equiv) in freshly distilled CH2Cl2 (10 mL/1 mmol
peptidomimetic) at 0 °C and the reaction mixture stirred at the same
temperature for 6 h. If needed, additional TFA (5 equiv) was added at 0
°C every 30 min until no starting material remained on TLC. The
mixture was then concentrated in vacuo, and the residual TFA salt was
dissolved in MeOH/H2O and brought to pH ≈ 7 using a saturated
NaHCO3 solution, followed by extraction with CH2Cl2 (3 × 50 mL),
and removal of solvent in vacuo to afford the pure free amine, which was
dried under high vacuum and used in the next reaction without further
purification.
Coupling. A round-bottom flask was charged with the appropriate
epoxide monoacid (1.0 equiv) dissolved in minimal DMF (∼2 mL/
mmol) with DIPEA (1.1 equiv) under argon and maintained at 0 °C.
EDCI (1.0 equiv) was then added in one portion and the suspension
stirred until homogeneous (typically <5 min), followed by the addition
of HOBt (1.2 equiv). After the mixture was stirred for an additional 15
min, the free amine (1.0 equiv) in DMF (∼3 mL/mmol) was added
dropwise, and the mixture was allowed to warm to room temperature,
monitored by TLC. After 12 h, the reaction was quenched acidified to
pH ≈ 4 with 1 N HCl, extracted with CH2Cl2 (3×). Combined organic
extracts were washed with 1 N HCl (2×), and brine (1×), dried over
Na2SO4, concentrated in vacuo, and purified by column chromatog-
raphy to give the desired peptidomimetic scaffolds.
Typically, the purity of the desired product reflects the purity of the ester
starting material. It was found that having sufficiently pure ester starting
material is essential to afford a pure acid upon hydrolysis.
(2S,3S)-3-((S)-4-Methyl-1-oxo-1-(phenylamino)pentan-2-
ylcarbamoyl)oxirane-2-carboxylic Acid (20). The general proce-
dure was followed using the corresponding peptidomimetic epoxide
ethyl ester 14a (70 mg, 0.2 mmol) and LiOH (4.8 mg, 0.2 mmol) and
after extraction afforded the desired product as a white solid (54 mg,
83.9%). 1H NMR (MeOD-d4, 400 MHz): δ 7.57−7.55 (d, 2H); 7.33−
7.29 (t, 2H); 7.13−7.09 (t, 1H); 4.64−4.61 (m, 1H); 3.71 (s, 1H); 3.57
(s, 1H); 1.76−1.65 (m, 3H); 1.02−0.98 (t, 6H). 13C NMR (MeOD-d4,
100 MHz): δ 171.22, 169.07, 167.14, 137.98, 128.41, 124.12, 120.14,
52.90, 52.58, 51.69, 40.70, 29.49, 24.64, 22.04, 20.59. ESI-HRMS (m/z)
[M − H+] calcd for C16H20N2O5: 319.3404. Observed: 319.1332. HPLC
method 1: tR = 21.0 min, purity = 96.1%.
(2S,3S)-3-((S)-1-(2,6-Difluorophenylamino)-4-methyl-1-oxo-
pentan-2-ylcarbamoyl)oxirane-2-carboxylic Acid (21a). The
general procedure was followed using the corresponding peptidomi-
metic epoxide ethyl ester 14b (28 mg, 0.07 mmol) and LiOH (1.7 mg,
0.07 mmol) and after extraction afforded the desired product as a white
solid (20 mg, 77.0%). 1H NMR (MeOD-d4, 400 MHz,): δ 7.91 (s, 1H);
7.36−7.31 (m, 1H); 7.07−7.02 (t, 2H); 4.72 (m, 1H); 3.65 (s, 1H); 3.52
(s, 1H); 1.74−1.68 (m, 3H); 1.01−0.99 (d, 6H). 13C NMR (DMSO-d6,
100 MHz): δ 171.26, 169.20, 165.81, 159.49 (d, J = 5.2 Hz); 157.00 (d, J
= 5.2 Hz); 128.98 (t, J = 19.7 Hz); 115.34 (t, J = 34.0 Hz); 113.10 (d, J =
5.2 Hz); 107.43, 106.22, 52.96, 51.51, 51.32, 41.30, 24.71, 23.81. ESI-
HRMS (m/z) [M − H]+ calcd for C16H20N2O5: 355.3216. Observed:
355.3101. HPLC method 1: tR = 19.7 min, purity = 95.0%.
(2R,3R)-3-((S)-1-(2,6-Difluorophenylamino)-4-methyl-1-oxo-
pentan-2-ylcarbamoyl)oxirane-2-carboxylic Acid (21b). The
general procedure was followed using the corresponding peptidomi-
metic epoxide ethyl ester 14c (85 mg, 0.22 mmol) and LiOH (5.2 mg,
0.22 mmol) and after extraction afforded the desired product as a white
solid (59 mg, 74.8%). 1H NMR (DMSO-d6, 400 MHz,): δ 9.88 (s, 1H);
8.81−8.79 (d, 1H, J = 8.21 Hz); 7.38−7.33 (m, 1H); 7.17−7.13 (t, 2H);
4.61−4.56 (q, 1H); 3.70−3.69 (d, 1H, J = 1.79 Hz); 3.52−3.51 (d, 1H, J
= 1.79 Hz); 1.66−1.58 (m, 3H); 0.93−0.88 (dd, 6H). 13C NMR
(DMSO-d6, 100 MHz): δ 171.35, 169.20, 165.79, 159.48 (d, J = 5.16
Hz); 157.01 (d, J = 5.21 Hz); 128.58 (t, 19.7 Hz); 114.63 (t, J = 34.0
Hz); 112.39 (d, J = 22.6 Hz); 53.05, 51.71, 51.62, 41.32, 24.71, 21.96.
ESI-HRMS (m/z) [M − H+] calcd for C16H20N2O5: 355.3216.
Observed: 355.3201. HPLC method 1: tR = 18.9 min, purity = 96.5%.
(2S,3S)-3-((S)-1-(4-(4-Fluorophenyl)thiazol-2-ylamino)-4-
methyl-1-oxopentan-2-ylcarbamoyl)oxirane-2-carboxylic Acid
(22a). The general procedure was followed using the corresponding
peptidomimetic epoxide ethyl ester 14d (203 mg, 0.45 mmol) and
LiOH (10.8 mg, 0.45 mmol) and after extraction afforded the desired
1
product as a white solid (150 mg, 78.8%). H NMR (DMSO-d6, 400
MHz): δ 12.51 (bs, 1H); 8.64−8.61 (d, 1H, J = 12.0 Hz); 7.95−7.92 (t,
2H); 7.62 (s, 1H); 7.28−7.24 (t, 2H); 4.63−4.58 (m, 1H); 3.51−3.50
(d, 1H, J = 1.60 Hz); 3.34−3.33 (d, 1H, J = 1.60 Hz); 165−1.54 (m,
3H); 0.91−0.88 (t, 6H). 13C NMR (MeOD-d4, 100 MHz): 172.44,
165.32, 162.87, 159.37, 150.45, 132.60, 132.57, 129.14, 129.06, 116.56,
116.34, 108.69, 54.41, 53.55, 53.19, 41.79, 26.21, 23.54, 22.02. ESI-
HRMS (m/z) [M + H+] calcd for C19H20FN3O5S: 422.1180. Observed:
422.1188. HPLC method 1: tR = 24.6 min, purity = 95.7%.
(2R,3R)-3-((S)-1-(4-(4-Fluorophenyl)thiazol-2-ylamino)-4-
methyl-1-oxopentan-2-ylcarbamoyl)oxirane-2-carboxylic Acid
(22b). The general procedure was followed using the corresponding
peptidomimetic epoxide ethyl ester 14e (76.4 mg, 0.16 mmol) and
LiOH (8 mg, 0.34 mmol) and after extraction afforded the desired
General Procedure for Peptidomimetic Epoxide Ester
Saponification. The peptidomimetic epoxide ester (1 equiv) was
dissolved in THF/MeOH/H2O (3:1:1) and cooled to 0 °C, and LiOH
(1 equiv) was added. The mixture was allowed to warm to room
temperature. Additional LiOH (0.2 equiv) was added at 0 °C every 3 h
as needed until no starting material remained on TLC. The resulting
solution was acidified with cold 1 N HCl and poured into CH2Cl2. In
some cases the acid precipitated out and was filtered through a sintered
funnel and washed consecutively with CH2Cl2 and H2O and isolated. In
instances when the compound stayed in solution, the aqueous phase was
extracted with CH2Cl2 (3 × 50 mL), and the combined organic extracts
were dried over Na2SO4 and concentrated to give the desired product.
