Cell-Based Optimization of Covalent Reversible Ketoamide Inhibitors Bridging the Unprimed to the Primed Site of the Proteasome β5 Subunit

Article abstract of DOI:10.1002/cmdc.201900472

The ubiquitin-proteasome system (UPS) is an established therapeutic target for approved drugs to treat selected hematologic malignancies. While drug discovery targeting the UPS focuses on irreversibly binding epoxyketones and slowly-reversibly binding boronates, optimization of novel covalent-reversibly binding warheads remains largely unattended. We previously reported α-ketoamides to be a promising reversible lead motif, yet the cytotoxic activity required further optimization. This work focuses on the lead optimization of phenoxy-substituted α-ketoamides combining the structure-activity relationships from the primed and the non-primed site of the proteasome β5 subunit. Our optimization strategy is accompanied by molecular modeling, suggesting occupation of P1′ by a 3-phenoxy group to increase β5 inhibition and cytotoxic activity in leukemia cell lines. Key compounds were further profiled for time-dependent inhibition of cellular substrate conversion. Furthermore, the α-ketoamide lead structure 27 does not affect escape response behavior in Danio rerio embryos, in contrast to bortezomib, which suggests increased target specificity.

Full text of DOI:10.1002/cmdc.201900472

Accepted Article
Title: Cell-Based Optimization of Covalent Reversible Ketoamide
Inhibitors Bridging the Unprimed to the Primed Site of
Proteasome´s β5 Subunit
Authors: Daniel Stubba, Dennis Bensinger, Janika Steinbacher, Lilia
Proskurjakov, Álvaro Salcedo Gómez, Uwe Schmidt, Stefan
Roth, Katja Schmitz, and Boris Schmidt
This manuscript has been accepted after peer review and appears as an
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the VoR from the journal website shown below when it is published
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To be cited as: ChemMedChem 10.1002/cmdc.201900472
Link to VoR: http://dx.doi.org/10.1002/cmdc.201900472
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Cell-Based Optimization of Covalent Reversible Ketoamide
Inhibitors Bridging the Unprimed to the Primed Site of
Proteasome´s β5 Subunit
Daniel Stubba,a# Dennis Bensinger,a# Janika Steinbacher,a Lilia Proskurjakov,a Álvaro Salcedo
Gómez,a Uwe Schmidt,b§ Stefan Roth,b Katja Schmitza and Boris Schmidta*
[a]
D.Stubba, Dr. D. Bensinger, J. Steinbacher, L. Proskurjakov, Á. Salcedo Gómez, Prof. Dr. K. Schmitz, Prof. Dr. B. Schmidt
Clemens-Schoepf-Institute for Organic Chemistry & Biochemistry
Technische Universität Darmstadt
Alarich-Weiss-Str. 4, 64287 Darmstadt (Germany)
E-mail: schmidt_boris@t-online.de
[b]
Dr. U. Schmidt, Prof. Dr. S. Roth
Visual Inference Lab, Department of Computer Science
Technische Universität Darmstadt
Huchschulstr. 10, 64289 Darmstadt (Germany)
Supporting information for this article is given via a link at the end of the document.
immunoproteasome.[4-5] As a consequence of deviant substrate
binding pockets in the β5 subunit, the iCP exerts different
recognition and cleavage patterns. The major structural difference
is an enlarged S1 pocket in β5i due to a different conformational
orientation of Met45. Additional alterations in the amino acid
sequence between cCP and iCP such as the thiol group of Cys48
pointing in the S4 pocket of the β5i subunit allow for selective
targeting of the immunoproteasome.[6-7]
Abstract: The Ubiquitin-proteasome system (UPS) is an established
therapeutic target for approved drugs to treat selected hematologic
malignancies. While drug discovery targeting the UPS focuses on
irreversibly binding epoxyketones and slowly-reversibly binding
boronates, optimization of novel covalent-reversibly binding warheads
remains largely unattended. We previously reported α-ketoamides to
be a promising reversible lead motif, yet the cytotoxic activity required
further optimization. This work focuses on the lead optimization of
phenoxy-substituted α-ketoamides combining the structure activity
relationships from the primed and the non-primed site of proteasome’s
β5 subunit. Our optimization strategy is accompanied by molecular
modeling, suggesting occupation of P1’ by a 3-phenoxy group to
increase β5 inhibition and cytotoxic activity in leukemia cell lines. Key
compounds were further profiled for time-dependent inhibition of
cellular substrate conversion. Furthermore, the α-ketoamide lead
structure 27 is not affecting escape response behavior in Danio rerio
embryos, in contrast to Bortezomib, which suggests increased target
specificity.
Defects in the UPS affects cell cycle progression as well as
immune response thus are recognized as an attractive target for
several diseases such as cancer indicating that inhibitors of this
pathway may prevent malignant cells from proliferation.[8-10]
Inhibition of the proteasome results in an imbalance between
proteasome load and proteasome capacity that serves as a
trigger for apoptosis[11] as well as for inhibition of NFκB signaling
by blocking the degradation of the NFκB-inhibitor IκB preventing
nuclear translocation of NFκB.[12] Bortezomib (1) was the first
proteasome inhibitor (PI) approved by the FDA in 2003 followed
by carfilzomib (3) in 2012 and ixazomib (2) in 2015, the first orally
available PI, for the treatment of multiple myeloma and mantle-
cell lymphoma (Figure 1).[7] Multiple clinical trials are still in
progress for the treatment of leukemia, non-Hodgkin lymphoma
and solid tumors. All approved PIs are covalently binding
inhibitors, bearing an electrophilic warhead which predominantly
binds to the catalytically active Thr1Oγ of the β5c subunit. The
first-generation PI bortezomib is a slowly reversible binding
peptidic boronic acid inhibiting the β5 and to a less extent the β2
and β1 subunits of the immuno as well as the constitutive
proteasome and numerous off-target proteases.[13] Insufficient
selectivity frequently results in a major adverse event upon
treatment with bortezomib: peripheral neuropathy, a dose limiting
neurotoxic effect occurring in 30 % to 39 % of the patients.[14]
Unlike reversibly binding boronates carfilzomib bears an
epoxyketone warhead that binds irreversibly to the hydroxy group
of Thr1Oγ forming a morpholino ring with Thr1N.[15-16] Recent
crystal structure analysis suggests formation of a seven-
membered 1,4-oxazepane ring due to a nucleophilic attack of
Thr1N on the β-carbon atom of the epoxide instead of the α-
carbon atom.[17] Irreversible inhibitors can overcome drug
resistance to reversible inhibitors but are less likely to penetrate
deep into tissue, resulting in impaired efficacy for solid, non-
vascularized tumors.[18] Ixazomib is formulated as a citrate
boronate prodrug improving distribution characteristics that allow
oral uptake.[19] It shows affinity to the β5 and β1 subunit
comparable to bortezomib combined with a shorter residence
Introduction
The Ubiquitin-proteasome-system (UPS) is the main non-
lysosomal proteolytic pathway for the degradation of misfolded,
altered or short-lived proteins in eukaryotes. It exerts a crucial role
in cellular protein turnover e.g. the production of building blocks
for the de novo synthesis of proteins and controls several cellular
functions such as protein homeostasis, proliferation, apoptosis,
signal transduction and antigen production.[1] The ultimate
proteolytic component of the UPS is the large, cylindric 26S
proteasome complex, consisting of two regulatory particles (19S
caps) and the 20S proteasome core particle (CP), being
responsible for the proteolysis of the designated proteins. The
20S proteasome consists of four heptameric rings (α1-7, β1-7, β1-
7, α1-7) bearing 28 subunits. Just three subunits per proteolytic
β-ring are catalytically active. The β1c subunit is referred to
caspase-like activity, the β2c to trypsin-like activity and the β5c is
found to show chymotrypsin- and elastase-like activity.[2-3]
Exposure to tumor necrosis factor α (TNFα) and/or interferon γ
(IFNγ) leads to substitution of those subunits by β1i (LMP2), β2i
(MECL-1) and β5i (LMP7) resulting in formation of the
1
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time that may account for deeper tissue penetration.[18] In general,
irreversible as well as slowly-reversible covalent inhibitors may
provide unique advantages when forming long-lived ties with their
target. In contrast, highly reactive inhibitors are likely to be
trapped by the high number of proteasomes in the red blood cells,
which limits the therapeutic availability to non-solid tumors.
Besides the approved drugs several inhibitors bearing reversible
warheads have been developed; the peptidic aldehydes MG132
(4), BSc2118 (5) and the peptidic α-ketoamides BSc2189 (6) and
BSc4999 (7).[20-22]
reaction of functionalized isocyanides and peptidic aldehydes with
trifluoro acetic acids to give α-hydroxy amides that were oxidized
using 2-iodoxybenzoic acid to provide the final ketoamides
(Scheme 1, SI chapter 1).[21] Phenoxy-substituted isocyanides
were prepared starting from commercially available fluoro nitro
benzenes that were reacted with phenol or by conversion of
nitrophenols with diphenyl iodonium chloride. Subsequent
reduction of the nitro group gave the corresponding anilines,
followed by N-formylation and subsequent dehydration leading to
isocyanides that were subjected to the Passerini reaction. The
peptidic aldehydes have been synthesized as reported
previously.[20-21]
Scheme 1. Synthesis outline for the development of phenoxy-substituted
tripeptidic ketoamides.
Our first goal was to evaluate whether substitutions in the 2-,
3- or 4- position are beneficial to bridge the inhibitor to substrate
pockets of the primed site. We decided to introduce a phenoxy
group as a substituent that is sufficiently bulky to occupy S1’ but
still flexible enough to allow an induced fit to the primed site.
Compound 7 from the cocrystal structure served as starting point
for the in silico analysis of analogues. The energy-minimization of
the virtual ligands revealed that all positions might tolerate such a
substitution (Figure 2b). However, the cytotoxic activity of 3-
phenoxy ketoamide 9 in MV4-11 leukemia cells is substantially
higher in comparison to the 2- and 4-phenoxy ketoamides 8 and
10, which correlates with the increased inhibition of the
proteasome. However, the inhibition of β5c by 9 is decreased (in
comparison to 7) while inhibition of β5i is increased resulting in
decreased selectivity β5c over β5i (2.6- to 4.3-fold, Table 1). We
therefore aimed to increase selectivity by re-introducing the
methyl groups in ortho or para position to the ketoamide NH.
Figure 1. Overview of approved and experimental proteasome inhibitors.
1: Bortezomib. 2: Ixazomib. 3: Carfilzomib. 4: MG132. 5: BSc2118. 6: BSc2189.
7: BSc4999.
Our previous work highlighted the α-ketoamide electrophile as
a highly active reversible lead motif with potential for the treatment
of solid tumors or autoimmune disorders.[21-22] However, the
reduced cytotoxic activity of 6 in malignant cell lines (in
comparison to carfilzomib) motivated us to optimize the potency
of ketoamide proteasome inhibitors targeting the primed site of
the β5 subunit.
Results and Discussion
Optimization of the ketoamide warhead started with the
examination of the yeast CP cocrystal structures of aldehyde 4
(PDB: 4NNN) as well as ketoamides 6 (PDB: 4NO8) and 7 (PDB:
4R02) that were obtained by our previous optimization efforts
(Figure 2a).[20-22]
All three inhibitors bind to the β5 subunit and adopt similar
conformations of the triple leucyl peptide residues as well as the
Cbz-protecting groups, located in P4, close to the β6 subunit.
However, the dimethyl phenyl group of ketoamide 7 is slightly
shifted (~ 0.8 Å) towards the primed site when compared to the
unsubstituted phenyl moiety in 6. While the hydroxyl group of the
hemiacetal adduct of 4 with Thr1Oγ is situated in the oxy anionic
hole of this site, the hemiketal hydroxyl group of 6 and 7 is
switched to the opposite site enabling the carbonyl oxygen to
constrain the conformation of the phenyl amide head group.
Peptidic phenyl ketoamides were synthesized using the Passerini
Introduction of methyl groups results in the 1,2,3,4-substituted
phenyl ketoamide 12 and decreases cellular activity substantially
whereas the 1,3,4,6-substituted ketoamide 11 retains the
cytotoxic activity of 9. Remarkably, the selectivity is now shifted
towards the inhibition of β5c over β5i. Mono-methyl substitution in
the 2- or 4-position decreases cytotoxic activity (13 and 14) as
well as selectivity over β5i. 6-Methyl substitution leading to
ketoamide 15 decreases inhibition of β5c and β5i while
cytotoxicity is slightly increased. In previous work, the occupation
of P2 by AspOtBu increased the selective inhibition of β5c by
peptidic aldehydes.[20] However, as proteasomes miss a defined
P2 pocket, the increased activity might be due to hydrophobic
interactions correlating with increased molecular weight and
resulting in the displacement of active site water molecules.
Figure 2. (a) Superposition of aldehyde 5 (green) and ketoamide 7 (blue). (b) Energy-minimized models of 2-, 3- and 4-phenoxy substituted ketoamides 8 – 10 and
(c) P4-substituted tripeptides 22 (purple), 23 (green) and 24 (blue) targeting the β5c subunit.