1
product as a white solid (51 mg, 71.5%). H NMR (DMSO-d6, 400
MHz): δ 12.52 (s, 1H); 8.78−8.76 (d, 1H, J = 8.0 Hz); 7.94−7.92 (t,
2H); 7.63 (s, 1H); 7.29−7.24 (t, 2H); 4.58−4.57 (m, 1H); 3.62−3.61
(d, 1H, J = 1.80 Hz); 3.41−3.40 (d, 1H, J = 1.80 Hz); 1.64−1.51 (m,
3H); 0.91−0.88 (dd, 6H). 13C NMR (DMSO-d6, 100 MHz): δ 171.02,
166.26, 162.88, 160.46, 157.72, 147.81, 130.80, 130.77, 127.62, 127.54,
115.59 (d, J = 21.6 Hz); 107.97, 52.36, 52.08, 51.37, 28.89, 24.25, 22.82,
21.19. ESI-HRMS (m/z) [M + H+] calcd for C19H20FN3O5S: 422.1180.
Observed: 422.1186. HPLC method 1: tR = 23.8 min, purity = 95.1%.
I
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(2S,3S)-3-((S)-1-(4-(4-Fluorophenylsulfonamido)-
butylamino)-4-methyl-1-oxopentan-2-ylcarbamoyl)oxirane-2-
carboxylic Acid (23). The general procedure was followed using the
corresponding peptidomimetic epoxide ethyl ester 14f (213 mg, 0.42
mmol) and LiOH (21 mg, 0.85 mmol) and after extraction afforded the
desired product as a white solid (110 mg, 54.7%). 1H NMR (DMSO-d6,
400 MHz): δ 8.15 (s, 1H); 7.89−7.85 (m, 2H); 7.38 (s, 1H); 7.21−7.17
(t, 2H); 6.14 (s, 1H); 3.68−3.60 (d, 2H); 3.38−3.37 (d, 1H, J = 1.60
Hz); 3.10−3.09 (d, 1H, J = 1.60 Hz); 2.97 (s, 1H); 2.85 (s, 1H); 1.65−
1.56 (m, 7H); 0.91−0.88 (m, 6H). 13C NMR (DMSO-d6, 100 MHz): δ
173.41, 170.47, 166.90, 166.52, 163.99, 135.75, 135.72, 130.03, 129.93,
116.71, 116.48, 53.83, 52.32, 43.08, 41.34, 39.65, 26.76, 26.22, 24.95,
22.94, 22.06. ESI-HRMS (m/z) [M + H+] calcd for C20H28FN3O7S:
474.1705. Observed: 474.1707. HPLC method 1: tR = 20.9 min, purity =
99.0%.
(2S,3S)-3-((2S)-1-(4-(5-(1,2-Dithiolan-3-yl)pentanamido)-
butylamino)-4-methyl-1-oxopentan-2-ylcarbamoyl)oxirane-2-
carboxylic Acid (24). 14g (53 mg, 0.1 mmol) was dissolved in 2 mL of
3:1:1 mixture of THF, methanol, and water and cooled to 0 °C, and
LiOH (5 mg, 0.2 mmol) was added. After the mixture was stirred at the
same temperature for 15 min the resulting solution was acidified with 1
N HCl and extracted with CH2Cl2. The organic layer was washed with
water, brine, dried over anhydrous Na2SO4, and concentrated in vacuo.
Column purification of the crude mixture (SiO2, 15% MeOH/CHCl3)
gave the title compound as a white solid. 1H NMR (CDCl3, 400 MHz):
δ 8.01−7.99 (d, 1H, J = 10.8 Hz); 7.26 (s, 1H); 6.25 (bs, 1H); 4.57−4.53
(q, 1H); 3.69 (s, 1H); 3.59 (s, 1H); 3.21−3.31 (m, 5H); 2.45−2.42 (m,
1H); 2.24−2.20 (t, 2H); 1.94−1.92 (m, 1H); 1.66−1.51 (m, 12H);
1.25−1.21 (m, 2H); 0.94−0.91 (m, 6H). 13C NMR (CDCl3, 100 MHz):
δ 174.12, 172.13, 168.92, 166.64, 56.52, 53.57, 52.57, 51.62, 41.19,
40.31, 39.09, 38.93, 38.50, 36.24, 34.63, 28.93, 26.56, 26.42, 25.55, 24.85,
22.80, 21.87. ESI-HRMS (m/z) [M + H+] calcd for C22H37N3O6S2:
504.2197. Observed: 504.2192. HPLC method 1: tR = 21.7 min, purity =
95.4%.
(2S,3S)-3-((S)-4-Methyl-1-oxo-1-(4-(5-((3aS,4S,6aR)-2-oxo-
hexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)-
butylamino)pentan-2-ylcarbamoyl)oxirane-2-carboxylic Acid
(25). The general saponification procedure was followed using the
corresponding peptidomimetic epoxide ethyl ester 14h (155 mg, 0.3
mmol), LiOH (14 mg, 0.58 mmol), and THF/MeOH/H2O (1.5 mL/
0.5 mL/0.5 mL), yielding 25 as a white solid (40 mg, 27.1%). 1H NMR
(DMSO-d6, 400 MHz): 8.56 (d, 1H); 8.06 (t, 1H); 7.76 (t, 1H); 6.41 (s,
1H), 6.35 (s, 1H); 4.57 (m, 2H); 4.31 (m, 1 H); 3.66 (d, 1H); 3.31 (d,
1H); 3.08 (m, 1H); 3.01 (m, 3H); 2.80 (dd, 1H); 2.56 (d, 1H); 2.04 (t,
2H); 1.53 (m, 6H); 1.43 (m, 3H); 1.36 (m, 2H); 0.89 (d, 3H); 0.84 (d,
3H). 13C NMR (DMSO-d6, 100 MHz): δ 171.80, 171.06, 168.78,
164.85, 162.68, 61.00, 59.16, 55.36, 52.65, 51.19, 41.14, 35.17, 28.17,
27.98, 26.59, 26.45, 25.28, 24.24, 22.87, 21.61. ESI-HRMS (m/z) no
ionization, mass not seen. HPLC method 1: tR = 14.8 min, purity =
95.6%.
(2S,3S)-3-((S)-3-(1H-Imidazol-4-yl)-1-oxo-1-(phenylamino)-
propan-2-ylcarbamoyl)oxirane-2-carboxylic Acid (26). The
general saponification procedure was followed using the corresponding
peptidomimetic epoxide ethyl ester 15a (155 mg, 0.3 mmol), LiOH (14
mg, 0.58 mmol), and THF/MeOH/H2O (1.5 mL/0.5 mL/0.5 mL),
yielding 26 as a white solid (40 mg, 27.1%). 1H NMR (MeOD-d4, 400
MHz): δ 8.66 (s, 1H); 7.57−7.55 (d, 2H, J = 7.86 Hz); 7.29−7.27 (t,
3H); 7.10−7.07 (t, 1H); 4.97−4.94 (q, 1H); 3.64−3.63 (d, 1H, J = 1.66
Hz); 3.529−3.525 (d, 1H, J =1.66 Hz); 3.36−3.15 (m, 2H). 13C NMR
(MeOD-d4, 100 MHz): δ 169.79, 167.26, 166.95, 137.90, 134.99,
128.37, 124.10, 120.11, 54.13, 52.94, 51.67. ESI-HRMS (m/z) [M + H+]
calcd for C16H16N4O5: 345.1194. Observed: 345.1185. HPLC method
1: tR = 18.2 min, purity = 95.2%.
3H); 2.07 (s, 6H). 13C NMR (MeOD-d4, 100 MHz): δ 174.40, 172.62,
170.80, 168.89, 136.54, 135.74, 135.11, 133.43, 131.25, 128.18, 117.26,
54.20, 53.82, 53.69, 52.80, 29.34, 19.55, 16.86. ESI-HRMS (m/z) [M −
H+] calcd for C19H22N4O5: 385.1517. Observed: 385.1516. HPLC
method 2: tR = 16.4 min, purity = 95.4%
(2S,3S)-3-(1-(4-(4-Fluorophenyl)thiazol-2-ylamino)-3-(1H-
imidazol-4-yl)-1-oxopropan-2-ylcarbamoyl)oxirane-2-carbox-
ylic Acid (28a). The general saponification procedure was followed
using the corresponding peptidomimetic epoxide ethyl ester 15c (184
mg, 0.39 mmol), LiOH (14 mg, 0.58 mmol), and THF/MeOH/H2O
(1.5 mL/0.5 mL/0.5 mL), yielding 28a as a white solid (40 mg, 27.1%).