2
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Table 1. Structure-activity relationship of Cbz-protected phenoxy-substituted ketoamides 8
̶
21 and previously reported ketoamide 7.[22]
Rel. Inhibition
(c =100 nM)
IC50 MV4-11
/ nM[a]
Compound
X
R1
R2
R3
R4
β5c
β5i
Bortezomib 1
-
-
-
-
-
-
-
-
-
34 ± 5
3.6 ± 1.8
19 ± 3
203 ± 22
18 ± 3
45 ± 3
19 ± 1
26 ± 8
23 ± 2
27 ± 8
16 ± 1
46 ± 6
46 ± 1
76 ± 14
36 ± 5
68 ± 9
297 ± 27
89%
86%
64%
12%
56%
25%
46%
20%
49%
41%
26%
57%
43%
28%
53%
41%
33%
85%
Carfilzomib 3
-
43%
33%
8%
7
Me
OPh
H
H
Me
H
H
8
H
H
9
OPh
H
H
H
23%
5%
10
11
12
13
14
15
16
17
18
19
20
21
H
OPh
Me
Me
Me
H
H
H
OPh
OPh
OPh
OPh
OPh
H
Me
H
12%
9%
Me
H
H
28%
29%
19%
48%
36%
18%
43%
40%
28%
Me
H
H
H
Me
H
H
H
Me
OPh
H
H
Me
H
H
H
H
OPh
H
H
H
H
OPh
Me
H
H
OPh
Me
[a] Values are the mean ±SD from two independent experiments, each carried out in technical triplicates.
As the cytotoxic activity of 5 (Asp(OtBu) in P2) is slightly higher
than of 4 (Leu in P2) correlating with higher β5c inhibition while
selectivity over β5i is retained, we investigated if this pattern can
be also applied to the ketoamide warhead. Subsequent synthesis
5 compared to ketoamides as visible in reported cocrystals. The
phenyl amide group hinders close vicinity of the prochiral carbon
to Thr1 leading to a changed induced fit of the ligand and possible
unfavorable placement of the bulky tBu residue in P2.
of ketoamides 16
̶
21 began with aldehyde 5. In general, the
The next optimization step was the replacement of the Cbz
protecting group occupying P4 of the β5c subunit. We
hypothesized that fewer rotatable bonds as well as additional
polar interactions might improve potency and cell permeability
and so allow alternative induced fit mechanisms to the active site.
We decided to introduce three different P4 groups that are
inspired by previously reported inhibitors (R1 – R3, Table 2).
Ixazomib 2 harbors a dichloro benzamide residue (R3) following
P2, comparable to the pyrazine-2-amide (R2) in Bortezomib 1 but
might act sterically and electronically distinct. Furthermore, we
aimed to probe if a bicyclic ring is tolerated and introduced a
quinazolinone group, that is linked to P3 leucine by an acetyl
group, similar to an approach reported by Micale et al (R1).[23] To
explore the plausibility of these substitutions, we modified 7 using
the cocrystal structure with subsequent energy minimization of
the ligand (Figure 2c). All three residues are able to fit into the S4
pocket close to the β6 subunit, similar to the carboxybenzyl group,
and form hydrogen bond interactions by the P4 amide NH with the
Asp126 side chain of β6. Additionally, R1 might form π-H
interactions with Pro127 of β6, while R2 might form two π-H
interactions with Val128. Exchange of the Cbz-protecting group to
cytotoxicity of all P2 = Asp(OtBu) derivatives in this series is lower
than the one for all compounds featuring leucine in P2.
Interestingly, the dimethyl-substituted ketoamide 17 shows similar
cytotoxic activity like the unsubstituted ketoamide 16 in MV4-11
cells. The trend in inhibition by phenoxy-substituted inhibitors
featuring leucine in P2 can also be observed for the AspOtBu
derivatives 18
̶ 20, where m-phenoxy ketoamide 19 is more
potent than o- and p-substituted phenoxy ketoamides 18 and 20.
While 18 and 20 are more potent inhibitors of β5c than their leucyl
counterparts, inhibition by 19 is comparable and the highest in
this series.
However, inhibition of β5i is increased relatively to β5c by m-
phenoxy ketoamide 19 leading to isoform unselective inhibition.
In strong contrast to 11, it was important to see that re-introduction
of the 4,6-dimethyl group giving the tetra substituted ketoamide
21 results in decreased cytotoxic activity as well as in β5i and β5c
inhibition compared to 19.
A possible reason for the opposing activity trend by AspOtBu-
substituted ketoamides and aldehydes might be due to a slight
shift in the positioning of the amide backbone in aldehydes 4 and
3
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amides was performed by catalytic hydrogenation to give peptidic
amino alcohols. These have been converted to the corresponding
amides using standard peptide coupling methods with the
functionalized acids and subsequent oxidation of the hydroxyl
group using IBX gave aldehydes that served as the second
starting material for the Passerini reaction.
Table 2. Structure-activity relationship of aldehydes 4, 5 and 22
̶
25 and dichloro benzamide-substituted m-phenoxy ketoamides 26 ̶ 29.
Rel. Inhibition
(c =100 nM)
IC50
Compound
Warhead
Y
Z
R4
MV4-11 / nM[a]
β5c
β5i
6%
4
Cbz
R1
-
131 ± 12
> 1000
43%
6%
22
23
24
5
-
11%
4%
R2
-
145 ± 23
16 ± 1
30%
76%
54%
64%
25%
83%
26%
26%
R3
-
-
60%
11%
38%
4%
Cbz
R3
123 ± 12
28 ± 5
25
26
27
28
29
-
R3
H
25 ± 5
R3
OPh
H
7.4 ± 2.6
47 ± 5
58%
35%
8%
R3
R3
OPh
58 ± 10
[a] Values are the mean ±SD from two independent experiments, each carried out in technical triplicates.
The dichloro benzamide-substituted aldehyde 24 exerts
strongly increased cytotoxic activity in comparison to 4, while the
bicyclic derivative 22 is inactive. The pyrazinyl moiety in 23
provides no improvement in cytotoxicity. Introduction of
Asp(OtBu) in P2 resulted in the R3-substituted aldehyde 25, which
leads to significantly increased cytotoxicity in comparison to the
Cbz-protected aldehyde 5, but remains less potent than the leucyl
counterpart similar to the ketoamide series.
potent compound in this series, displaying the highest cytotoxic
activity and inhibition of β5c (IC50 = 23 nM, Table 3) as well as
selectivity over β5i (IC50 = 96 nM, 4.2-fold). Combination of the
dimethyl phenyl ketoamide with the dichloro benzamide group in
P4 provides no benefit in cytotoxicity. Furthermore substitution of
leucine in P2 with Asp(OtBu) (28) decreases cytotoxic activity
favoring inhibition of β5i. Introduction of the m-phenoxy group
resulting in the Asp(OtBu)-analogue of 27 decreases cytotoxic
activity even more. For all compounds the inhibition of β2c and
β1c is low at 1 µM of inhibitor while they strongly inhibit β5c (Table
S1).
Table 3. Comparison of cytotoxicity in leukemia cell lines and inhibition of β5i
and β5c by lead compounds 7, 9 and 27 as well as carfilzomib 3.
Covalent Docking of BSc4999 (7) and lead compound 27
Due to the surprising advantage of the m-phenoxy substituted
dichloro benzamide ketoamide 27 which our initial binding model
could not show any explanation for we challenged our original
binding hypothesis using covalent docking to explain increased
cellular and proteasomal activity of 27. During the last decade
extensive approaches of the computational supported drug
discovery have been applied for the modelling of proteasome
inhibitors.[24] These include docking of dipeptidyl boronates
starting from known conformations similar to Bortezomib[25] as
well as non-peptidic boronates[26]. However, true de novo docking
of more complex peptides as tri- and tetra peptides remains rare
as ligand flexibility, size and solvent-exposed binding pockets
increase computational time exponentially and generate a large
number of possible poses. Therefore most approaches use
simplifications. Examples include covalent docking of peptidyl
ketoamides targeting the HCV serine protease in which the
common peptidic core needs to be constraint;[27] as well as
docking approaches in which the covalent attachment point is not
Cell viability IC50 / nM[a]
IC50 / nM
Fold-IC50
β5i/β5c
17.8
Compound
MV4-11
THP-1
135 ± 39
66 ± 1
Jurkat
β5c
β5i
142
96
3
27
9
3.6 ± 1.8
7.4 ± 2.6
18 ± 3
14 ± 3
11 ± 1
8
23
66
48
4.2
30 ± 10
24 ± 0.1
24 ± 4
170
204
2.6
7
19 ± 3
30 ± 0.3
4.3
[a] Values are the mean ±SD from two independent experiments, each carried
out in technical triplicates.
Then we combined our SAR efforts exploring phenoxy-
substituted ketoamides and P4 variation to give hybrid
̶ 29. In line with the SAR described so far, 3-
phenoxy-4,6-dimethylphenyl ketoamide 27 resulted in the most
ketoamides 26
4
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included in modelling[28] or only the non-peptidic warhead is
this approach is not straightforward for prospective docking of
new ligands with unknown bound conformations. This is due to
the fact that only random conformations can be chosen as input.
To illustrate this issue, we performed unpretentious redocking of
7 using a random, energy-minimized input conformation that only
gave high root-mean-square deviation (RMSD) poses (no pose <
2.0 Å, top-scored RMSD = 5.2 Å; top-RMSD pose on scoring rank
17, see SI chapter 3 for details). This probably happens as LU-
122 contains polar amino acids engaging in hydrogen bonds with
β5 residues, that are energetically favored by scoring functions
compared to simple hydrophobic interactions. Occurrence of
three leucyl residues in P1 – P3 in 7 as well as the lead compound
27 increases ligand size as well as the number of rotatable bonds,
thus hampering reliable modeling.
modelled.[22]
In 2014, our group reported the development of DOCKTITE, a
covalent docking workflow, that closed this knowledge gap and
allowed precise redocking of proteasome inhibitors such as the
tripeptide vinyl sulfonamides LU-122.[29]
In enhancement of this methodology we performed de novo
covalent docking of tripeptidic phenyl ketoamides simultaneously
targeting the primed and unprimed site of the β5 subunit. Covalent
redocking of 7 was successful using the conformation extracted
from the yCP cocrystal structure as input conformation for docking
only changing the hemiketal to the parental ketoamide warhead
(PDB: 4r02, top-scored pose RMSD = 1.3 Å) which is similar to
redocking of vinyl sulfonamide LU-122 (PDB: 4int).[29] However
Figure 3. (a) Pharmacophore features of 7 used for covalent docking. (b) Top-scored binding pose of compound 27. (c) Ligand interaction map of compound 7 and
(d) compound 27.
We therefore applied a novel screening strategy to address this
problem, that is, in principle, customizable to other scaffolds and
protein targets.
input conformation fitting the pharmacophore, up to 100 top-
scored poses were subjected to forcefield refinement. Using this
strategy, 9.625 poses resulted from covalent docking that were
subsequently cleaved from the nucleophile and rescored using
the knowledge-based DSX scoring function.[30] Among the top 1%
DSX-scored poses, 7 poses had a RMSD value of less than 2 Å
compared to the native structure. Interestingly, between 0 and 30
docking poses resulted per input conformation (mean: 7.7 poses),
suggesting that the conformational pre-sampling of the tripeptides
increased the success rates of covalent docking.
This method was applied to de novo docking of the lead
compound 27 in the β5 subunit using the pharmacophore model
created before. Stochastic conformational search showed 343
unbound conformations of 27 that were subsequently tagged and
attached to the side chain giving 686 input conformations for
covalent docking. Approximately 32.000 poses resulted and were
cleaved again from the nucleophile and rescored by DSX. The
top1% of poses (320, DSX score < -200) were analyzed for RMSD
values to the top-scored pose (Figure 3b, d), giving two
ensembles of poses (Figure 4a).
In contrast to redocking of native poses, where the
conformation obtained from the cocrystal structure is used as
input, conformational sampling of manually sketched structures
prior to docking is necessary as no definite starting points are
available. We therefore performed a stochastic conformational
search, subsequently tagged the ketoamide warhead and
performed ligand attachment. For redocking of 7, 624
conformations have been obtained that gave 1248 input
conformations after sidechain attachment as the warhead is
prochiral creating R and S enantiomeric adducts with Thr1Oγ. An
automated pharmacophore defining the heavy chain atoms of
Thr1 was generated (d = 0.4 Å) during the side-chain attachment
step of DOCKTITE. In order to constrain docking to the substrate
binding channel we introduced an aromatic pharmacophore
feature as well as a donor and an acceptor feature, defined by the
amide bond between P2 and P3 of 7 (each d = 2.0 Å, Figure 3a,
c). Then up to 5.000 conformations were sampled again from
each input conformation during pharmacophore placement prior
to energy-minimization steps. MOE gave approximately 6 million
conformations that were screened based on the defined
pharmacophore features. From all docking conformations of one
Ensemble 1 gave 158 poses with RMSD values smaller than
2.5 Å compared to the top-scored pose of 27 (Figure 4b). In this
group, the dichloro benzamide tail is situated in S3 instead of
5
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leucine. This was already observed in the reported cocrystal
structures of 6 and 7 (Figure 2a).
Figure 4. (a) Pose distribution sorted by increasing RMSD to top DSX-scored pose of 27. (b) Random selection of poses for ensemble 1 (pose 1 to 158; RMSD <
2.5 Å) and the subgroups of ensemble 2 comprised of poses 201-297 (4.14 Å – 4.95 Å) consisting of (c) group 1 (51 of 97 poses) and (d) group 2 (46 of 97 poses).
This might be of advantage since entropy is increased by
displacement of three water molecules due to the size of this
group. As the dimethyl phenyl group of 27 obtains a similar
orientation as in the cocrystal structure of 7, the phenoxy group is
allowed to reside in the S1’ pocket. Superposition of the top-
scored pose with the cocrystal structure of bis-benzyl protected
homobelactosin c (PDB: 4j70) shows similar occupation of the
phenoxy group in S1’ as the benzyl protecting group in
homobelactosin c.
The occupation of S1 and S2 by the leucine residues agrees
well in comparison to ensemble 2 (RMSD-ranked pose 201-297).
However, two distinct pose clusters can be distinguished further.