1H NMR (DMSO-d6, 400 MHz): δ 12.50 (bs, 1H); 8.84−8.73 (dd, 1H, J
= 7.3 Hz, 7.3 Hz); 7.95−7.91 (q, 2H); 7.73 (s, 1H); 7.63 (s, 1H); 7.29−
7.24 (t, 2H); 6.90 (s, 1H); 4.81−4.74 (m, 1H); 3.65−3.64 (dd, 2H, J =
1.68 Hz, 1.68 Hz); 3.46−3.42 (dd, 2H, J = 1.65 Hz, 1.65 Hz); 3.09−3.01
(m, 2H). 13C NMR (DMSO-d6, 100 MHz): δ 169.81, 165.89, 163.95,
157.92, 147.96, 134.99, 132.93, 132.91, 127.89, 127.72, 115.77, 115.56,
108.17, 99.59, 52.69, 51.85, 51.77. ESI-HRMS (m/z) [M + H+] calcd for
C19H16FN5O5S: 446.0929. Observed: 446.0922. HPLC method 2: tR =
16.2 min (S-isomer) and tR = 12.8 min (R-isomer), purity = 98.2%. For
characterization purposes, isomeric identity was determined by
resynthesis with the use of HOBT in the initial peptide coupling. This
led to isolation of the enantiomerically pure S-isomer, which elutes at tR
= 16.2 min, corresponding to the S-isomer in the enantiomeric mixture.
Only the racemic mixture was tested for potency and selectivity.
(2R,3R)-3-((S)-1-(4-(4-Fluorophenyl)thiazol-2-ylamino)-3-
(1H-imidazol-4-yl)-1-oxopropan-2-ylcarbamoyl)oxirane-2-car-
boxylic Acid (28b). The general saponification procedure was
followed using the corresponding peptidomimetic epoxide ethyl ester
15d (200 mg, 0.42 mmol), LiOH (12 mg, 0.50 mmol), and THF/
MeOH/H2O (2.5 mL/1.5 mL/1.5 mL), yielding 28b as a white solid
1
(85 mg, 45.2%). H NMR (DMSO-d6, 400 MHz,): δ 12.52 (bs, 1H);
8.85−8.75 (dd, 2H, J = 6.9 Hz; J = 43.1 Hz); 7.94−7.91 (t, 2H); 7.80 (s,
1H); 7.62 (s, 1H); 7.28−7.24 (t, 2H); 6.93 (s, 1H); 4.80−4.76 (q, 1H);
3.65−3.64 (d, 1H, J = 4.40 Hz); 3.46−3.42 (d, 1H, J = 14.0 Hz); 3.09−
2.96 (m, 2H). 13C NMR (DMSO-d6, 100 MHz): δ 169.5, 168.8, 165.9,
162.9, 160.5, 157.6, 147.8, 134.6, 132.1, 130.7, 127.6, 116.4, 115.5 (d, J =
21.4 Hz), 108.0, 52.9, 52.7, 52.6, 51.9, 28.6. ESI-HRMS (m/z) [M + H+]
calcd for C19H16FN5O5S: 446.0928. Observed: 446.0939; HPLC
method 2: tR = 15.8 min, purity = 97.2%.
(2S,3S)-3-((S)-1-(6-Fluorobenzo[d]thiazol-2-ylamino)-3-(1H-
imidazol-4-yl)-1-oxopropan-2-ylcarbamoyl)oxirane-2-carbox-
ylic Acid (29a). The general saponification procedure was followed
using the corresponding peptidomimetic epoxide ethyl ester 15e (153
mg, 0.32 mmol), LiOH (7.7 mg, 0.32 mmol), and THF/MeOH/H2O
(3.5 mL/1.5 mL/1.5 mL), yielding 29a as a white solid (103 mg, 71.5%).
1H NMR (DMSO-d6, 400 MHz): δ 14.55 (bs, 1H); 14.36 (bs, 1H);
9.18−9.02 (m, 2H); 7.93−7.91 (m, 1H); 7.79−7.75 (m, 1H); 7.44 (s,
1H); 7.31−7.29 (t, 1H); 4.96−4.91 (q, 1H); 3.72−3.71 (d, 1H, J = 1.62
Hz); 3.51−3.50 (d, 1H, J = 1.62 Hz); 3.29−3.16 (m, 2H). %). 13C NMR
(DMSO-d6, 100 MHz): δ 169.50, 168.51, 165.84, 165.84, 159.82,
157.53, 157.44, 145.02, 133.67, 132.68, 132.57, 128.56, 121.63, 117.01,
114.40, 114.16, 108.27, 108.00, 52.67, 52.19, 51.34, 26.20. ESI-HRMS
(m/z) [M + H+] calcd for C17H14FN5O5S: 420.0773. Observed:
420.0777.
(2R,3R)-3-((S)-1-(6-Fluorobenzo[d]thiazol-2-ylamino)-3-(1H-
imidazol-4-yl)-1-oxopropan-2-ylcarbamoyl)oxirane-2-carbox-
ylic Acid (29b). The general saponification procedure was followed
using the corresponding peptidomimetic epoxide ethyl ester 15f (125
mg, 0.26 mmol), LiOH (6.2 mg, 0.26 mmol), and THF/MeOH/H2O
(1.5 mL/1.0 mL/0.5 mL), yielding 29b as a white solid (46 mg, 39.1%).
1H NMR (400 MHz, MeOD-d4): δ 11.88 (bs, 1H), 8.89−8.83 (dd, 1H, J
(2S,3S)-3-((S)-3-(1H-Imidazol-5-yl)-1-(mesitylamino)-1-oxo-
propan-2-ylcarbamoyl)oxirane-2-carboxylic Acid (27). The
general saponification procedure was followed using the corresponding
peptidomimetic epoxide ethyl ester 15b (51.6 mg, 0.1 mmol), LiOH (5
mg, 0.2 mmol), and THF/MeOH/H2O (1.5 mL/0.5 mL/0.5 mL),
yielding 27 as a white solid (26.2 mg, 54.2%). 1H NMR (MeOD-d4, 400
MHz): δ 7.62 (s. 1H); 6.93 (s, 1H); 6.87 (s, 2H); 4.84−4.82 (m, 1H);
3.53−3.49 (d, 1H); 3.38−3.35 (d, 1H); 3.25−3.22 (m, 2H); 2.25 (s,
= 7.4 Hz, J = 7.4 Hz); 7.91−7.89 (d, 1H, J = 2.5 Hz); 7.78- 7.72 (q, 1H);
7.56 (s, 1H); 7.30 (td, 1H, J = 2.6 Hz); 6.85 (s, 1H); 4.81−4.76 (m, 1H);
3.76−3.74 (dd, 1H, J = 1.68 Hz); 3.45−3.43 (dd, 1H, J = 1.65 Hz);
3.11−3.00 (m, 2H). 13C NMR (DMSO-d6, 100 MHz): δ 171.13, 167.48,
165.83, 160.32, 158.21, 145.64, 135.40, 133.23, 122.17, 114.83, 108.74,
53.86, 53.40, 51.86. ESI-HRMS (m/z) [M + H+] calcd for
C17H14FN5O5S: 420.0773. Observed: 420.0769.
J
dx.doi.org/10.1021/jm4006719 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(2S,3S)-3-((S)-1-Oxo-1-(phenylamino)-3-(thiazol-4-yl)-
propan-2-ylcarbamoyl)oxirane-2-carboxylic Acid (30). The
general saponification procedure was followed using the corresponding
peptidomimetic epoxide ethyl ester 16a (112 mg, 0.28 mmol), LiOH
(6.9 mg, 0.29 mmol), and THF/MeOH/H2O (2.5 mL/1.0 mL/1.0
mL), yielding 30 as a white solid (45 mg, 43.3%). 1H NMR (MeOD-d4,
400 MHz,): δ 8.97 (s, 1H); 7.52−7.50 (d, 2H); 7.32 (s, 1H); 7.30−7.26
(t, 3H); 4.94−4.92 (t, 1H); 3.62 (s, 1H); 3.52−3.45 (q, 2H); 3.43 (s,
1H). 13C NMR (DMSO-d6, 100 MHz): δ 172.48, 169.65, 168.87,
153.83, 152.41, 138.16, 128.33, 124.04, 120.73, 115.89, 54.49, 53.60,
52.89 32.87. ESI-HRMS (m/z) [M + H+] calcd for C16H15N3O5S:
360.0660. Observed: 360.0673. HPLC method 2: tR = 16.4 min; purity =
95.2%.
Preparation of the Alkynyl Epoxide Ester for Click Derivatiza-
tion, 19. 19 was generated using the optimized peptide coupling
procedure using Boc-protected peptidomimetic (740 mg, 3.5 mmol),
(2S,3S)-epoxysuccinic acid monoester (564 mg, 3.52 mmol), DIPEA
(1.53 mL, 8.8 mmol), EDCI (740 mg, 3.9 mmol), and HOBT (520 mg,
3.9 mmol) in DMF (10 mL), affording the title compound (960 mg,
77.6%) as a white solid.
(2S,3S)-Ethyl 3-((S)-1-Oxo-1-(prop-2-ynylamino)-3-(thiazol-4-
1
yl)propan-2-ylcarbamoyl)oxirane-2-carboxylate (19). H NMR
(CDCl3, 400 MHz): δ 8.78 (s, 1H); 7.65−7.63 (d, 1H, J = 1.00 Hz); 7.14
(s, 1H); 7.04 (bs, 1H); 4.80−4.75 (q, 1H); 4.29−4.24 (m, 2H); 3.99−
3.97 (q, 2H); 3.72−3.71 (d, 1H, J = 1.6 Hz); 3.53−3.52 (d, 1H, J = 1.6
Hz); 3.34−3.15 (m, 2H); 2.19 (bs, 1H); 1.33−1.30 (t, 3H). 13C NMR
(CDCl3, 100 MHz): δ 169.59, 166.49, 166.45, 153.22, 152.42, 116.20,
78.98, 71.59, 62.31, 53.77, 52.81, 52.44, 32.65, 29.20, 14.02.
3-((S)-1-(4-(4-Fluorophenyl)thiazol-2-ylamino)-1-oxo-3-
(thiazol-4-yl)propan-2-ylcarbamoyl)oxirane-2-carboxylic Acid
(31). The general saponification procedure was followed using the
corresponding peptidomimetic epoxide ethyl ester 16b (974 mg, 2.0
mmol), LiOH (47.5 mg, 2.0 mmol), and THF/MeOH/H2O (25 mL/5
General Procedure for Huisgen Cycloaddition To Give the
Intermediate Esters (35E−44E). The appropriate arylazide (1.2
equiv) and 19 (1.0 equiv) were dissolved in t-BuOH/EtOH/H2O
(1:1:0.5). CuSO4 (0.2 equiv), sodium ascorbate (0.4 equiv), and a
catalytic amount of TBTA (0.01 equiv) were added sequentially, and the
mixture was stirred for 12 h. The resulting precipitate was filtered off,
dissolved in CH2Cl2, filtered through Celite, and concentrated in vacuo
to afford the desired product in yields and quantities as follows.
(2S,3S)-Ethyl 3-((S)-1-Oxo-1-(1-phenyl-1H-1,2,3-triazol-4-yl)-
methylamino)-3-(thiazol-4-yl)propan-2-ylcarbamoyl)oxirane-
2-carboxylate (35E). The general click procedure was used
substituting the following quantities 19 (25.0 mg, 0.08 mmol),
phenylazide (9.5 mg, 0.07 mmol), CuSO4 (2.0 mg, 0.01 mmol),
NaAsc (6.0 mg, 0.03 mmol), and TBTA (5.0 mg, 0.01 mmol) in t-
BuOH/EtOH/H2O (2:1:0.5), affording 35E as a white solid (32 mg,
92.6%). 1H NMR (CDCl3, 400 MHz): δ 8.74−7.73 (d, 1H, J = 1.90 Hz),
7.93 (s, 1H); 7.73−7.71 (d, 3H, J = 8.68 Hz); 7.55−7.52 (t, 2H); 7.47−
7.39 (m, 2H); 7.07−7.06 (d, 1H, J = 1.71 Hz); 4.84−4.79 (q, 1H);
4.56−4.54 (d, 2H, J = 5.87 Hz); 4.32−4.21 (m, 2H); 3.71−3.70 (d, 1H, J
= 1.78 Hz); 3.57−3.56 (d, 1H, J = 1.78 Hz); 3.39−3.18 (m, 2H); 1.33−
1.31 (t, 3H). 13C NMR (CDCl3, 100 MHz): δ 170.06, 166.52, 166.39,
153.25, 152.36, 145.11, 136.91, 129.75, 128.81, 120.57, 120.46, 115.97,
62.27, 53.80, 52.79, 52.58, 34.98, 32.76, 14.01.
(2S,3S)-Ethyl 3-((S)-1-((1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-
yl)methylamino)-1-oxo-3-(thiazol-4-yl)propan-2-ylcarbamoyl)-
oxirane-2-carboxylate (36E). The general click procedure was used
substituting the following quantities 19 (27.3 mg, 0.08 mmol), 1-azido-
4-fluorobenzene (10.4 mg, 0.08 mmol), CuSO4 (2.0 mg, 0.01 mmol),
NaAsc (6.0 mg, 0.03 mmol), and TBTA (5.0 mg, 0.01 mmol) in t-
BuOH/EtOH/H2O (2:1:0.5), affording 36E as a white solid (35 mg,
92.2%). 1H NMR (CDCl3, 400 MHz): δ 8.73−8.72 (d, 1H, J = 1.91 Hz);
7.90 (s, 1H); 7.74−7.67 (m, 3H); 7.58−7.55 (t, 1H); 7.24−7.18 (t, 2H);
7.07−7.06 (d, 1H, J = 1.71 Hz); 4.84−4.79 (q, 1H); 4.54−4.50 (d, 2H, J
= 5.90 Hz); 4.29−4.21 (m, 2H); 3.68−3.67 (d, 1H, J = 1.78 Hz); 3.53−
3.52 (d, 1H, J = 1.78 Hz); 3.37−3.18 (m, 2H); 1.30−1.26 (t, 3H). 13C
NMR (CDCl3, 100 MHz): δ 169.82, 166.17, 166.03, 163.28, 160.81,
152.86, 152.00, 144.94, 132.78, 122.10, 122.01, 120.48, 116.45, 116.22,
115.59, 61.91, 53.43, 52.40, 52.24, 34.55, 32.42, 13.63.
(2S,3S)-Ethyl 3-((S)-1-Oxo-1-((1-(4-(piperidin-1-ylsulfonyl)-
phenyl)-1H-1,2,3-triazol-4-yl)methylamino)-3-(thiazol-4-yl)-
propan-2-ylcarbamoyl)oxirane-2-carboxylate (37E). The general
click procedure was used substituting the following quantities 19 (28.1
mg, 0.08 mmol), 4-piperidinophenylazide (10.7 mg, 0.08 mmol),
CuSO4 (3.0 mg, 0.02 mmol), NaAsc (6.0 mg, 0.03 mmol), and TBTA
(5.0 mg, 0.01 mmol) in t-BuOH/EtOH/H2O (2:1:0.5), affording 37E
as a white solid (31 mg, 79.8%). 1H NMR (CDCl3, 400 MHz): δ 8.76−
8.75 (d, 1H, J = 1.84 Hz); 8.05 (s, 1H); 7.93 (s, 4H); 7.76−7.74 (d, 1H, J
= 7.04 Hz); 7.38−7.36 (t, 1H); 7.10−7.09 (d, 1H, J = 1.70 Hz); 4.79−
4.76 (q, 1H); 4.58−4.55 (q, 2H); 4.30−4.25 (m, 2H); 3.72−3.71 (d,
1H, J = 1.78 Hz); 3.55−3.54 (d, 1H, J = 1.78 Hz); 3.38−3.17 (abq, 2H);
3.07−304 (t, 3H); 1.75−1.60 (m, 1H); 1.31−1.26 (t, 3H). 13C NMR
(CDCl3, 100 MHz): δ 170.31, 169.65, 166.52, 166.44, 153.30, 152.40,
152.34, 145.93, 139.64, 136.68, 129.40, 120.61, 120.41, 116.20, 116.02,
79.01, 71.57, 62.31, 53.77, 52.80, 52.45, 46.94, 32.70, 29.19, 25.11, 23.42,
14.02.
1
mL/2 mL), yielding 31 as a white solid (685 mg, 74.6%). H NMR
(MeOD-d4, 400 MHz,): δ 8.99−8.98(d, 1H, J = 1.67 Hz); 7.94−7.91 (q,
2H); 7.37 (s, 1H); 7.36 (s, 1H); 7.15−7.10 (t, 2H); 5.06−5.02 (q, 1H);
3.64−3.63 (d, 1H, J = 1.57 Hz); 3.49−3.45 (m, 3H). 13C NMR (MeOD-
d4, 100 MHz): δ 170.05, 169.01, 166.19, 163.43, 158.19, 154.25, 152.66,
148.36, 131.30, 128.18, 116.63, 116.15, 115.93, 108.62, 53.12, 53.05,
52.21, 33.09. ESI-HRMS (m/z) [M + H+] calcd for C19H15FN4O5S2:
463.0541. Observed: 463.0545. HPLC method 2: tR = 13.7 min; purity =
96.3%.