In cluster one (51 poses, Figure 4c) 180° rotation around the
phenyl-nitrogen bond leads to reorientation of the phenoxy group
to the opposite direction of S1’ while binding of the dichloro
benzamide group in S3 is changed in group two (46 poses, Figure
4d).
Ensemble 1 comprises ~ 3-fold more poses as the two
subgroups in ensemble 2, furthermore the phenyl groups of 27
and homobelactosin C display similar binding in S1’, and so this
model was favored for further structure-based optimization.
The R-configuration dominates the stereochemistry of adduct
formation in ensemble 1 of 27 (77%). This fact is observed for 7
in the cocrystal structure. This preference is decreased regarding
the remaining top-scored poses including ensemble 2 (62% R).
Interestingly, while aldehydes are attacked by Thr1Oγ to give the
hemi-acetal hydroxyl group residing in the oxyanion hole,
ketoamides are attacked while the amide carbonyl group resides
in the oxyanion hole having the hemiketal hydroxide in the
opposite direction, which was confirmed by our docking model.
Noteworthy, there is no correlation in pose scoring (DSX and
London dG) to the RMSD to the native pose, suggesting that this
conformational clustering approach applied here can obtain a
clearer picture than scoring alone. However, the ultimate proof of
the binding mode of 27 reside in proteasome crystallography.
Cellular inhibition of proteasome activity and time-
dependent inhibition of cell viability
In 2016, our group reported the development of subunit-
specific, fluorogenic proteasome substrate 30, benefiting from an
AspOtBu residue in P2 (Z-LD(OtBu)A-AMC, Figure 5a). This
indicates increased selectivity and catalytic efficiency for small P1
residues, such as alanine, while inhibition performance shows the
opposite trend, favoring spacious P1 residues.[2] In this work, we
want to apply this methodology for the determination of
intracellular proteasome inhibition among different cell lines.
First, we benchmarked the fluorogenic substrate 30 towards
the standard substrate 31 (Suc-LLVY-AMC) using varying
substrate concentrations and cell numbers of MV4-11 cells
(Figure 5b). The highest substrate conversion was detected using
100 µM 30 in 50.000 cells (135 rfu/min) in MV4-11 cells and was
converted ~4 times faster than 31 (33.3 rfu/min). There was an
increase of differences in conversion rates with smaller number of
cells (10.000 cells, 100 µM substrate: 10-fold conversion) and
lower substrate concentration (50.000 cells, 25 µM substrate: 15-
fold conversion).
Interestingly, while THP-1 cells show similar turnover of 30 as
MV4-11 cells, conversion in Jurkat cells is ~2-fold increased.
Then we used this optimized cellular proteasome activity assay to
determine proteasome inhibition with our benchmark inhibitors 7,
9, 27 as well as reference compound carfilzomib 3, using different
pre-incubation times (0 h – 13 h) of inhibitor with cells, prior to
addition of substrate conversion and fluorescence detection
(Figure 5c, d).
6
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Figure 5. (a) Structure of proteasome substrates 30 and 31. (b) Comparison of substrate conversion by MV4-11 cells (50.000 cells/well). (c) Inhibition of substrate
30 conversion displays increased time-dependency by ketoamide inhibitor 27 compared to epoxyketone 3. (d) Overview of time-dependent inhibition of substrate
30 conversion by 7, 9, 27 and 3 in different leukemia cell lines. (e) Comparison of time-dependent cytotoxicity by compounds 27 and 3 and (f) relative cytotoxic
ꢀꢁ
ꢂꢃ
activity (Fold-IC50
=
ꢄꢆꢅ; Table S2).
ꢀꢁ
ꢂꢃ
In MV4-11 cells, 3 shows a rapid, almost complete inhibition of
substrate conversion in the single-digit nanomolar range. This
happens even though substrate and inhibitor are added at the
same time and incubated for only 30 min, prior to fluorescence
detection. Inhibition of cellular conversion is even more
pronounced for 1 h and 13 h pre-incubation. Compound 3 is
characterized by a fast onset of inhibition by 3, whereas
ketoamides show a slower onset of inhibition increasing with
incubation time with regard to phenoxy-substituted ketoamides 9
and 27, and decreasing at 13 h for 7 only. In line with the cytotoxic
activity, 27 is more potent than 9 and 7 in MV4-11 cells after 13 h
pre-incubation. Interestingly, 7 is 4-fold more potent than 9 without
pre-incubation of inhibitor and cells, suggesting that the on-rate of
inhibitor binding is decreased or cell permeability of 9 is slower. A
similar trend can be observed for the inhibition of proteasome
substrate conversion in Jurkat cells. Carfilzomib 3 is the most
potent compound displaying fast onset and sustained inhibition at
13 h pre-incubation time while the ketoamides show slower onset
of inhibition highest for 27 and lower for 9 and 7. However, in THP-
1 cells the inhibitors show altered potency in inhibiting conversion
of 30, presumably as iCP expression is higher than in MV4-11 and
Jurkat cells.[31] After short incubation times 3 is the most potent
compound, while inhibition is decreased with incubation time,
leading to lower inhibition after 13 h incubation time for 9 and 27.
Inhibition by 9 and 27 increases with incubation time, while, in
contrast to Jurkat and MV4-11 cells 9 is more potent than 27 after
13 h pre-incubation. 7 displays the same trend as in MV4-11 cells
being the least potent compound displaying increasing inhibition
after 1 h compared to 0 h that is decreasing after 13 h incubation
time.
We further investigated time-dependent cytotoxicity comparing
our lead compound 27 to Carfilzomib 3 using a continuous
luminescent cell viability assay over 18 h (Figure 5e, f). In line with
the results for cytotoxicity determined after 72 h treatment of cell
lines as well as cellular substrate conversion, 3 shows stronger
cytotoxicity in MV4-11 cells and comparable cytotoxicity in Jurkat
cells having a faster onset of cytotoxic activity (Table S2).
Remarkably, in THP-1 cells the initial cytotoxicity is increasing for
3 compared to 27 until 14 h treatment (9-fold) but is approaching
to the end of the assay. As the 72-h endpoint cytotoxicity assay
shows increased cytotoxicity for 27 than for 3 we assume that this
trend is continuing and reversing relative inhibition favoring 27.
It was interesting to find that after 13 h incubation cytotoxicity
is about 50 nM to 400 nM for 3 and 300 nM to 600 nM for 27
suggesting cell death becomes relevant to the cellular conversion
assay after this time scale. This effect might lead to artifacts, for
example by leading to competing proteolysis in the media and
increasing substrate influx through disrupted membranes.
Development of BODIPY-conjugated ketoamide inhibitors
We further aimed to visualize distribution of ketoamide
proteasome inhibitors in MV4-11 cells and in Danio rerio embryos
using BODIPY conjugations to 9 connected by different linking
groups (Figure 6a). Synthesis was performed starting with the
7
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hydroxyamide precursor of 9 being deprotected by catalytic
hydrogenation and coupled with acetyl, acetyl-glycine or acetyl-β-
alanine substituted BODIPYs that were subsequently oxidized
using IBX to give ketoamides 32 – 34. BODIPY-conjugated
ketoamides show similar inhibition of cellular conversion of
631 nM) in MV4-11 cells. The highly flexible β-alanine linking
group in 34 might facilitate ligand binding leading to most
pronounced inhibition (IC50 = 519 nM). In MV4-11 cells treated
with 32 – 34 an emission spectrum similar to isolated BODIPY
fluorescence detection can be recorded (Excitation maximum
~540 nm, Figure 6b).
substrate 30 (1 h incubation) as the parent compound 9 (IC50
=
Figure 6. (a) Structure of BODIPY-conjugated ketoamides 32 – 34 and inhibition of cellular substrate conversion of 30 in MV4-11 cells. (b) Exemplary spectrum of
BODIPY-conjugated inhibitors analyzed using the Nuance Fx Imaging system on Zeiss AxioScope A.1. Fluorescence is measured using AF488 fluorescence filter
sampling from 500 nm to 720 nm. Fluorescence intensity is normalized to highest fluorescence signal. (c) Staining of MV4-11 cells with compounds 32 – 34 with or
without Hoechst33342 DNA staining (2500 ms exposure time; Filter 365 nm or AF488, 40x magnification). (d) Uptake of fluorescent inhibitors 32 – 34 in Danio rerio
embryos (3225.5 ms exposure time, Filter AF488, 5x magnification).
Localization of all BODIPYs is mainly observed in the
cytoplasm, while the nucleus shows lower fluorescence (Figure
6c). Furthermore fluorescence is concentrated in clusters, which
are most likely aggresomes consisting of misfolded
polyubiquitinated proteins aggregating with proteasomes, which
is reported to be a result of proteasome inhibition.[32] These results
are in line with previous reports of green[33] as well as red-
27). In general, toxicity in treated, but still chorion-protected
embryos was low for both compounds (80% to 100% survival,
Table S3). All control embryos, either dechorionated or in chorion
were alive up to the end of the experiment. As we aimed to
compare toxic effects between ketoamide 27 and bortezomib 1 to
control, kinematic analysis was focused on treatment groups
where at least 50% of bortezomib- as well as 27-treated embryos
survived (Tables S4, S5).
shifted[34] fluorescent derivatives of peptidic aldehyde
5
colocalizing with proteasome in aggresomes and allowing
selective proteasome-staining. The uptake of fluorescent
ketoamides in Danio rerio embryos is most intense upon
treatment with 33, while 32 and 34 show only slightly increased
fluorescence compared to control embryos (Figure 6d).
Fluorescence is predominantly localized in the intestine and colon
for 32 and 34 whereas 33 shows increased fluorescence in the
yolk sac suggesting increased uptake.
The characteristic escape response of control as well as
unaffected treated embryos starts with a large body bend (C-
bend) pointing away from the stimulus followed by a counter bend
for reorientation and the escape movement itself (Figure 7b, c). In
control embryos, the mean C-bend amplitude is 129 ± 13,3° (3
embryos, 3 replicates of each embryos) and the C-bend duration
19.7 ± 1.4 ms. While mean C-bend amplitude (131.6 ± 21.3°) as
well as duration (32.7 ± 6.5 ms) is unchanged for 24 h / 25 µM
bortezomib-treated embryos (each p > 0.05) a huge decrease of
C-bend amplitude (6.9 ± 5.6°, p < 0.0001, Figure 7d) is observed
in 48 h / 25 µM bortezomib-treated embryos, while mean C-bend
duration is not statistically significant changed but apparently
highly variable (95% CI = -26 to +3 ms compared to control).
Interestingly, 24 h / 50 µM bortezomib-treated embryos show no
response to touch stimuli (50 stimuli per embryo, 150 stimuli total,
0 responses). Therefore we decided to set body amplitudes to
zero, describing an absent escape response (0 movements, p <
0.0001, n = 9). Accordingly, C-bend amplitude (0±0°, p < 0.0001)
as well as C-bend duration (0±0 ms, p < 0.0001) are absent and
thus massively decreased compared to control.
Danio rerio neurotoxicity assay
As the primary dose-limiting side effect of bortezomib is
peripheral neuropathy, we further characterized our lead
compound 27 in a Danio rerio embryo escape response assay
established in our group similar to reported approaches.[35-36] For
the determination of neurotoxic effects embryos were
dechorionated at 24 hpf and treated with two escalating doses of
bortezomib 1 and ketoamide 27 (25 µM, 50 µM) for 24 h to 72 h.
Subsequently, escape responses of surviving embryos were
analyzed at 96 hpf evoked by touch stimulation and tracked with
a high-speed camera at 500 fps to allow quantification of embryo
movement characteristics (Figure 7a).
Toxicity of bortezomib was high at 50 µM and 48 h to 72 h
incubation time as well as 25 µM inhibitor and 72 h incubation time
(100% of bortezomib-treated embryos dead) while embryos
treated with 27 showed a higher survival rate (25% to 75%).
However, overall survival in 27-treated embryos was lower than
for bortezomib at low doses (92% in Bortezomib group, 58% with
In contrast, 27-treated embryos showed similar escape
responses to control. However, a slight decrease in C-bend
duration can be observed for 25 µM / 24 h treated embryos
(14 ± 0.8 ms, p < 0.01) as well as 25 µM / 48 h treated embryos
(15.6 ± 0.5 ms, p < 0.05).
8
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Figure 7. (a) Example for the touch-evoked escape response of a control embryo and determination of body-bend amplitude α. (b) Comparison of the body-bend
amplitude over time observed after treatment with bortezomib or 27 and control embryos after 48 h. (c) Analysis of treatment effects using different incubation
parameters. Comparison of the C-bend amplitude, C-bend duration, reflex duration, number of movement and number of movements per 100 ms. Yellow: Kinematic
parameters of control embryos. Grey: no statistically significant differences in mean compared to control. Green: Statistically significant differences of mean value.
* = p < 0.05. ** = p < 0.01. *** = p < 0.001. **** = p < 0.0001. n.s. = p > 0.05. (d) Comparison of the C-Bend amplitude in degrees and number of movements per
100 ms observed in bortezomib or 27 treated embryos after 48 h.
We also observed a strong difference with regard to the touch
responses of the other treatment groups, a hallmark of sensory
neuropathy. In the control group, the number of mean reflexes per
stimuli is 47%. In bortezomib-treated groups a strong decrease
can be observed (8% for 24 h incubation time, 2% for 48 h; each
25 µM bortezomib) while an increase in touch responsiveness is
observed for 27 (100% for 24 h treated embryos with 25 µM and
50 µM) as well as a comparable responsiveness to control in 25
µM 48 h treated embryos (55%).