(2S,3S)-3-((S)-1-(4-(4-Ethynylphenyl)thiazol-2-ylamino)-1-
oxo-3-(thiazol-4-yl)propan-2-ylcarbamoyl)oxirane-2-carbox-
ylic Acid (32). The general saponification procedure was followed using
16c (115 mg, 0.23 mmol), LiOH (6.7 mg, 0.28 mmol), and THF/
MeOH/H2O (4.5 mL/1.5 mL/1.5 mL), yielding 32 as a white solid (48
mg, 44.2%). 1H NMR (DMSO-d6, 400 MHz): δ 12.57 (s, 1H); 9.06−
9.05 (d, 1H, J = 1.92 Hz); 8.81−8.79 (d, 1H, J = 7.8 Hz); 7.93−7.91 (d,
2H, J = 8.4 Hz); 7.76 (s, 1H); 7.55−7.53 (d, 2H, J = 8.4 Hz); 7.45−7.44
(d, 1H, 1.8 Hz); 4.98−4.93 (q, 1H); 4.25 (s, 1H); 3.66−3.65 (d, 1H, J =
1.8 Hz); 3.42−3.41 (d, 1H, J = 1.8 Hz); 3.35−3.22 (m, 2H). 13C NMR
(DMSO-d6, 100 MHz): δ 170.08, 169.03, 165.98, 158.25, 154.33,
152.54, 148.47, 134.95, 132.61, 126.26, 121.32, 116.75, 110.20, 83.87,
81.99, 53.15, 53.08, 51.88, 33.05. ESI-HRMS (m/z) [M + H+] calcd for
C21H16N4O5S2: 469.0635; observed, 469.0627. HPLC method 1: tR =
12.5 min, purity = 95.7%.
(2S,3S)-3-((S)-1-(4-(4-Fluorophenyl)thiazol-2-ylamino)-3-(1-
methyl-1H-imidazol-5-yl)-1-oxopropan-2-ylcarbamoyl)-
oxirane-2-carboxylic Acid (33). The general saponification proce-
dure was followed using the corresponding peptidomimetic epoxide
ethyl ester 17 (129 mg, 0.26 mmol), LiOH (6.3 mg, 0.26 mmol), and
THF/MeOH/H2O (2.5 mL/1.5 mL/1.5 mL), yielding 33 as a white
solid (56 mg, 46.1%). 1H NMR (DMSO-d6, 400 MHz): δ 8.92−8.90 (d,
1H); 7.96−7.91 (m, 2H); 7.68 (s, 1H); 7.35−7.24 (m, 3H); 4.94−4.87
(m, 1H); 4.64−4.56 (m, 2H); 3.82−3.50 (m, 5H). 13C NMR (400 MHz,
DMSO-d6): δ 171.52, 167.50, 165.57, 162.45, 161.02, 148.42, 138.62,
131.27, 128.18, 128.01, 127.83, 127.05, 116.17, 115.99, 106.70, 53.24,
52.58, 51.90, 31.29, 29.52. ESI-HRMS (m/z) [M + H+] calcd for
C20H18FN5O5S: 460.1085; observed, 460.1092. HPLC method 1: tR =
17.8 min, purity = 93.6%.
(2S,3S)-3-((S)-1-(4-(4-Fluorophenyl)thiazol-2-ylamino)-3-(1-
methyl-1H-imidazol-4-yl)-1-oxopropan-2-ylcarbamoyl)-
oxirane-2-carboxylic Acid (34). The general saponification proce-
dure was followed using the corresponding peptidomimetic epoxide
ethyl ester 18 (93 mg, 0.19 mmol), LiOH (4.5 mg, 0.19 mmol), and
THF/MeOH/H2O (1.5 mL/0.5 mL/0.5 mL), yielding 34 as a white
1
solid (29 mg, 33.1%). H NMR (400 MHz, DMSO-d6): δ 12.50 (bs,
1H), 8.82−8.80 (d, 1H); 7.93−7.91 (m, 2H); 7.76 (s, 1H); 7.63 (s, 1H);
7.46−7.24 (t, 2H); 6.99 (s, 1H); 4.79−4.74 (m, 1H); 3.67−3.66 (d,
1H); 3.62 (s, 3H); 3.49−3.48 (d, 1H); 3.12−2.90 (m, 2H). 13C NMR
(400 MHz, DMSO-d6): δ 169.72, 168.60, 165.56, 162.97, 160.54,
157.78, 147.88, 137.17, 130.84, 127.71, 127.63, 118.35, 115.68, 115.47,
108.08, 53.07, 52.98, 51.56, 33.11, 29.60. ESI-HRMS (m/z) [M + H+]
calcd for C20H18FNO5S: 460.1085. Observed: 460.1090. HPLC method
1: tR = 18.1 min, purity = 95.3%.
K
dx.doi.org/10.1021/jm4006719 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(2S,3S)-Ethyl 3-((S)-1-(1-(Benzo[d][1,3]dioxol-5-yl)-1H-1,2,3-
triazol-4-yl)methylamino)-1-oxo-3-(thiazol-4-yl)propan-2-
ylcarbamoyl)oxirane-2-carboxylate (38E). The general click
procedure was used substituting the following quantities 19 (25.0 mg,
0.07 mmol), 5-azidobenzo[d][1,3]dioxole (9.5 mg, 0.07 mmol), CuSO4
(2.0 mg, 0.01 mmol), NaAsc (6.0 mg, 0.03 mmol), and TBTA (5.0 mg,
0.01 mmol) in t-BuOH/EtOH/H2O (2:1:0.5), affording 38E as a white
8.77−8.74 (t, 1H); 8.73 (s, 1H); 8.63−8.61 (d, 1H, J = 8.17 Hz); 8.47−
8.44 (d, 2H, J = 9.13 Hz); 8.21−8.19 (d, 2H, J = 9.13 Hz); 7.36 (d, 1H, J
= 1.78 Hz); 4.71−4.65 (q, 1H); 4.42−4.40 (d, 2H, J = 5.50 Hz); 4.20−
4.09 (m, 2H); 3.66−3.65 (d, 1H, J = 1.75 Hz); 3.52−3.51 (d, 1H, J =
1.75 Hz); 3.28−3.06 (m, 2H); 1.22−1.17 (t, 3H). 13C NMR (CDCl3,
100 MHz): δ 170.25, 167.14, 165.00, 153.70, 152.87, 146.77, 146.68,
140.89, 125.70, 121.59, 120.42, 115.86, 61.60, 53.09, 52.61, 51.40, 34.32,
33.12, 13.94.
1
solid (31 mg, 89.7%). H NMR (CDCl3, 400 MHz): δ 8.76−8.75 (d,
1H, J = 1.86 Hz); 7.79 (s, 1H); 7.73−7.71 (d, 1H, J = 7.16 Hz); 7.24−
7.21 (dd, 2H); 7.14−7.11 (dd, 1H); 7.08−7.07 (d, 1H, J = 1.63 Hz);
6.93−6.91 (d, 1H, J = 8.32 Hz); 6.09 (s, 2H); 4.79−4.76 (q, 1H); 4.54−
4.53 (d, 2H, J = 5.90 Hz); 4.31−4.25 (m, 2H); 3.72−3.71 (d, 1H, J =
1.76 Hz); 3.55−3.54 (d, 1H, J = 1.76 Hz); 3.38−3.15 (ab, 2H, J = 92.8
Hz, 57.57 Hz); 1.35−1.30 (t, 3H). 13C NMR (CDCl3, 100 MHz): δ
169.49, 169.05, 164.22, 155.01, 153.30, 148.84, 147.92, 148.27, 130.98,
121.72, 116.26, 114.17, 109.12, 102.60, 102.33, 62.14, 52.95, 52.54,
51.90, 34.78, 33.49, 14.23.
(2S,3S)-Ethyl 3-((S)-1-Oxo-1-((1-(4-sulfamoylphenyl)-1H-
1,2,3-triazol-4-yl)methylamino)-3-(thiazol-4-yl)propan-2-
ylcarbamoyl)oxirane-2-carboxylate (39E). The general click
procedure was used substituting the following quantities 19 (109.0
mg, 0.31 mmol), 4-azidobenzenesulfonamide (61.5 mg, 0.31 mmol),
CuSO4 (10.0 mg, 0.06 mmol), NaAsc (20.0 mg, 0.03 mmol), and TBTA
(10.0 mg, 0.02 mmol) in t-BuOH/EtOH/H2O (4:4:2), affording 39E as
a white solid (119 mg, 69.8%). 1H NMR (CDCl3, 400 MHz): δ 8.93 (s,
1H); 8.45 (s, 1H); 8.19−8.05 (q, 4H); 7.33 (s, 1H); 4.81−4.77 (t, 1H);
4.57−4.48 (q, 2H); 4.28−4.23 (q, 2H); 3.64 (s, 1H); 3.50 (s, 1H);
3.40−3.21 (m, 2H); 1.28−1.23 (t, 3H). 13C NMR (MeOD-d4, 100
MHz): δ 170.94, 166.80, 166.72, 153.45, 151.92, 145.70, 143.49, 138.85,
127.31, 120.74, 119.76, 115.56, 61.42, 52.72, 52.67, 51.47, 33.93, 31.91,
12.52.