Besides the C-bend and touch responsiveness, the total
duration of response as well as the total number of movements
and ultimately, the ratio of number of movements per time is
another important kinematic parameter. This parameter
demonstrates a statistically significant decrease of movements
per 100 ms in all Bortezomib treated groups compared to control
(3.5 ± 0.32) which effects approximately half for 25 µM / 24 h
treated embryos (1.92 ± 0.35, p < 0.01). It is almost completely
diminished for 25 µM / 48 h (0.24 ± 0.32, p < 0.0001) as well as
for non-responding 50 µM / 24 h treated embryos (0 ± 0, p <
0.0001). In strong contrast, mean values of movements per 100
ms for 27-treated embryos are not changed statistically significant
for 25 µM at 24 h (3.9 ± 0.25, p > 0.05) as well as for 48 h
incubation time (4.0 ± 0.26, p > 0.05). Interestingly, embryos
treated with 50 µM 27 for 24 h showed an increased reflex
duration (799 ± 0.92 ms, p < 0.01) as well as an increased number
of movements (30.7 ± 3.3, p < 0.01) but a comparable movement
relative to time (3.9 ± 0.22, p > 0.05).
These results suggest that 27 does not cause off-target effects
in vivo that lead to alterations in touch responsiveness or escape
reflex behavior suggesting the assay as an additional preclinical
safety screen in the development of proteasome and protease
inhibitors.
Conclusions and Outlook
Despite of covalent-reversible inhibitors of the proteasome
represented the first class of proteasome inhibitors, e.g. peptidic
boronic acids and aldehydes, the drug development of reversible
binding warheads remained underexplored during the last
decade, focusing on irreversible acting warheads such as
epoxyketones and sulfonyl fluorides instead.[37-39] Furthermore,
explorations of structure-activity relationships of inhibitors
9
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limit of 100; RMS gradient of 0.001, iteration limit of 1·106 and MM iteration
limit of 500. Amide bond rotation was not allowed and RMSD limit was set
to 0.15. Conformations were then filtered for electrophilic warheads and
tagged as reported in the standard protocol for using DOCKTITE.[29] Thr1
was marked as covalent attachment point and ligands were attached to
targeting the primed site of β5 have been reported occasionally
only. Our group previously reported the identification of ketoamide
7
that displays increased inhibitory activity compared to
unsubstituted phenyl ketoamide 6 and allows for subsequent
modification occupying the S1’ pocket.[20-22]
this nucleophile.
A pharmacophore model of the nucleophile was
This work focuses on bridging the tripeptidic backbone in P1 –
P3 to the primed site P1’ pocket, increasing proteasome inhibition
as well as cytotoxic activity in leukemia cell lines. Phenoxy
substitutions in meta position to the phenyl ketoamide are most
beneficial while reintroduction of the dimethyl group increases
further proteasome inhibition. Remarkably, the occupation of P2
by AspOtBu instead of leucine is, in contrast to reversible peptidic
aldehydes, not beneficial for proteasome inhibition. Inhibition by
ketoamides increases strongly upon substitution of the
carboxybenzyl group by dichloro benzamide in P4. Merging of the
P1’-, P2- and P4-SAR yielded compound 27, the most potent
ketoamide inhibitor in this series. To our surprise, cytotoxic activity
of the inhibitors investigated in this study varies strongly by cell
line. Carfilzomib 3 is the most potent compound tested in the
acute monocytic leukemia-derived cell line MV4-11, whereas 27
is more potent in Jurkat cells than 3, a cell line derived from T-cell
leukemia. Interestingly, both inhibitors show much reduced
activity in THP-1 cells, also derived from acute monocytic
leukemia expressing presumably iCP[31] correlating with more
potent inhibition of β5c over β5i by 3 and 27. Furthermore,
inhibition of cellular conversion of substrate 30 correlates with
altered sensitivity in these cell lines. Our lead compound 27 was
further assessed in an escape response assay using Danio rerio
embryos. While bortezomib leads to a strongly impaired escape
response and movement behavior in treated embryos, behavior
in 27-treated embryos remains largely unchanged. This indicates
that neurotoxicity resulting from off-target inhibition such as
Htra2/Omi, as observed for bortezomib 1, is reduced.
Further work expanding the field of reversible covalent
inhibition by targeting the primed site of proteasome subunits
should focus on substitution of the amino acid side chains in P1
and P2 as well as substitution of the phenoxy group in P1’. As
reported recently, small hydrophobic P1 residues such as alanine
favor selectivity of binding to β5c rather than β5i while potency is
decreasing.[40] We therefore assume that selectivity to either β5i
or β5c might be achieved most effectively if the non-conserved
residues in P1’ are targeted as well (β5c: Ser116, Glu117; β5i:
Glu116, His117).
Altogether the approach we refer to in this work uses iterative
molecular modeling, synthesis and cellular profiling that highlight
the use of covalent reversible inhibitors targeting the proteasome
as effective as irreversible inhibitors enabling pharmacokinetic
and pharmacodynamic altered drug development approaches in
principle.
generated automatically defining the position of Thr1 by elements (d = 0.4
Å, 7 atoms). Thr1 as well as the neighboring amine in Thr2 were deleted
to avoid steric clash during docking of sidechain-attached ligands. The
pharmacophore was enhanced by 3 features defined by inhibitor 7 placed
in PDB: 4r02. Docking was performed using the pharmacophore
placement method with prior conformational sampling of the input structure
(up to 5000 conformations), pharmacophore filtering and refinement of up
to 100 top-scored poses (London dG scoring). Further refinement was
performed using the GBVI/WSA dG scoring function (force constant 1e9).
Docked poses were cleaved from the nucleophilic side chain and rescored
using the DSX standalone version in Linux as described before.[29]
Chemistry
General remarks. All reactions requiring anhydrous conditions were
performed in dried glassware under argon atmosphere. All reagents and
solvents were obtained from commercial suppliers without further
purification. IBX, MG132 4 and BSc2118 5 were synthesized as described
before.[20-21] NMR spectra were recorded with a Bruker AR 300 (300 MHz
1H and 75 MHz 13C) and a Bruker DRX 500 (500 MHz 1H, 126 MHz 13C,
471 MHz 19F and 160 MHz 11B). Deuterated solvents were used as internal
standard. The δ values are reported in parts per million (ppm) downfield
from TMS and were referenced to the residual solvent signal (CDCl3,
DMSO-d6). Coupling constants J are given in Hertz (Hz). The spectra were
analyzed using MestReNova 11 (Mestrelab Research). ESI-MS spectra
were recorded on a Bruker Daltonics qTOF spectrometer. Ionization was
achieved by an electron-spray-ionization source (ESI). HPLC was
performed using an Agilent 1100 system with a Phenomenex synergi polar
reversed phase column (4 µm particle size, 150 x 3.0 mm, pore size 80Å)
connected to a variable wavelength detector. The mobile phase consists
of water/acetonitrile + 0.1% trifluoro acetic acid forming a linear gradient
starting with 30% water (held for 1 min) increased to 90% acetonitrile within
10 min and held for
1 min with a constant flow of 1 mL/min.
Chromatography was performed using flash chromatography of the
indicated solvent system on 40–63 µm silica gel (VWR). Thin-layer
chromatography was carried out on 0.2 mm silica gel 60 plates (F-254
Merck). They were detected by UV light (254 and 365 nm). All compounds
used in biochemical assays have a purity of more than 95% as determined
by HPLC and are reported at the corresponding experiments.
Synthesis of ꢇ -Ketophenylamides (GP i). The corresponding
peptidic aldehyde (1.0 equiv) and aromatic isocyanides (1.5 equiv) were
dissolved in a minimal amount of CH2Cl2 and cooled to 0°C under an
atmosphere of argon. A mixture of trifluoroacetic acid (2.0 equiv) in CH2Cl2
was added dropwise and the mixture was stirred for 2 h at 0°C and 24 h at
room temperature. Completion of the reaction was monitored by HPLC and
TLC. CH2Cl2 was added and the organic layer was washed with 0.1 N aq.
HCl (3x), sat. aq. NaHCO3 (3x) and sat. aq. NaCl. The organic extract was
dried over Na2SO4 and the solvent was evaporated under reduced
pressure. The resulting solid was dissolved in DMSO and IBX (2.0 equiv)
was added. The reaction mixture was stirred 24 h at rt. After completion
CH2Cl2 was added and the mixture was washed with water (3x), sat. aq.
NaHCO3 (3x) and sat. aq. NaCl. The organic extract was dried over
Na2SO4 and the solvent was evaporated under reduced pressure.
Purification was done by column chromatography of the residue on silica
gel (cyclohexane / AcOEt 2:1).
Experimental Section
Molecular Modeling
Structural analysis of BSc4999 (8) and derivatization. Molecular
modeling was performed using the Molecular Operating Environment
(Version 2016.0802, Chemical Computing Group). BSc4999 (7) in
complex with the yeast proteasome was derived from the protein data bank
(PDB: 4r02) and prepared using the LigX function without refinement
(Figure S26 – S34). Subunits β4 (chain L), β5 (chain K) and β6 (chain J)
as well as ligands and waters in these subunits were isolated.
Derivatization of the initial N-terminal Cbz-group as well as 2-, 3-, and 4-
phenoxy substitution of the phenyl ketoamide were built starting from the
BSc4999-CP complex and energy-minimized while placed the CP β5
subunit using the Amber12:EHT forcefield.
Oxidation of alcohols via IBX (GP ii). The corresponding peptidic
alcohol (1.0 equiv) was dissolved in DMSO and IBX (1.5 equiv) was added.
The mixture was stirred overnight at rt. Completion of the reaction was
monitored by HPLC and TLC. CH2Cl2 was added and the organic layer
was washed with water (3x), sat. aq. NaHCO3 (3x) and sat. aq. NaCl. The
organic extract was dried over Na2SO4 and the solvent was evaporated
under reduced pressure.
Peptide synthesis via HATU (GP iii). The carboxylic acid (1.0 equiv)
was dissolved in DMF and HATU (1.1 equiv) was added. The mixture was
stirred for 20 min. at rt before the amine (1.0 equiv) and DIPEA (2.9 equiv)
were added. The reaction mixture was stirred overnight at rt. Completion
of the reaction was monitored by HPLC and TLC. CH2Cl2 was added and
the organic layer was washed with 0.1 N aq. HCl (5x), 0.1 N aq. NaOH (3x)
and sat. aq. NaCl. The organic extract was dried over Na2SO4 and the
solvent was evaporated under reduced pressure. If necessary, purification
was done by column chromatography of the residue on silica gel.
Covalent Docking. The lead compound 27 was docked using a
methodology that was validated by docking of 7 (Figure S35 – S38).
Conformational search of compound 7 and 27 was performed using the
built-in
conformational
search
method
of
MOE2016.0802.[41]
Conformations were sampled using the stochastic method with a rejection
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equiv), trifluoroacetic acid (111 μL, 164 mg, 1.44 mmol, 2.0 equiv) and IBX
(404 mg, 1.44 mmol, 2.0 equiv) were reacted according to GP i to afford
12 (168 mg, 0.235 mmol, 33%) as a pale yellow amorphous solid. Rf = 0.32
(cyclohexane / AcOEt = 2:1). HPLC (254 nm, VWD): tR = 9.09 min
(96.18%). 1H-NMR (500 MHz, CDCl3): d = 8.66 – 8.57 (m, 1H), 7.91 – 7.81
(m, 1H), 7.37 – 7.28 (m, 5H), 7.28 – 7.22 (m, 2H), 7.15 – 7.11 (m, 1H),
6.98 (t, J = 7.3 Hz, 1H), 6.88 – 6.83 (m, 1H), 6.75 – 6.71 (m, 2H), 6.51 –
6.42 (m, 1H), 5.45 – 5.37 (m, 1H), 5.25 – 5.18 (m, 1H), 5.14 – 5.06 (m,
2H), 4.56 – 4.45 (m, 1H), 4.23 – 4.13 (m, 1H), 2.12 – 2.03 (m, 6H), 1.82 –
1.57 (m, 6H), 1.57 – 1.48 (m, 3H), 1.04 – 0.99 (m, 3H), 0.92 (s, 15H). 13C-
NMR (126 MHz, CDCl3): d = 196.8, 172.5, 171.7, 157.8, 156.9, 151.2,
136.2, 133.5, 129.9, 129.1, 128.7, 128.4, 128.2, 121.7, 118.9, 114.8, 67.6,
67.4, 54.3, 53.9, 53.0, 51.7, 51.5, 41.3, 40.6, 40.3, 25.4, 25.0, 24.9, 23.4,
23.1, 23.0, 22.2, 21.6, 16.5, 10.7. ESI-MS: m/z = 715.41 [M+H]+.
Benzyl-((S)-4-methyl-1-(((S)-4-methyl-1-(((S)-5-methyl-1,2-dioxo-1-
((2-phenoxyphenyl)-amino)hexan-3-yl)amino)-1-oxopentan-2-
yl)amino)-1-oxopentan-2-yl)carbamate (8) Cbz-Leu-Leu-Leu-al (338
mg, 0.711 mmol, 1.0 equiv), 1-isocyano-2-phenoxybenzene (209 mg,
1.07 mmol, 1.5 equiv), trifluoroacetic acid (109 μL, 162 mg, 1.42 mmol, 2.0
equiv) and IBX (398 mg, 1.42 mmol, 2.0 equiv) were reacted according to
GP i to afford 8 (184 mg, 0.268 mmol, 35%) as a pale yellow amorphous
solid. Rf = 0.10 (cyclohexane / AcOEt = 5:1).
HPLC (254 nm, VWD): tR = 9.06 min (98.03%). 1H-NMR (300 MHz, CDCl3):
d = 9.32 (s, 1H), 8.52 – 8.44 (m, 1H), 7.41 – 7.28 (m, 7H), 7.19 – 6.98 (m,
6H), 6.88 – 6.81 (m, 1H), 6.64 – 6.44 (m, 1H), 5.52 – 5.39 (m, 1H), 5.39 –
5.24 (m, 1H), 5.14 – 5.01 (m, 2H), 4.58 – 4.43 (m, 1H), 4.25 – 4.08 (m, 1H),
1.81 – 1.60 (m, 6H), 1.55 – 1.45 (m, 3H), 1.02 – 0.84 (m, 18H). 13C-NMR
(75 MHz, CDCl3): d = 196.1, 172.5, 171.9, 171.7, 156.8, 156.1, 147.0,
136.1, 130.1, 128.7, 128.4, 128.2, 127.9, 125.4, 124.4, 123.8, 120.8, 119.3,
119.2, 117.5, 67.4, 54.2, 52.8, 51.6, 51.5, 41.4, 40.8, 40.6, 40.3, 40.2, 39.9,
27.1, 25.4, 24.9, 23.3, 23.0, 22.2, 22.0, 21.5. ESI-MS: m/z = 687.39 [M+H]+.
Benzyl((S)-4-methyl-1-(((S)-4-methyl-1-(((S)-5-methyl-1-((4-methyl-
3-phenoxyphenyl)-amino)-1,2-dioxohexan-3-yl)amino)-1-oxopentan-
2-yl)amino)-1-oxopentan-2-yl)carbamate (13) Cbz-Leu-Leu-Leu-al (250
mg, 0.526 mmol, 1.0 equiv), 4-isocyano-1-methyl-2-phenoxybenzene (165
mg, 0.789 mmol, 1.5 equiv), trifluoroacetic acid (81 μL, 120 mg,
1.05 mmol, 2.0 equiv) and IBX (295 mg, 1.05 mmol, 2.0 equiv) were
reacted according to GP i to afford 13 (146 mg, 0.208 mmol, 40%) as a
pale yellow amorphous solid. Rf = 0.33 (cyclohexane / AcOEt = 2:1). HPLC
(254 nm, VWD): tR = 9.09 min (98.02%). 1H-NMR (500 MHz, CDCl3): d =
8.59 – 8.55 (m, 1H), 7.39 – 7.28 (m, 8H), 7.21 (dd, J = 8.2, 3.4 Hz, 1H),
7.18 – 7.16 (m, 1H), 7.08 – 7.03 (m, 1H), 6.92 – 6.88 (m, 2H), 6.78 (d, J =
7.3 Hz, 1H), 6.48 – 6.41 (m, 1H), 5.36 – 5.30 (m, 1H), 5.24 – 5.16 (m, 1H),
5.09 (s, 2H), 4.53 – 4.42 (m, 1H), 4.21 – 4.14 (m, 1H), 2.23 – 2.19 (m, 3H),
1.78 – 1.55 (m, 6H), 1.54 – 1.44 (m, 3H), 1.01 – 0.84 (m, 18H). 13C-NMR
(126 MHz, CDCl3): d = 196.6, 172.4, 171.7, 157.7, 156.7, 155.0, 135.3,
131.9, 129.9, 128.7, 128.4, 128.2, 127.3, 122.9, 117.6, 117.5, 115.6,
111.5, 67.6, 67.4, 52.9, 51.7, 41.3, 40.6, 40.5, 40.2, 40.0, 25.5, 25.4, 25.0,
24.9, 23.3, 23.1, 22.9, 22.2, 22.0, 21.5, 16.0. ESI-MS: m/z = 701.39
[M+H]+.
Benzyl-((S)-4-methyl-1-(((S)-4-methyl-1-(((S)-5-methyl-1,2-dioxo-1-
((3-phenoxyphenyl)-amino)hexan-3-yl)amino)-1-oxopentan-2-
yl)amino)-1-oxopentan-2-yl)carbamate (9) Cbz-Leu-Leu-Leu-al (275
mg, 0.578 mmol, 1.0 equiv), 1-isocyano-3-phenoxybenzene (170 mg,
0.871 mmol, 1.5 equiv), trifluoroacetic acid (89 μL, 132 mg, 1.16 mmol, 2.0
equiv) and IBX (325 mg, 1.16 mmol, 2.0 equiv) were reacted according to
GP i to afford 9 (123 mg, 0.179 mmol, 29%) as a pale yellow amorphous
solid. Rf = 0.10 (cyclohexane / AcOEt = 5:1). HPLC (205 nm, VWD): tR
=
9.91 min (97.83%). 1H-NMR (500 MHz, CDCl3): d = 8.64 (s, 1H), 7.39 –
7.27 (m, 10H), 7.15 – 7.11 (m, 1H), 7.03 – 7.00 (m, 2H), 6.82 – 6.76 (m,
2H), 6.47 – 6.41 (m, 1H), 5.35 – 5.30 (m, 1H), 5.17 (d, J = 7.7 Hz, 1H),
5.11 (s, 2H), 4.49 – 4.42 (m, 1H), 4.21 – 4.14 (m, 1H), 1.80 – 1.56 (m, 6H),
1.55 – 1.46 (m, 3H), 1.01 – 0.86 (m, 18H). 13C-NMR (126 MHz, CDCl3): d
= 196.5, 172.5, 171.7, 158.2, 156.9, 137.7, 136.2, 130.4, 130.0, 128.8,
128.5, 128.2, 123.8, 119.3, 115.7, 114.7, 110.6, 67.4, 53.9, 53.0, 51.7,
41.3, 40.5, 40.2, 25.4, 24.9, 23.3, 23.1, 22.9, 22.2, 22.1, 21.5. ESI-MS: m/z
= 687.39 [M+H]+.
Benzyl((S)-4-methyl-1-(((S)-4-methyl-1-(((S)-5-methyl-1-((2-methyl-
3-phenoxyphenyl)-amino)-1,2-dioxohexan-3-yl)amino)-1-oxopentan-
2-yl)amino)-1-oxopentan-2-yl)carbamate (14) Cbz-Leu-Leu-Leu-al (250
mg, 0.526 mmol, 1.0 equiv), 1-isocyano-2-methyl-3-phenoxybenzene
(165 mg, 0.789 mmol, 1.5 equiv), trifluoroacetic acid (81 μL, 120 mg,
1.05 mmol, 2.0 equiv) and IBX (295 mg, 1.05 mmol, 2.0 equiv) were
reacted according to GP i to afford 14 (152 mg, 0.217 mmol, 41%) as a
pale yellow amorphous solid. Rf = 0.35 (cyclohexane / AcOEt = 2:1). HPLC
(254 nm, VWD): tR = 8.98 min (97.81%). 1H-NMR (500 MHz, CDCl3): d =
8.73 – 8.67 (m, 1H), 7.91 – 7.84 (m, 1H), 7.36 – 7.27 (m, 7H), 7.21 – 7.16
(m, 1H), 7.06 (t, J = 7.4 Hz, 1H), 7.00 – 6.97 (m, 1H), 6.92 – 6.87 (m, 2H),
6.80 – 6.76 (m, 1H), 6.61 – 6.51 (m, 1H), 5.45 – 5.38 (m, 1H), 5.34 (t, J =
8.6 Hz, 1H), 5.14 – 5.06 (m, 2H), 4.57 – 4.50 (m, 1H), 4.22 – 4.14 (m, 1H),
2.22 – 2.15 (m, 3H), 1.83 – 1.58 (m, 6H), 1.58 – 1.47 (m, 3H), 1.01 (dt, J
= 11.2, 6.2 Hz, 3H), 0.97 – 0.86 (m, 15H). 13C-NMR (126 MHz, CDCl3): d
= 196.7, 172.6, 171.9, 171.8, 157.8, 156.9, 155.0, 135.8, 129.9, 128.7,
128.4, 128.2, 127.3, 122.9, 121.2, 117.7, 117.5, 117.2, 67.4, 54.2, 53.1,
53.0, 51.7, 51.5, 40.8, 40.2, 25.5, 25.4, 25.0, 24.9, 24.9, 23.3, 23.1, 22.2,
22.0, 21.6, 10.2. ESI-MS: m/z = 701.40 [M+H]+.
Benzyl-((S)-4-methyl-1-(((S)-4-methyl-1-(((S)-5-methyl-1,2-dioxo-1-
((4-phenoxyphenyl)-amino)hexan-3-yl)amino)-1-oxopentan-2-
yl)amino)-1-oxopentan-2-yl)carbamate (10) Cbz-Leu-Leu-Leu-al (275
mg, 0.578 mmol, 1.0 equiv), 1-isocyano-4-phenoxybenzene (170 mg,
0.871 mmol, 1.5 equiv), trifluoroacetic acid (89 μL, 132 mg, 1.16 mmol, 2.0
equiv) and IBX (325 mg, 1.16 mmol, 2.0 equiv) were reacted according to
GP i to afford 10 (106 mg, 0.154 mmol, 25%) as a pale yellow amorphous
solid. Rf = 0.11 (cyclohexane / AcOEt = 5:1). HPLC (205 nm, VWD): tR =
9.23 min (97.85%). 1H-NMR (500 MHz, CDCl3): d = 8.66 (s, 1H), 7.61 –
7.57 (m, 2H), 7.38 – 7.30 (m, 7H), 7.13 – 7.08 (m, 1H), 7.03 – 6.97 (m,
4H), 6.83 – 6.78 (m, 1H), 6.49 – 6.45 (m, 1H), 5.42 – 5.32 (m, 1H), 5.18
(d, J = 7.7 Hz, 1H), 5.11 (s, 2H), 4.51 – 4.43 (m, 1H), 4.18 (br s, 1H), 1.81
– 1.59 (m, 6H), 1.56 – 1.45 (m, 3H), 1.03 – 0.83 (m, 18H). 13C-NMR (126
MHz, CDCl3): d = 196.7, 172.5, 171.8, 157.3, 156.8, 154.7, 136.2, 131.7,
130.0, 128.8, 128.5, 128.2, 123.5, 121.7, 119.7, 118.9, 67.4, 53.1, 51.7,
41.3, 40.6, 40.3, 29.9, 25.4, 24.9, 23.4, 23.1, 22.9, 22.2, 22.1, 21.6. ESI-
MS: m/z = 687.39 [M+H]+.
Benzyl((S)-1-(((S)-1-(((S)-1-((2,4-dimethyl-5-
phenoxyphenyl)amino)-5-methyl-1,2-dioxo-hexan-3-yl)amino)-4-
methyl-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-
Benzyl((S)-4-methyl-1-(((S)-4-methyl-1-(((S)-5-methyl-1-((2-methyl-
5-phenoxyphenyl)-amino)-1,2-dioxohexan-3-yl)amino)-1-oxopentan-
2-yl)amino)-1-oxopentan-2-yl)carbamate (15) Cbz-Leu-Leu-Leu-al (250
mg, 0.526 mmol, 1.0 equiv), 2-isocyano-1-methyl-4-phenoxybenzene (165
mg, 0.789 mmol, 1.5 equiv), trifluoroacetic acid (81 μL, 120 mg,
1.05 mmol, 2.0 equiv) and IBX (295 mg, 1.05 mmol, 2.0 equiv) were
reacted according to GP i to afford 15 (151 mg, 0.215 mmol, 41%) as a
pale yellow amorphous solid. Rf = 0.35 (cyclohexane / AcOEt = 2:1). HPLC
(254 nm, VWD): tR = 9.01 min (98.44%). 1H-NMR (500 MHz, CDCl3): d =
8.62 (d, J = 9.2 Hz, 1H), 7.87 (dd, J = 7.4, 2.5 Hz, 1H), 7.37 – 7.28 (m, 7H),
7.15 – 7.11 (m, 1H), 7.10 – 7.06 (m, 1H), 7.00 – 6.97 (m, 2H), 6.88 – 6.83
(m, 1H), 6.77 – 6.72 (m, 1H), 6.49 – 6.44 (m, 1H), 5.38 – 5.32 (m, 1H),
5.24 (d, J = 7.7 Hz, 1H), 5.13 – 5.06 (m, 2H), 4.55 – 4.43 (m, 1H), 4.20 –
4.12 (m, 1H), 2.28 (s, 1.5H), 2.24 (s, 1.5H), 1.81 – 1.57 (m, 6H), 1.57 –
1.45 (m, 3H), 1.00 – 0.97 (m, 3H), 0.96 – 0.85 (m, 15H). 13C-NMR (126
MHz, CDCl3): d = 196.6, 172.5, 171.9, 171.7, 157.5, 156.7, 155.9, 136.2,
136.1, 135.4, 131.5, 129.8, 128.7, 128.4, 128.2, 128.1, 123.3, 123.1,
118.6, 116.2, 112.7, 112.7, 67.4, 53.1, 53.0, 51.7, 51.5, 40.6, 40.2, 25.5,
25.4, 25.0, 24.9, 24.9, 23.3, 23.3, 23.1, 23.0, 23.0, 22.2, 22.1, 22.0, 21.6,
21.5, 17.0, 16.9. ESI-MS: m/z = 701.40 [M+H]+.
yl)carbama-te (11) Cbz-Leu-Leu-Leu-al (250 mg, 0.527 mmol, 1.0 equiv),
1-isocyano-2,4-dimethyl-5-phenoxybenzene (176 mg, 0.788 mmol,
1.5 equiv), trifluoroacetic acid (81 μL, 120 mg, 1.05 mmol, 2.0 equiv) and
IBX (295 mg, 1.05 mmol, 2.0 equiv) were reacted according to GP i to
afford 11 (122 mg, 0.171 mmol, 32%) as a pale yellow amorphous solid.