(2S,3S)-Ethyl 3-((S)-1-((1-(3,5-Bis(trifluoromethyl)phenyl)-1H-
1,2,3-triazol-4-yl)methylamino)-1-oxo-3-(thiazol-4-yl)propan-
2-ylcarbamoyl)oxirane-2-carboxylate (40E). The general click
procedure was used substituting the following quantities 19 (93.7 mg,
0.27 mmol), 1-azido-3,5-bis(trifluoromethyl)benzene (82.5 mg, 0.32
mmol), CuSO4 (0.6 mg, 0.01 mmol), NaAsc (3.5 mg, 0.02 mmol), and
TBTA (15.0 mg, 0.03 mmol) in t-BuOH/EtOH/H2O (4:4:2) with an
additional 3 drops of DMF, affording 40E as a white solid (142 mg,
87.8%). 1H NMR (CDCl3, 400 MHz): δ 8.77 (s, 1H); 8.25 (s, 2H); 8.09
(s, 1H); 7.96 (s, 1H); 7.77−7.75 (d, 1H, J = 7.09 Hz); 7.34−7.32 (t,
1H); 7.11 (s, 1H); 4.81−4.76 (q, 1H); 4.59−4.57 (d, 2H, J = 5.90 Hz);
4.31−4.25 (m, 2H); 3.72−3.711 (d, 1H, J = 1.64 Hz); 3.55−3.54 (d, 1H,
J = 1.64 Hz); 3.41−3.17 (m, 2H); 1.01−0.99 (t, 3H). 13C NMR
(DMSO-d6, 100 MHz): δ 170.62, 167.49, 165.37, 154.06, 153.24,
146.92, 138.31, 124.57, 122.37, 121.85, 121.09, 116.21, 61.97, 53.44,
52.97, 51.74, 34.64, 33.49, 14.29.
(2S,3S)-Ethyl 3-((S)-1-((1-(4-Bromophenyl)-1H-1,2,3-triazol-4-
yl)methylamino)-1-oxo-3-(thiazol-4-yl)propan-2-ylcarbamoyl)-
oxirane-2-carboxylate (41E). The general click procedure was used
substituting the following quantities 19 (60 mg, 0.17 mmol), 1-azido-4-
bromobenzene (51 mg, 0.26 mmol), CuSO4 (4.4 mg, 0.03 mmol),
NaAsc (22 mg, 0.11 mmol), and TBTA (20.0 mg, 0.04 mmol) in t-
BuOH/EtOH/H2O (2:2:1), affording 41E as a white solid (40 mg,
42.6%). 1H NMR (DMSO-d6, 400 MHz): δ 9.00−8.99 (d, 1H, J = 1.66
Hz); 8.74−8.71 (t, 1H); 8.63−8.61 (d, 1H, J = 8.22 Hz); 8.54 (s, 1H);
7.60−7.56 (d, 2H, J = 14.85 Hz); 7.345−7.341 (d, 1H, J = 1.63 Hz);
7.11−7.07 (d, 2H, J = 14.85 Hz); 4.69−4.66 (q, 1H); 4.40−4.38 (d, 2H,
J = 5.59 Hz); 4.20−4.13 (m, 2H); 3.66−3.66 (d, 1H, J = 1.73 Hz); 3.52−
3.51 (d,1H, J = 1.73 Hz); 3.33−3.12 (m, 2H); 1.23−1.20 (t, 3H). 13C
NMR (100 MHz, DMSO-d6): δ 170.57, 167.51, 165.36, 154.07, 153.27,
146.56, 139.39, 136.25, 122.30, 121.54, 117.51, 116.23, 61.97, 53.46,
52.99, 51.76, 34.75, 33.50, 14.32.
(2S,3S)-Ethyl 3-((S)-1-((1-(2,6-Difluorophenyl)-1H-1,2,3-tria-
zol-4-yl)methylamino)-1-oxo-3-(thiazol-4-yl)propan-2-
ylcarbamoyl)oxirane-2-carboxylate (43E). The general click
procedure was used substituting the following quantities 19 (48 mg,
0.14 mmol), 2-azido-1,3-difluorobenzene (24 mg, 0.15 mmol), CuSO4
(3.5 mg, 0.02 mmol), NaAsc (18 mg, 0.09 mmol), and TBTA (12 mg,
0.01 mmol) in t-BuOH/EtOH/H2O (1:1:0.5), affording 43E as a white
solid (20 mg, 31.6%). 1H NMR (DMSO-d6, 400 MHz): δ 8.74−8.73 (d,
1H, J = 1.79 Hz); 7.74−7.72 (m, 2H); 7.55−7.45 (m, 1H); 7.34−7.32
(m, 1H); 7.17−7.13 (t, 2H); 7.04−7.03 (d, 1H, J = 1.72 Hz); 4.82−4.80
(q, 1H); 4.57−4.56 (d, 2H, J = 5.97 Hz); 4.29−4.24 (m, 2H); 3.71−3.70
(d, 1H, J = 1.78 Hz); 3.55−3.54 (d, 1H, J = 1.78 Hz); 3.35−3.18 (m,
2H); 1.33−1.30 (t, 3H). 13C NMR (CDCl3, 100 MHz): δ 170.16,
166.65, 166.30, 158.01, 155.46, 153.29, 152.24, 144.36, 131.48, 129.14,
125.10, 116.01, 112.64, 62.24, 53.81, 52.74, 52.62, 34.74, 32.91, 13.99.
(2S,3S)-Ethyl 3-((S)-1-((1-Mesityl-1H-1,2,3-triazol-4-yl)-
methylamino)-1-oxo-3-(thiazol-4-yl)propan-2-ylcarbamoyl)-
oxirane-2-carboxylate (44E). The general click procedure was used
substituting the following quantities 19 (48 mg, 0.14 mmol), 2-azido-
1,3-difluorobenzene (24 mg, 0.15 mmol), CuSO4 (3.5 mg, 0.02 mmol),
NaAsc (18 mg, 0.09 mmol), and TBTA (12 mg, 0.01 mmol) in t-BuOH/
EtOH/H2O (1:1:0.5), affording 44E as a white solid (30 mg, 42.8%). 1H
NMR (400 MHz, DMSO-d6): δ 8.79−8.74 (d, 1H, J =1.79 Hz); 7.68−
7.64 (m, 2H); 7.50 (s, 1H); 7.36−7.34 (d, 2H); 7.28−7.26 (d, 2H),
7.15−7.10 (m, 2H), 6.98 (s, 1H); 5.50 (s, 1H); 4.83−4.78 (m, 1H);
4.59−4.58 (d, 1H); 4.29−4.25 (m, 2H); 3.99−3.97 (q, 1H); 3.81 (s,
3H); 3.69−3.68 (m, 3H); 3.55−3.54 (d, 1H); 3.37−3.19 (abq, 2H);
2.35 (s, 3H); 1.93 (s, 3H) 1.33−1.29 (t, 3H). 13C NMR (DMSO-d6, 100
MHz): δ 169.25, 165.62, 153.65, 152.98, 144.93, 140.11, 134.51, 134.50,
128.12, 124.69, 115.88, 63.51, 52.69, 52.78, 51.98, 48.66, 34.54, 33.10,
20.69, 16.89, 13.52.
(2S,3S)-3-((S)-1-Oxo-1-((1-phenyl-1H-1,2,3-triazol-4-yl)-
methylamino)-3-(thiazol-4-yl)propan-2-ylcarbamoyl)oxirane-
2-carboxylic Acid (35). The general procedure was followed using the
corresponding peptidomimetic epoxide ethyl ester 35E (23 mg, 0.47
mmol) and LiOH (1.2 mg, 0.05 mmol) and after extraction afforded the
desired product as a white solid (18 mg, 84.9%). 1H NMR (DMSO-d6,
400 MHz): δ 9.00−8.99 (d, 1H, J = 1.76 Hz); 8.73−8.70 (t, 1H); 8.58−
8.56 (d, 1H, J = 8.24 Hz); 8.50 (s, 1H); 7.87−7.85 (d, 2H, J = 7.76 Hz);
7.61−7.49 (m, 3H); 7.344−7.341 (d, 1H, J = 1.21 Hz); 4.69−4.66 (q,
1H); 4.41−4.39 (d, 2H, J = 5.49 Hz); 3.59−3.59 (d, 1H, J = 1.58 Hz);
3.35−3.10 (m, 3H). 13C NMR (DMSO-d6, 100 MHz): δ 170.62, 169.06,
165.75, 154.05, 153.31, 146.36, 137.07, 130.38, 129.06, 131.48, 120.41,
116.19, 53.25, 52.96, 51.90, 34.80, 29.42. HRMS-ESI m/z [M + H+]
calcd for C18H16N6O5S: 443.1137. Observed: m/z 443.1135 (M + H+);.
HPLC method 2: tR = 16.7 min; purity = 97.9%.
(2S,3S)-3-((S)-1-((1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)-
methylamino)-1-oxo-3-(thiazol-4-yl)propan-2-ylcarbamoyl)-
oxirane-2-carboxylic Acid (36). The general procedure was followed
using the corresponding peptidomimetic epoxide ethyl ester 36E (25
mg, 0.51 mmol) and LiOH (1.3 mg, 0.06 mmol) and after extraction
afforded the desired product as a white solid (18 mg, 76.2%). 1H NMR
(DMSO-d6, 400 MHz): δ 8.98 (s, 1H); 8.72−8.70 (t, 1H); 8.57−8.55
(d, 1H, J = 8.06); 8.48 (s, 1H); 7.93−7.90 (q, 2H); 7.48−7.44 (t, 2H);
7.34 (s, 1H); 4.70−4.65 (q, 1H); 4.40−4.38 (d, 2H; J = 5.52); 3.66 (s,
1H); 3.54−3.06 (m, 3H). 13C NMR (DMSO-d6, 100 MHz): δ 170.62,
165.75, 163.24, 160.80, 154.06, 153.32, 146.40, 133.62, 122.81, 122.72,
121.76, 117.33, 117.10, 53.24, 53.01, 52.94, 34.77, 33.50. HRMS-ESI m/
z [M + H+] calcd for C19H17FN6O5S: 461.1043. Observed: m/z
461.1049 (M + H+). HPLC method 2: tR = 17.9 min; purity = 97.2%.
(2S,3S)-3-((S)-1-Oxo-1-((1-(4-(piperidin-1-ylsulfonyl)phenyl)-
1H-1,2,3-triazol-4-yl)methylamino)-3-(thiazol-4-yl)propan-2-
ylcarbamoyl)oxirane-2-carboxylic Acid (37). The general proce-
(2S,3S)-Ethyl 3-((S)-1-((1-(4-Nitrophenyl)-1H-1,2,3-triazol-4-
yl)methylamino)-1-oxo-3-(thiazol-4-yl)propan-2-ylcarbamoyl)-
oxirane-2-carboxylate (42E). The general click procedure was used
substituting the following quantities 19 (60 mg, 0.17 mmol), 1-azido-4-
nitrobenzene (42 mg, 0.26 mmol), CuSO4 (4.4 mg, 0.03 mmol), NaAsc
(22 mg, 0.11 mmol), and TBTA (20.0 mg, 0.04 mmol) in t-BuOH/
EtOH/H2O (2:2:1), affording 42E as an orange solid (40 mg, 45.6%).
1H NMR (DMSO-d6, 400 MHz): δ 9.00−8.99 (d, 1H, J = 1.8 Hz);
L
dx.doi.org/10.1021/jm4006719 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
dure was followed using the corresponding peptidomimetic epoxide
ethyl ester 37E (26 mg, 0.42 mmol) and LiOH (1.0 mg, 0.04 mmol) and
after extraction afforded the desired product as a white solid (12 mg,
48.3%). 1H NMR (acetone-d6, 400 MHz): δ 8.98 (bs, 1H); 8.48 (s, 1H);
8.16−8.12 (d, 2H, 8.48); 8.02−8.00 (d, 2H, J = 8.48 Hz); 7.37 (bs, 1H);
4.81 (bs, 1H); 4.52 (s, 2H); 3.85−3.79 (m, 4H); 3.61 (s, 1H); 3.55 (s,
1H); 1.82−1.60 (m, 6H). 13C NMR (acetone-d6, 100 MHz): δ 170.07,
169.01, 165.12, 153.52, 139.36, 136.19, 129.47, 120.84, 120.21, 107.39,
107.25, 66.62, 53.44, 52.80, 52.77, 46.83, 32.52, 30.07, 25.02, 23.13.
HRMS-ESI m/z [M + H+] calcd for C24H27N7O7S2: 590.6440.
Observed: m/z 590.1050 (M + H+). HPLC method 2: tR = 21.7 min;
purity = 96.3%.
(2S,3S)-3-((S)-1-((1-(Benzo[d][1,3]dioxol-5-yl)-1H-1,2,3-tria-
zol-4-yl)methylamino)-1-oxo-3-(thiazol-4-yl)propan-2-
ylcarbamoyl)oxirane-2-carboxylic Acid (38). The general proce-
dure was followed using the corresponding peptidomimetic epoxide
ethyl ester 38E (10 mg, 0.16 mmol) and LiOH (0.35 mg, 0.02 mmol)
and after extraction afforded the desired product as a white solid (7 mg,
73.1%). 1H NMR (DMSO-d6, 400 MHz): δ 9.00−8.99 (d, 1H, J = 1.88
Hz); 8.71−8.68 (t, 1H); 8.57−8.55 (d, 1H, J = 8.28 Hz); 8.38 (s, 1H);
7.46−7.45 (d, 1H, J = 2.15 Hz); 7.33−7.31 (m, 2H); 7.11 (s, 1H); 7.09
(s,1H); 6.15 (s, 2H); 4.67−4.65 (m, 1H); 4.38−4.36 (d, 2H); 3.60−
3.58 (d, 1H, J = 1.72 Hz); 3.50−3.17 (m, 3H). 13C NMR (DMSO-d6,
100 MHz): δ 170.59, 169.06, 165.75, 154.05, 153.31, 148.65, 147.81,
146.07, 131.53, 121.69, 116.19, 114.15, 109.11, 102.58, 102.33, 53.24,
52.94, 51.90, 34.78, 33.49. HRMS-ESI m/z [M + H+] calcd for
C20H18N6O7S: 487.4643. Observed: m/z 487.1050 (M + H+); HPLC
method 2: tR = 18.1 min; purity = 97.7%.
(2S,3S)-3-((S)-1-Oxo-1-((1-(4-sulfamoylphenyl)-1H-1,2,3-tria-
zol-4-yl)methylamino)-3-(thiazol-4-yl)propan-2-ylcarbamoyl)-
oxirane-2-carboxylic Acid (39). The general procedure was followed
using the corresponding peptidomimetic epoxide ethyl ester 39E (30
mg, 0.55 mmol) and LiOH (1.3 mg, 0.055 mmol). The product goes
into the aqueous layer during workup. The aqueous layer was washed
with CH2Cl2 and lyophilized to give the desired product as a white solid
(8 mg, 45.1%). 1H NMR (DMSO-d6, 400 MHz): δ 9.00−8.99 (d, 1H, J
= 1.92 Hz); 8.79−8.76 (t, 1H); 8.70−8.68 (d, 1H, J = 8.0 Hz); 8.66 (s,
1H); 8.12−8.01 (m, 4H); 7.55 (s, 2H); 7.36 (s, 1H); 4.70−4.65 (m,
1H); 4.41−4.40 (d, 2H, J = 5.20 Hz); 3.61 (s, 1H); 3.44−3.10 (m, 3H).
13C NMR (DMSO-d6, 100 MHz): δ 172.25, 170.66, 169.06, 165.74,
169.08, 165.74, 154.06, 153.29, 146.57, 136.25, 133.27, 122.31, 121.67,
121.54, 116.20, 53.25, 52.94, 51.89, 34.76, 33.49. HRMS-ESI m/z [M +
H+] calcd for C19H17BrN6O5S: 521.34. Observed: m/z 523.0229 (M +
H+); m/z 519.0123 (M − H+). HPLC method 2: tR = 21.8 min; purity =
97.2%
(2S,3S)-3-((S)-1-((1-(4-Nitrophenyl)-1H-1,2,3-triazol-4-yl)-
methylamino)-1-oxo-3-(thiazol-4-yl)propan-2-ylcarbamoyl)-
oxirane-2-carboxylic Acid (42). The general procedure was followed
using the corresponding peptidomimetic epoxide ethyl ester 42E (27
mg, 0.05 mmol) and LiOH (1.2 mg, 0.05 mmol) and after extraction
afforded the desired product as a white solid (20 mg, 78.3%). 1H NMR
(DMSO-d6, 400 MHz): δ 9.00−8.99 (d, 1H, J = 1.79 Hz); 8.74−8.71 (t,
1H); 8.59 (s, 1H); 8.48−8.47 (d, 1H, J = 8.42 Hz); 8.47−8.45 (d, 2H, J =
9.15 Hz); 8.21−8.19 (d, 2H, J = 9.15 Hz); 7.34−7.33 (d, 1H, J = 1.78
Hz); 4.69−4.66 (q, 1H); 4.42−4.41 (d, 2H, J = 5.60 Hz); 3.60−3.59 (d,
1H, J = 1.79 Hz); 3.50−3.10 (m, 3H). 13C NMR (DMSO-d6, 100 MHz):
δ 170.68, 169.07, 165.75, 154.10, 153.25, 147.11, 147.05, 141.26, 126.08,
121.95, 120.94, 116.23, 53.24, 52.95, 51.85, 34.69, 33.44. HRMS-ESI m/
z [M + H+] calcd for C19H17N7O7S: 488.4543. Observed: m/z 488.0986
(M + H+); m/z 486.0864 (M − H+). HPLC method 2: tR = 20.4 min;
purity = 93.4%.