Rf = 0.27 (cyclohexane / AcOEt = 2:1). HPLC (254 nm, VWD): tR = 9.13
min (97.81%). 1H-NMR (500 MHz, CDCl3): d = 8.58 – 8.54 (m, 1H), 7.75 –
7.71 (m, 1H), 7.37 – 7.24 (m, 7H), 7.09 – 7.05 (m, 1H), 7.03 – 6.99 (m,
1H), 6.94 – 6.76 (m, 3H), 6.45 – 6.36 (m, 1H), 5.36 – 5.29 (m, 1H), 5.21 –
5.14 (m, 1H), 5.10 (s, 2H), 4.54 – 4.42 (m, 1H), 4.19 – 4.12 (m, 1H), 2.29
– 2.21 (m, 3H), 2.18 – 2.14 (m, 3H), 1.80 – 1.55 (m, 6H), 1.55 – 1.44 (m,
3H), 1.02 – 0.84 (m, 18H). 13C-NMR (126 MHz, CDCl3): d = 196.5, 172.3,
171.7, 171.5, 158.0, 156.4, 152.3, 136.0, 133.1, 132.8, 129.6, 128.6,
128.3, 128.1, 127.7, 124.2, 122.1, 116.6, 113.9, 67.4, 67.2, 54.1, 52.9,
51.5, 40.4, 40.2, 40.0, 39.9, 25.2, 24.7, 23.2, 22.9, 22.0, 21.9, 21.3, 16.8,
15.8. ESI-MS: m/z = 715.41 [M+H]+.
Benzyl((S)-1-(((S)-1-(((S)-1-((2,4-dimethyl-3-
phenoxyphenyl)amino)-5-methyl-1,2-dioxo-hexan-3-yl)amino)-4-
methyl-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-
yl)carbama-te (12) Cbz-Leu-Leu-Leu-al (343 mg, 0.721 mmol, 1.0 equiv),
1-isocyano-2,4-dimethyl-3-phenoxybenzene (242 mg, 1.08 mmol, 1.5
tert-Butyl-(S)-3-((S)-2-(((benzyloxy)carbonyl)amino)-4-
methylpentanamido)-4-(((S)-5-methyl-1,2-dioxo-1-
(phenylamino)hexan-3-yl)amino)-4-oxobutanoate
(16)
Cbz-Leu-
Asp(OtBu)-Leu-al (265 mg, 0.497 mmol, 1.0 equiv), isocyanobenzene (77
11
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10.1002/cmdc.201900472
ChemMedChem
FULL PAPER
mg, 0.746 mmol, 1.5 quiv), trifluoroacetic acid (77 μL, 113 mg, 0.994
mmol, 2.0 equiv) and IBX (278 mg, 0.994 mmol, 2.0 equiv) were reacted
according to GP i to afford 16 (133 mg, 0.204 mmol, 41%) as a pale yellow
amorphous solid. Rf = 0.29 (cyclohexane / AcOEt = 2:1). HPLC (254 nm,
VWD): tR = 8.27 min (98.52%). 1H-NMR (500 MHz, CDCl3): d = 8.65 (s,
1H), 7.64 – 7.60 (m, 2H), 7.38 – 7.25 (m, 9H), 7.19 – 7.15 (m, 1H), 5.44 –
5.37 (m, 1H), 5.18 – 5.14 (m, 1H), 5.13 (d, J = 2.8 Hz, 2H), 4.81 – 4.74 (m,
1H), 4.20 (s, 1H), 2.98 – 2.89 (m, 1H), 2.59 – 2.51 (m, 1H), 1.84 – 1.60 (m,
4H), 1.58 – 1.49 (m, 2H), 1.45 (s, 9H), 1.04 – 0.99 (m, 3H), 0.97 – 0.92 (m,
9H). 13C-NMR (126 MHz, CDCl3): d = 196.6, 172.2, 171.7, 170.5, 156.8,
136.4, 136.1, 129.3, 128.7, 128.5, 128.2, 125.5, 120.0, 82.2, 67.5, 54.2,
53.2, 49.3, 41.4, 40.2, 36.8, 28.2, 25.4, 25.0, 23.4, 23.1, 21.9, 21.5. ESI-
MS: m/z = 653.35 [M+H]+.
were reacted according to GP i to afford 20 (130 mg, 0.175 mmol, 33%)
as a pale yellow amorphous solid. Rf = 0.29 (CH2Cl2 / AcOEt = 2:1). HPLC
(254 nm, VWD): tR = 9.05 min (97.53%). 1H-NMR (500 MHz, CDCl3): d =
8.65 (s, 1H), 7.62 – 7.56 (m, 2H), 7.41 – 7.26 (m, 9H), 7.13 – 7.08 (m, 1H),
7.02 – 6.97 (m, 4H), 5.43 – 5.37 (m, 1H), 5.19 – 5.14 (m, 1H), 5.13 (d, J =
2.8 Hz, 2H), 4.80 – 4.75 (m, 1H), 4.21 (s, 1H), 2.94 (d, J = 17.4 Hz, 1H),
2.60 – 2.52 (m, 1H), 1.82 – 1.61 (m, 4H), 1.56 – 1.49 (m, 2H), 1.45 (s, 9H),
1.03 – 0.99 (m, 3H), 0.97 – 0.92 (m, 9H). 13C-NMR (126 MHz, CDCl3): d =
196.6, 172.2, 171.7, 170.5, 157.4, 156.7, 154.6, 136.1, 131.8, 129.9,
128.7, 128.5, 128.2, 123.5, 121.6, 119.7, 118.8, 82.2, 67.5, 54.2, 53.2,
49.3, 41.4, 40.2, 36.8, 28.2, 25.4, 25.0, 23.4, 23.1, 21.9, 21.5. ESI-MS: m/z
= 745.39 [M+H]+.
tert-Butyl(S)-3-((S)-2-(((benzyloxy)carbonyl)amino)-4-
methylpentanamido)-4-(((S)-1-((2,4-dime-thyl-5-
tert-Butyl-(S)-3-((S)-2-(((benzyloxy)carbonyl)amino)-4-
methylpentanamido)-4-(((S)-1-((2,4-dime-thylphenyl)amino)-5-
methyl-1,2-dioxohexan-3-yl)amino)-4-oxobutanoate (17) Cbz-Leu-
Asp(OtBu)-Leu-al (196 mg, 0.367 mmol, 1.0 equiv), 1-isocyano-2,4-
dimethylbenzene (72 mg, 0.549 mmol, 1.5 equiv), trifluoroacetic acid (56
μL, 83 mg, 0.734 mmol, 2.0 equiv) and IBX (206 mg, 0.734 mmol, 2.0
equiv) were reacted according to GP i to afford 17 (96 mg, 0.141 mmol,
40%) as a pale yellow amorphous solid. Rf = 0.33 (CH2Cl2 / MeOH = 50:1).
HPLC (254 nm, VWD): tR = 9.47 min (97.38%). 1H-NMR (500 MHz, DMSO-
d6): d = 9.96 (s, 1H), 8.22 (d, J = 8.1 Hz, 1H), 8.05 (d, J = 7.3 Hz, 1H), 7.42
(d, J = 8.0 Hz, 1H), 7.39 – 7.27 (m, 6H), 7.22 (d, J = 8.0 Hz, 1H), 7.06 (d,
J = 1.9 Hz, 1H), 7.93 – 6.08 (m, 1H), 5.17 – 5.11 (m, 1H), 5.02 (d, J = 4.9
Hz, 2H), 4.67 – 4.59 (m, 1H), 4.08 – 4.01 (m, 1H), 2.69 (dd, J = 16.1, 5.3
Hz, 1H), 2.26 (s, 3H), 2.14 (s, 3H), 1.74 – 1.41 (m, 6H), 1.38 (s, 9H), 0.92
– 0.82 (m, 12H). 13C-NMR (126 MHz, DMSO-d6): d = 196.9, 172.1, 170.4,
169.1, 159.4, 155.9, 136.9, 135.5, 132.6, 132.0, 130.9, 127.7, 127.6,
126.5, 125.4, 80.2, 65.3, 53.1, 52.2, 49.1, 40.7, 38.4, 37.2, 27.6, 24.4,
24.1, 23.0, 22.9, 21.4, 21.1, 20.5, 17.5. ESI-MS: m/z = 681.37 [M+H]+.
phenoxyphenyl)amino)-5-methyl-1,2-dioxohexan-3-yl)amino)-4-
oxobutanoate (21) Cbz-Leu-Asp(OtBu)-Leu-al (250 mg, 0.468 mmol, 1.0
equiv),
1-isocyano-2,4-dimethyl-5-phenoxybenzene
(157 mg,
0.702 mmol, 1.5 equiv), trifluoroacetic acid (72 μL, 107 mg, 0.936 mmol,
2.0 equiv) and IBX (262 mg, 0.936 mmol, 2.0 equiv) were reacted
according to GP i to afford 21 (207 mg, 0.263 mmol, 56%) as a pale yellow
amorphous solid. Rf = 0.27 (cyclohexane / AcOEt = 2:1). HPLC (254 nm,
VWD): tR = 9.34 min (97.86%). 1H-NMR (500 MHz, CDCl3): d = 8.61 (s,
1H), 7.77 (s, 1H), 7.42 (d, J = 8.2 Hz, 1H), 7.36 – 7.31 (m, 5H), 7.30 – 7.25
(m, 3H), 7.08 (s, 1H), 7.03 – 6.99 (m, 1H), 6.89 – 6.86 (m, 2H), 5.42 – 5.35
(m, 1H), 5.32 (d, J = 7.2 Hz, 1H), 5.12 (s, 2H), 4.82 – 4.75 (m, 1H), 4.24 –
4.19 (m, 1H), 2.95 – 2.87 (m, 1H), 2.61 – 2.52 (m, 1H), 2.27 (s, 3H), 2.16
(s, 3H), 1.80 – 1.64 (m, 4H), 1.58 – 1.49 (m, 2H), 1.45 (s, 9H), 1.00 – 0.97
(m, 3H), 0.96 – 0.92 (m, 9H). 13C-NMR (126 MHz, CDCl3): d = 196.6, 172.2,
171.5, 170.3, 158.1, 156.4, 152.3, 136.1, 133.2, 133.0, 129.7, 128.6,
128.3, 128.1, 127.7, 124.2, 122.2, 116.6, 114.0, 82.0, 67.3, 54.1, 53.0,
49.2, 41.3, 40.0, 36.7, 28.1, 25.3, 24.9, 23.3, 23.0, 21.8, 21.4, 16.9, 15.8.
ESI-MS: m/z = 773.41 [M+H]+.
tert-Butyl-(S)-3-((S)-2-(((benzyloxy)carbonyl)amino)-4-
methylpentanamido)-4-(((S)-5-methyl-1,2-dioxo-1-((2-
(S)-4-Methyl-N-((S)-4-methyl-1-(((S)-4-methyl-1-oxopentan-2-
yl)amino)-1-oxopentan-2-yl)-2-(2-(4-oxoquinazolin-3(4H)-
phenoxyphenyl)amino)hexan-3-yl)amino)-4-oxobutanoate (18) Cbz-
Leu-Asp(OtBu)-Leu-al (180 mg, 0.337 mmol, 1.0 equiv), 1-isocyano-2-
phenoxybenzene (209 mg, 0.506 mmol, 1.5 equiv), trifluoroacetic acid (53
μL, 80.0 mg, 0.675 mmol, 2.0 equiv) and IBX (189 mg, 0.675 mmol, 2.0
equiv) were reacted according to GP i. Column chromatography was done
with a mixture of CH2Cl2 / MeOH (400:1) to afford 18 (28.0 mg,
yl)acetamido)pentanamide (22) (S)-N-((S)-1-Hydroxy-4-methylpentan-
2-yl)-4-methyl-2-((S)-4-methyl-2-(2-(4-oxoquinazolin-3(4H)yl)acet-
amido)pen-tanamido)pentanamide (130 mg, 0.245 mmol, 1.0 equiv) and
IBX (103 mg, 0.368 mmol, 1.5 equiv) in 6 mL of DMSO were reacted
according to GP ii to afford 22 (109 mg, 0.207 mmol, 84%) as a colorless
amorphous solid. Rf = 0.13 (cyclohexane / AcOEt = 1:5). HPLC (254 nm,
VWD): tR = 4.97 min (97.80%). 1H-NMR (500 MHz, DMSO-d6): d = 9.32 (s,
1H), 8.61 (d, J = 7.8 Hz, 1H), 8.29 (s, 1H), 8.11 (dd, J = 8.0, 1.5 Hz, 1H),
8.03 (d, J = 7.3 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.84 (ddd, J = 8.5, 7.1,
1.6 Hz, 1H), 7.70 (dd, J = 8.2, 1.0 Hz, 1H), 7.56 (ddd, J = 8.1, 7.2, 1.2 Hz,
1H), 4.71 (s, 2H), 4.36 – 4.26 (m, 2H), 4.04 – 3.98 (m, 1H), 1.69 – 1.58 (m,
2H), 1.57 – 1.41 (m, 6H), 1.36 – 1.29 (m, 1H), 0.91 – 0.89 (m, 6H), 0.87 –
0.84 (m, 6H), 0.80 – 0.78 (m, 3H), 0.75 – 0.73 (m, 3H). 13C-NMR (126
MHz, DMSO-d6): d = 201.2, 172.3, 171.4, 166.9, 160.3, 148.5, 148.1,
134.4, 127.2, 127.0, 125.9, 121.4, 56.4, 51.4, 51.1, 48.2, 40.8, 40.4, 36.2,
24.2, 24.1, 23.8, 23.0, 22.9, 22.8, 21.7, 21.1. ESI-MS: m/z = 528.33
[M+H]+.
0.038 mmol, 11%) as a colorless amorphous solid. Rf = 0.31 (CH2Cl2
/
MeOH = 50:1). HPLC (254 nm, VWD): tR = 9.27 min (99.12%). 1H-NMR
(500 MHz, CDCl3): d = 9.33 (s, 1H), 8.49 (dd, J = 8.1, 1.7 Hz, 1H), 7.38 –
7.30 (m, 8H), 7.23 (d, J = 7.7 Hz, 1H), 7.18 – 7.14 (m, 1H), 7.12 (td, J =
7.8, 1.5 Hz, 1H), 7.07 (dd, J = 8.0, 1.7 Hz, 1H), 7.05 – 7.02 (m, 2H), 6.86
(dd, J = 8.1, 1.5 Hz, 1H), 5.48 – 5.40 (m, 1H), 5.16 – 5.08 (m, 2H), 4.78 –
4.73 (m, 1H), 4.23 – 4.17 (m, 1H), 2.93 (d, J = 17.2 Hz, 1H), 2.58 – 2.50
(m, 1H), 1.80 – 1.60 (m, 6H), 1.58 – 1.45 (m, 2H), 1.44 (s, 9H), 1.03 – 0.99
(m, 3H), 0.98 – 0.90 (m, 9H). 13C-NMR (126 MHz, CDCl3): d = 196.1, 172.1,
171.7, 170.4, 156.7, 156.2, 145.0, 136.1, 130.1, 128.7, 128.4, 128.2,
128.0, 125.4, 124.4, 123.9, 120.8, 119.2, 117.6, 82.2, 67.4, 54.1, 53.1,
49.2, 41.5, 40.3, 36.8, 28.1, 25.4, 25.0, 23.4, 23.1, 21.9, 21.5. ESI-MS: m/z
= 745.40 [M+H]+.
N-((S)-4-Methyl-1-(((S)-4-methyl-1-(((S)-4-methyl-1-oxopentan-2-
yl)amino)-1-oxopentan-2-yl)amino)-1-oxopentan-2-yl)pyrazine-2-
carboxamide (23) N-((S)-1-(((S)-1-(((S)-1-Hydroxy-4-methylpentan-2-
yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-
yl)pyrazine-2-carboxamide (120 mg, 0.267 mmol, 1.0 equiv) and IBX
(112 mg, 0.400 mmol, 1.5 equiv) in 6 mL of DMSO were reacted according
to GP ii to afford 23 (107 mg, 0.239 mmol, 90%) as a colorless amorphous
solid. Rf = 0.34 (cyclohexane / AcOEt = 1:5). HPLC (254 nm, VWD): tR =
tert-Butyl-(S)-3-((S)-2-(((benzyloxy)carbonyl)amino)-4-
methylpentanamido)-4-(((S)-5-methyl-1,2-dioxo-1-((3-
phenoxyphenyl)amino)hexan-3-yl)amino)-4-oxobutanoate (19) Cbz-
Leu-Asp(OtBu)-Leu-al (280 mg, 0.525 mmol, 1.0 equiv), 1-isocyano-3-
phenoxybenzene (154 mg, 0.787 mmol, 1.5 equiv), trifluoroacetic acid (81
μL, 120 mg, 1.05 mmol, 2.0 equiv) and IBX (294 mg, 1.05 mmol, 2.0 equiv)
were reacted according to GP i to afford 19 (153 mg, 0.205 mmol, 39%)
as a pale yellow amorphous solid. Rf = 0.23 (cyclohexane / AcOEt = 2:1).
HPLC (254 nm, VWD): tR = 9.12 min (99.08%). 1H-NMR (500 MHz, CDCl3):
d = 8.63 (s, 1H), 7.38 – 7.27 (m, 11H), 7.15 – 7.10 (m, 1H), 7.03 – 6.99 (m,
2H), 6.82 – 6.78 (m, 1H), 5.39 – 5.33 (m, 1H), 5.18 – 5.13 (m, 1H), 5.12
(d, J = 2.0 Hz, 2H), 4.80 – 4.73 (m, 1H), 4.19 (s, 1H), 2.97 – 2.88 (m, 1H),
2.58 – 2.50 (m, 1H), 1.79 – 1.63 (m, 4H), 1.56 – 1.47 (m, 2H), 1.45 (s, 9H),
1.00 – 0.97 (m, 3H), 0.97 – 0.91 (m, 9H). 13C-NMR (126 MHz, CDCl3): d =
196.3, 172.0, 171.5, 170.3, 158.0, 156.7, 156.6, 137.6, 136.0, 130.2,
129.8, 128.6, 128.3, 128.1, 123.6, 119.1, 115.5, 114.6, 110.5, 82.0, 67.3,
54.0, 52.9, 49.1, 41.3, 39.9, 36.6, 28.0, 25.2, 24.8, 23.2, 23.0, 21.7, 21.4.
ESI-MS: m/z = 745.39 [M+H]+.
1
4.62 min (98.99%). H-NMR (500 MHz, CDCl3): d = 9.54 (s, 1H), 9.37 (d,
J = 1.5 Hz, 1H), 8.77 (d, J = 2.5 Hz, 1H), 8.55 – 8.53 (m, 1H), 8.15 (d, J =
8.2 Hz, 1H), 6.77 – 6.69 (m, 2H), 4.69 – 4.61 (m, 1H), 4.50 – 4.45 (m, 2H),
1.80 – 1.65 (m, 6H), 1.63 – 1.43 (m, 3H), 0.98 – 0.92 (m, 12H), 0.90 – 0.86
(m, 3H), 0.86 – 0.83 (m, 3H). 13C-NMR (126 MHz, CDCl3): d = 199.5, 172.0,
171.9, 163.5, 147.8, 144.6, 144.0, 142.9, 57.4, 52.2, 52.1, 41.1, 40.6, 37.9,
25.0, 24.9, 24.9, 23.2, 23.0, 22.9, 22.2, 22.0. ESI-MS: m/z = 482.24
[M+H]+.
2,5-Dichloro-N-((S)-4-methyl-1-(((S)-4-methyl-1-(((S)-4-methyl-1-
oxopentan-2-yl)-amino)-1-oxo-pentan-2-yl)amino)-1-oxopentan-2-
yl)benzamide
(24)
2,5-Dichloro-N-((S)-1-(((S)-1-(((S)-1-hydroxy-4-
tert-Butyl-(S)-3-((S)-2-(((benzyloxy)carbonyl)amino)-4-
methylpentanamido)-4-(((S)-5-methyl-1,2-dioxo-1-((4-
phenoxyphenyl)amino)hexan-3-yl)amino)-4-oxobutanoate (20) Cbz-
Leu-Asp(OtBu)-Leu-al (270 mg, 0.525 mmol, 1.0 equiv), 1-isocyano-4-
phenoxybenzene (154 mg, 0.787 mmol, 1.5 equiv), trifluoroacetic acid (81
μL, 130 mg, 1.14 mmol, 2.0 equiv) and IBX (294 mg, 1.05 mmol, 2.0 equiv)
methylpentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-
oxopentan-2-yl)benzamide (820 mg, 1.59 mmol, 1.0 equiv) and IBX
(667 mg, 2.38 mmol, 1.5 equiv) in 20 mL of DMSO were reacted according
to GP ii to afford 24 (722 mg, 1.40 mmol, 88%) as a colorless amorphous
solid. Rf = 0.60 (cyclohexane / AcOEt = 1:2). HPLC (205 nm, VWD): tR =
7.20 min (97.85%). 1H-NMR (500 MHz, CDCl3): d = 9.50 (s, 1H), 7.47 (t, J
12
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900472
ChemMedChem
FULL PAPER
= 1.4 Hz, 1H), 7.30 (d, J = 1.5 Hz, 2H), 7.15 – 7.10 (m, 2H), 7.01 (d, J =
7.5 Hz, 1H), 4.82 – 4.73 (m, 1H), 4.67 – 4.59 (m, 1H), 4.39 – 4.32 (m, 1H),
1.78 – 1.54 (m, 8H), 1.44 – 1.37 (m, 1H), 0.98 – 0.85 (m, 18H). 13C-NMR
(126 MHz, CDCl3): d = 199.6, 172.2, 171.7, 165.4, 136.1, 133.3, 131.5,
129.9, 129.3, 57.4, 52.6, 51.9, 41.4, 41.2, 37.6, 25.1, 25.0, 24.8, 23.2,
23.0, 22.9, 22.5, 21.9. ESI-MS: m/z = 514.23 [M+H]+.
CDCl3): d = 196.7, 171.7, 171.3, 170.2, 165.9, 156.6, 135.9, 135.5, 133.4,
131.8, 131.6, 131.4, 131.4, 130.2, 129.0, 128.6, 127.5, 121.6, 82.2, 53.3,
53.2, 49.4, 41.0, 40.2, 36.7, 28.1, 25.3, 25.1, 23.3, 23.1, 21.8, 21.3, 21.0,
17.5. ESI-MS: m/z = 719.30 [M+H]+.
tert-Butyl
(S)-3-((S)-2-(2,5-dichlorobenzamido)-4-
methylpentanamido)-4-(((S)-1-((2,4-dimethyl-5-
tert-Butyl
(S)-3-((S)-2-(2,5-dichlorobenzamido)-4-
phenoxyphenyl)amino)-5-methyl-1,2-dioxohexan-3-yl)amino)-4-
oxobuta-noate (29) Compound 25 (260 mg, 0.454 mmol, 1.0 equiv), 1-
isocyano-2,4-dimethyl-5-phenoxybenzene (152 mg, 0.681 mmol, 1.5
equiv), trifluoroacetic acid (70 μL, 104 mg, 0.908 mmol, 2.0 equiv) in 8 mL
of CH2Cl2 and IBX (254 mg, 0.908 mmol, 2.0 equiv) in 5 mL of DMSO were
reacted according to GP i to afford 29 (184 mg, 0.227 mmol, 50%) as a
pale yellow amorphous solid. Rf = 0.21 (cyclohexane / AcOEt = 2:1). HPLC
(254 nm, VWD): tR = 9.49 min (99.28%).
1H-NMR (500 MHz, CDCl3): d = 8.64 (s, 1H), 7.76 (s, 1H), 7.71 – 7.69 (m,
1H), 7.61 (d, J = 8.3 Hz, 1H), 7.34 (d, J = 1.4 Hz, 2H), 7.32 – 7.26 (m, 3H),
7.08 (s, 1H), 7.03 – 6.98 (m, 1H), 6.90 – 6.86 (m, 2H), 6.84 (d, J = 6.9 Hz,
1H), 5.46 – 5.39 (m, 1H), 4.84 – 4.78 (m, 1H), 4.70 – 4.63 (m, 1H), 2.97
(dd, J = 17.2, 4.2 Hz, 1H), 2.59 (dd, J = 17.2, 6.5 Hz, 1H), 2.27 (s, 3H),
2.16 (s, 3H), 1.84 – 1.77 (m, 2H), 1.77 – 1.67 (m, 3H), 1.55 – 1.49 (m, 1H),
1.45 (s, 9H), 1.02 – 0.96 (m, 9H), 0.88 – 0.85 (m, 3H). 13C-NMR (126 MHz,
CDCl3): d = 196.6, 171.6, 171.3, 170.1, 165.9, 158.1, 156.4, 152.3, 135.9,
133.3, 133.1, 133.0, 131.6, 131.4, 130.1, 129.7, 129.0, 127.7, 124.3,
122.1, 116.6, 114.0, 82.2, 53.3, 53.0, 49.4, 41.0, 40.0, 36.6, 28.0, 25.2,
25.0, 23.2, 23.1, 21.7, 21.3, 16.9, 15.8. ESI-MS: m/z = 811.32 [M+H]+.
methylpentanamido)-4-(((S)-4-methyl-1-oxopen-tan-2-yl)amino)-4-
oxobutanoate (25) tert-Butyl (S)-3-((S)-2-(2,5-dichlorobenzamido)-4-
methylpentanamido)-4-(((S)-1-hydroxy-4-methylpentan-2-yl)amino)-4-
oxobutanoate (700 mg, 1.22 mmol, 1.0 equiv) and IBX (512 mg,
1.83 mmol, 1.5 equiv) in 8 mL of DMSO were reacted according to GP ii
to afford 25 (537 mg, 0.938 mmol, 77%) as a colorless amorphous solid.
Rf = 0.15 (cyclohexane / AcOEt = 2:1). HPLC (205 nm, VWD): tR = 7.53
min (96.63%). 1H-NMR (500 MHz, CDCl3): d = 9.53 (s, 1H), 7.75 – 7.74
(m, 1H), 7.58 (d, J = 8.3 Hz, 1H), 7.37 – 7.36 (m, 2H), 7.18 (d, J = 7.7 Hz,
1H), 6.58 (d, J = 6.4 Hz, 1H), 4.84 – 4.78 (m, 1H), 4.59 – 4.54 (m, 1H),
4.44 – 4.39 (m, 1H), 3.05 (dd, J = 17.3, 3.9 Hz, 1H), 2.58 (dd, J = 17.2, 6.2
Hz, 1H), 1.87 – 1.75 (m, 2H), 1.72 – 1.59 (m, 4H), 1.44 (s, 9H), 1.03 – 0.99
(m, 6H), 0.91 – 0.89 (m, 3H), 0.86 (d, J = 6.4 Hz, 3H). 13C-NMR (126 MHz,
CDCl3): d = 199.9, 172.0, 171.2, 170.7, 166.2, 135.7, 133.6, 131.9, 131.6,
130.4, 129.0, 82.6, 57.6, 53.7, 49.6, 40.9, 37.5, 36.5, 28.2, 25.2, 24.7,
23.2, 21.7. ESI-MS: m/z = 572.23 [M+H]+.