(2S,3S)-3-((S)-1-((1-(2,6-Difluorophenyl)-1H-1,2,3-triazol-4-
yl)methylamino)-1-oxo-3-(thiazol-4-yl)propan-2-ylcarbamoyl)-
oxirane-2-carboxylic Acid (43). The general procedure was followed
using the corresponding peptidomimetic epoxide ethyl ester 43E (24
mg, 0.05 mmol) and LiOH (1.1 mg, 0.05 mmol) and after extraction
afforded the desired product as a white solid (13 mg, 57.3%). 1H NMR
(DMSO-d6, 400 MHz): δ 9.00−8.99 (d, 1H, J = 1.90 Hz); 8.78−8.76 (t,
1H); 8.60−8.58 (d, 1H, J = 8.29 Hz); 8.27 (s, 1H); 7.77−7.69 (m, 1H);
7.49−7.45 (t, 2H); 7.36−7.27 (m, 2H); 4.71−4.65 (m, 1H); 4.44−4.42
(d, 2H, J = 5.63 Hz); 3.598−3.593 (d, 1H, J = 1.78); 3.360−3.355 (d,
1H, J = 1.78); 3.27−3.22 (m, 2H). 13C NMR (DMSO-d6, 100 MHz): δ
170.69, 169.07, 165.69, 158.07, 158.04, 155.56, 155.53, 154.05, 153.31,
145.67, 136.62, 129.17, 128.48, 126.17, 116.17, 53.23,, 52.93, 51.83,
34.67, 33.51. HRMS-ESI m/z [M + H+] calcd for C19H16F2N6O5S:
479.4343. Observed: m/z 479.0960 (M + H+). HPLC method 2: tR =
17.32 min; purity = 95.9%.
(2S,3S)-3-((S)-1-((1-Mesityl-1H-1,2,3-triazol-4-yl)-
methylamino)-1-oxo-3-(thiazol-4-yl)propan-2-ylcarbamoyl)-
oxirane-2-carboxylic Acid (44). The general procedure was followed
using the corresponding peptidomimetic epoxide ethyl ester 44E (24
mg, 0.05 mmol) and LiOH (1.1 mg, 0.05 mmol) and after extraction
afforded the desired product as a white solid (15 mg, 66.1%). 1H NMR
(DMSO-d6, 400 MHz): δ 8.98−8.97 (d, 1H, J = 1.87 Hz); 8.72−8.70 (t,
1H, J = 11.2 Hz); 8.55−8.53 (d, 1H, J = 8.23 Hz); 7.93 (s, 1H); 7.34 (s,
1H); 7.08 (s, 1H); 4.68−4.63 (m, 1H); 4.42−4.40 (d, 2H, J = 5.50 Hz);
3.61−3.50 (m, 4H); 2.32 (s, 3H); 1.86 (s, 6H). 13C NMR (DMSO-d6,
100 MHz): δ 170.29, 165.60, 153.65, 152.98, 144.93, 139.46, 134.51,
133.50, 128.90, 124.69, 115.78, 52.79, 52.58, 48.66, 34.54, 33.10, 20.69,
16.89. HRMS-ESI m/z [M + H+] calcd for C22H24N6O5S: 485.5343.
Observed: m/z 485.1622 (M + H+); m/z 483.1600 (M − H+). HPLC
method 2: tR = 23.6 min; purity = 97.4%.
Modeling. Coordinates of calpain protein structures were down-
loaded from the Protein Data Bank (PDB).56 Visual inspection of the
crystal structures 1KXR and 2ARY was performed using Chimera.57
Conserved water molecules near the binding site were included and
allowed to be toggled on and off during docking. Ligands and the
binding sites were prepared in MOE.58 GOLD59 was used for docking/
scoring and employing default parameters and settings.
154.10, 152.52, 138.29, 146.80, 144.19, 128.01, 121.75, 120.59, 116.29,
53.25, 52.98, 51.84, 49.01, 33.43. HRMS-ESI m/z [M + H+] calcd for
C19H19N7O7S2: 522.0865. Observed: m/z 522.0858 (M + H+). HPLC
method 2: tR = 14.8 min; purity = 95.1%.
(2S,3S)-3-((S)-1-((1-(3,5-Bis(trifluoromethyl)phenyl)-1H-1,2,3-
triazol-4-yl)methylamino)-1-oxo-3-(thiazol-4-yl)propan-2-
ylcarbamoyl)oxirane-2-carboxylic Acid (40). The general proce-
dure was followed using the corresponding peptidomimetic epoxide
ethyl ester 40E (94 mg, 0.15 mmol) and LiOH (3.7 mg, 0.15 mmol) and
after extraction afforded the desired product as a white solid (72 mg,
80.3%). 1H NMR (DMSO-d6, 400 MHz): δ 8.99−8.98 (d, 1H, J = 1.92
Hz); 8.92 (s, 1H); 8.79−8.77 (t, 1H); 8.63 (s, 2H); 8.57−8.55 (d, 1H, J
= 8.27 Hz); 8.27 (s, 1H); 7.34−7.33 (d, 1H, J = 1.85 Hz); 4.71−4.66 (m,
1H); 4.43−4.42 (d, 2H, J = 5.67 Hz); 3.58−3.57 (d, 1H, J = 1.73 Hz);
3.36−3.35 (d, 1H, J = 1.70 Hz); 3.28−3.10 (m, 2H). 13C NMR (DMSO-
d6, 100 MHz): δ 170.67, 169.07, 165.80, 154.06, 153.29, 146.93, 138.32,
132.48, 132.14, 122.38, 121.11, 116.18, 53.22, 52.90, 51.96, 34.64, 33.51.
HRMS-ESI m/z [M + H+] calcd for C21H16F6N6O5S: 579.4443.
Observed: m/z 579.0890 (M + H+); m/z 577.0775 (M − H+). HPLC
method 2: tR = 24.5 min; purity = 95.3%.
(2S,3S)-3-((S)-1-((1-(4-Bromophenyl)-1H-1,2,3-triazol-4-yl)-
methylamino)-1-oxo-3-(thiazol-4-yl)propan-2-ylcarbamoyl)-
oxirane-2-carboxylic Acid (41). The general procedure was followed
using the corresponding peptidomimetic epoxide ethyl ester 41E (25
mg, 0.04 mmol) and LiOH (1.1 mg, 0.04 mmol) and after extraction
afforded the desired product as a white solid (18 mg, 75.9%). 1H NMR
(DMSO-d6, 400 MHz): δ 8.99−8.98 (d, 1H, J = 1.87 Hz); 8.74−8.71 (t,
1H); 8.58−8.56 (d, 1H, J = 8.27 Hz); 8.54 (s, 1H); 7.86−7.79 (q, 4H);
7.34−7.33 (d, 1H, J = 1.75 Hz); 4.70−4.65 (q, 1H); 4.40−4.38 (d, 2H, J
= 5.59 Hz); 3.60−3.59 (d, 1H, J = 1.77 Hz); 3.38−3.37 (d, 1H, J = 1.77
Hz); 3.28−3.10 (m, 2H). 13C NMR (DMSO-d6, 100 MHz): δ 170.62,
ASSOCIATED CONTENT
■
S
* Supporting Information
Further details of synthesis, characterization, protein expression;
LC−MS; and selectivity assay. This material is available free of
charge via the Internet at http://pubs.acs.org.
M
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AUTHOR INFORMATION
Corresponding Author
*Phone: 312-355-5282. Fax: 312 996 7107. E-mail: thatcher@
uic.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The work was supported by NIH Grant U01 AG028713.
Molecular modeling was in part conducted using free academic
license for the UCSF Chimera package from the Resource for
Biocomputing, Visualization, and Informatics at the University of
California, San Francisco (supported by NIH Grant P41 RR-
01081).
ABBREVIATIONS USED
■
AD, Alzheimer’s disease; ADMET, absorption, distribution,
metabolism, excretion, toxicity; Cal1, calpain; Cal1cat, calpain 1
recombinant catalytic domain; Cath, cathepsin; CathB, cathepsin
B; DET, diethyl tartrate; EIC, extracted ion chromatogram;
EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide); GSH,
glutathione; HOBT, hydroxybenzotriazole; IAA, iodoacetamide;
S/N, signal to noise; TIC, total ion chromatogram; TBTA,
tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine
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