2,5-Dichloro-N-((S)-1-(((S)-1-(((S)-1-((2,4-dimethylphenyl)amino)-5-
methyl-1,2-dioxohexan-3-yl)amino)-4-methyl-1-oxopentan-2-
yl)amino)-4-methyl-1-oxopentan-2-yl)-benzamide (26) Compound 24
(425 mg, 0.826 mmol, 1.0 equiv), 1-isocyano-2,4-dimethylbenzene (163
mg, 1.24mmol, 1.5 equiv), trifluoroacetic acid (127 μL, 188 mg, 1.65 mmol,
2.0 equiv) in 10 mL of CH2Cl2 and IBX (462 mg, 1.65 mmol, 2.0 equiv) in
5 mL of DMSO were reacted according to GP 4 to afford 26 (184 mg, 0.268
mmol, 17%) as a colorless amorphous solid. Rf = 0.32 (cyclohexane /
AcOEt = 2:1). HPLC (254 nm, VWD): tR = 8.43 min (97.54%). 1H-NMR (500
MHz, CDCl3): d = 9.95 (s, 1H), 8.69 (d, J = 8.0 Hz, 1H), 8.25 (d, J = 7.0 Hz,
1H), 7.94 (d, J = 8.3 Hz, 1H), 7.53 (s, 2H), 7.43 (s, 1H), 7.22 (d, J = 8.0
Hz, 1H), 7.06 (s, 1H), 7.00 (d, J = 8.1 Hz, 1H), 5.16 – 5.07 (m, 1H), 4.51 –
4.40 (m, 2H), 2.26 (s, 3H), 2.15 (s, 3H), 1.75 – 1.62 (m, 3H), 1.61 – 1.43
(m, 6H), 0.96 – 0.83 (m, 18H). 13C-NMR (126 MHz, CDCl3): d = 197.1,
172.0, 171.1, 164.8, 159.6, 138.0, 135.4, 132.5, 132.0, 131.4, 131.3,
130.8, 130.5, 128.7, 128.5, 126.5, 125.3, 52.1, 51.7, 50.5, 41.0, 39.9, 38.2,
24.5, 24.2, 24.0, 23.0, 23.0, 21.7, 21.5, 21.0, 20.5, 17.5. ESI-MS: m/z =
661.30[M+H]+.
(S)-3-((S)-2-((S)-2-(2-(4-(2,8-Diethyl-5,5-difluoro-1,3,7,9-
tetramethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-
yl)phenoxy)acetamido)-4-methylpentanami-do)-4-
methylpentanamido)-5-methyl-2-oxo-N-(3-
phenoxyphenyl)hexanamide
(32)
(3S)-3-((S)-2-((S)-2-Amino-4-
methylpentanamido)-4-methylpentanamido)-2-hydroxy-5-methyl-N-(3-
phenoxyphenyl)hexanamide (87 mg, 0.159 mmol, 1.1 equiv), 2-(4-(2,8-
diethyl-5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-
f][1,3,2]diazaborinin-10-yl)phe-noxy)acetic acid (66 mg, 0.145 mmol, 1.0
equiv), HATU (60 mg, 0.159 mmol, 1.1 equiv), and DIPEA (72 μL, 54 mg,
0.421 mmol, 2.9 equiv) in 10 mL of DMF were reacted according to GP iii.
The crude coupling product was directly taken up in 4 mL of DMSO und
oxidized with IBX (81 mg, 0.290 mmol, 2.0 equiv). Column
chromatography of the residue on silica (cyclohexane / AcOEt 2:1)
afforded 32 (48 mg, 0.0485 mmol, 33%) as a purple amorphous solid. Rf =
0.47 (cyclohexane / AcOEt = 1:1). HPLC (360 nm, VWD): tR = 10.59 min
(99.29%). 1H-NMR (500 MHz, CDCl3): d = 8.65 (s, 1H), 7.39 – 7.34 (m,
2H), 7.34 – 7.31 (m, 2H), 7.30 – 7.27 (m, 1H), 7.25 – 7.22 (m, 2H), 7.15 –
7.10 (m, 1H), 7.05 (d, J = 8.6 Hz, 2H), 7.03 – 6.98 (m, 3H), 6.83 – 6.78 (m,
1H), 6.64 (d, J = 7.4 Hz, 1H), 6.53 (d, J = 8.0 Hz, 1H), 5.39 – 5.32 (m, 1H),
4.60 – 4.53 (m, 3H), 4.51 – 4.43 (m, 1H), 2.53 (s, 6H), 2.30 (q, J = 7.6 Hz,
4H), 1.82 – 1.67 (m, 5H), 1.67 – 1.58 (m, 2H), 1.57 – 1.49 (m, 2H), 1.31
(s, 6H), 1.03 – 0.88 (m, 24H). 13C-NMR (126 MHz, CDCl3): d = 196.5,
171.7, 171.6, 168.4, 168.2, 158.3, 157.5, 156.8, 154.0, 139.5, 138.3,
137.6, 133.0, 131.2, 130.4, 130.1, 130.0, 123.9, 119.3, 119.3, 115.7,
115.4, 114.7, 110.6, 67.3, 53.1, 51.8, 51.6, 41.1, 40.7, 40.4, 25.6, 25.5,
25.1, 25.0, 24.9, 23.3, 23.1, 23.0, 22.9, 22.3, 22.3, 22.2, 22.1, 21.6, 17.2,
14.8, 12.7, 12.0. 19F NMR (471 MHz, CDCl3): d = -145.81 (dd, J = 66.5,
32.1 Hz). ESI-MS: m/z = 969.53 [M-F]+.
2,5-Dichloro-N-((S)-1-(((S)-1-(((S)-1-((2,4-dimethyl-5-
phenoxyphenyl)amino)-5-methyl-1,2-dioxo-hexan-3-yl)amino)-4-
methyl-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-
yl)benzamide (27) Compound 24 (294 mg, 0.572 mmol, 1.0 equiv), 1-
isocyano-2,4-dimethyl-5-phenoxybenzene (200 mg, 0.857 mmol, 1.5
equiv), trifluoroacetic acid (127 μL, 188 mg, 1.65 mmol, 2.0 equiv) in 8 mL
of CH2Cl2 and IBX (462 mg, 1.65 mmol, 2.0 equiv) in 5 mL of DMSO were
reacted according to GP i to afford 27 (136 mg, 0.180 mmol, 31%) as a
pale yellow amorphous solid. Rf = 0.33 (cyclohexane / AcOEt = 2:1). HPLC
(254 nm, VWD): tR = 9.27 min (99.05%). 1H-NMR (500 MHz, CDCl3): d =
8.56 (s, 1H), 7.69 (s, 1H), 7.50 (t, J = 1.5 Hz, 1H), 7.30 – 7.26 (m, 5H),
7.08 (s, 1H), 7.03 – 6.99 (m, 2H), 6.96 (d, J = 7.5 Hz, 1H), 6.89 – 6.86 (m,
2H), 5.32 – 5.26 (m, 1H), 4.79 – 4.72 (m, 1H), 4.63 – 4.56 (m, 1H), 2.27
(s, 3H), 2.16 (s, 3H), 1.77 – 1.60 (m, 7H), 1.58 – 1.50 (m, 1H), 1.49 – 1.41
(m, 1H), 0.98 – 0.87 (m, 18H). 13C-NMR (126 MHz, CDCl3): d = 196.7,
165.4, 158.1, 156.6, 152.5, 136.0, 133.3, 133.2, 133.0, 131.5, 130.1,
129.8, 129.2, 127.9, 124.4, 122.3, 116.8, 114.1, 53.1, 52.6, 51.7, 41.3,
41.1, 40.1, 25.4, 25.0, 24.9, 23.3, 23.0, 22.9, 22.4, 22.4, 21.5, 17.0, 15.9.
ESI-MS: m/z = 753.33 [M+H]+.
(S)-3-((S)-2-((S)-2-(2-(2-(4-(2,8-diethyl-5,5-difluoro-1,3,7,9-
tetramethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-
yl)phenoxy)acetamido)acetamido)-4-methyl-pentanamido)-4-
methylpentan-amido)-5-methyl-2-oxo-N-(3-
phenoxyphenyl)hexanamide
(33)
(3S)-3-((S)-2-((S)-2-Amino-4-
methylpentan-amido)-4-methylpentanamido)-2-hydroxy-5-methyl-N-(3-
phenoxyphenyl)hexanamide (207 mg, 0.373 mmol, 1.5 equiv), (2-(4-(2,8-
Diethyl-5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-
f][1,3,2]diazabo-rinin-10-yl)phenoxy)acetyl)glycine (127 mg, 0.249 mmol,
1.0 equiv), HATU (104 mg, 0.274 mmol, 1.1 equiv), and DIPEA (123 μL,
93 mg, 0.721 mmol, 2.9 equiv) in 10 mL of DMF were reacted according
to GP iii. The crude coupling product was directly taken up in 10 mL of
DMSO und oxidized with IBX (139 mg, 0.498 mmol, 2.0 equiv). Column
chromatography of the residue on silica (cyclohexane / AcOEt 1:1)
afforded 33 (122 mg, 0.117 mmol, 47%) as a purple amorphous solid. Rf =
0.31 (cyclohexane / AcOEt = 1:2). HPLC (360 nm, VWD): tR = 10.20 min
(99.48%). 1H-NMR (500 MHz, DMSO-d6): d = 10.68 – 10.55 (m, 1H), 8.37
– 8.29 (m, 1H), 8.28 – 8.20 (m, 1H), 8.09 – 8.02 (m, 1H), 7.94 (d, J = 8.2
Hz, 1H), 7.60 – 7.55 (m, 1H), 7.53 (t, J = 2.3 Hz, 1H), 7.44 – 7.35 (m, 2H),
7.35 – 7.30 (m, 1H), 7.28 – 7.20 (m, 2H), 7.18 – 7.12 (m, 3H), 7.03 – 6.98
(m, 2H), 6.79 – 6.74 (m, 1H), 5.09 – 4.91 (m, 1H), 4.63 – 4.57 (m, 2H),
4.36 – 4.30 (m, 2H), 3.87 – 3.78 (m, 2H), 2.43 (s, 6H), 2.35 – 2.23 (m, 4H),
1.74 – 1.64 (m, 1H), 1.62 – 1.49 (m, 4H), 1.49 – 1.37 (m, 4H), 1.32 – 1.28
tert-Butyl
(S)-3-((S)-2-(2,5-dichlorobenzamido)-4-
methylpentanamido)-4-(((S)-1-((2,4-dimethyl-phenyl)amino)-5-
methyl-1,2-dioxohexan-3-yl)amino)-4-oxobutanoate (28) Compound
25 (250 mg, 0.437 mmol, 1.0 equiv), 1-isocyano-2,4-dimethylbenzene (86
mg, 0.655 mmol, 1.5 equiv), trifluoroacetic acid (67 μL, 100 mg, 0.874
mmol, 2.0 equiv) in 8 mL of CH2Cl2 and IBX in 5 mL of DMSO (245 mg,
0.874mmol, 2.0 equiv) were reacted according to GP i to afford 28 (138
mg, 0.192 mmol, 44%) as a pale yellow amorphous solid. Rf = 0.19
(cyclohexane / AcOEt = 2:1). HPLC (254 nm, VWD): tR = 8.64 min
(97.46%). 1H-NMR (500 MHz, CDCl3): d = 8.58 (s, 1H), 7.87 (d, J = 8.0 Hz,
1H), 7.68 (t, J = 1.5 Hz, 1H), 7.58 (d, J = 8.3 Hz, 1H), 7.32 (d, J = 1.4 Hz,
2H), 7.30 (d, J = 7.9 Hz, 1H), 7.01 – 6.97 (m, 2H), 6.79 (d, J = 6.9 Hz, 1H),
5.51 – 5.41 (m, 1H), 4.82 – 4.77 (m, 1H), 4.69 – 4.60 (m, 1H), 2.97 (dd, J
= 17.2, 4.1 Hz, 1H), 2.58 (dd, J = 17.2, 6.5 Hz, 1H), 2.27 (s, 3H), 2.23 (s,
3H), 1.82 – 1.72 (m, 4H), 1.72 – 1.64 (m, 1H), 1.56 – 1.48 (m, 1H), 1.43
(s, 9H), 1.00 – 0.97 (m, 9H), 0.88 – 0.86 (m, 3H). 13C-NMR (126 MHz,
13
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900472
ChemMedChem
FULL PAPER
(m, 6H), 0.97 – 0.91 (m, 6H), 0.91 – 0.81 (m, 14H), 0.81 – 0.76 (m, 4H).
13C-NMR (126 MHz, DMSO-d6): d = 196.8, 172.1, 171.6, 168.3, 167.7,
159.7, 158.1, 156.9, 156.3, 152.9, 140.4, 139.0, 138.1, 132.4, 130.3,
130.0, 129.3, 127.4, 123.6, 118.7, 115.4, 115.2, 114.4, 110.4, 66.9, 52.1,
51.0, 50.5, 41.7, 40.9, 40.7, 38.1, 24.4, 24.1, 24.0, 23.0, 22.7, 21.8, 21.6,
21.0, 16.4, 14.5, 12.2, 11.5. 19F NMR (471 MHz, DMSO-d6): d = -143.01
(dd, J = 66.5, 28.6 Hz). 11B NMR (160 MHz, DMSO-d6): d = 3.69 (t, J =
33.4 Hz). ESI-MS: m/z = 1026.56 [M-F]+.
Bottom)/(1+10(X-LogIC50))” using GraphPad Prism 7.02. IC50 values are
determined from technical triplicates as mean value of two independent
experiments. Standard deviations (Table S1) as well as dose response
curves (SI chapter 2b) are given in the supplementary information.
In-cell determination of proteasome IC50. Cellular inhibition of
conversion of proteasome substrate 30 was determined by plating 25.000
cells per well in RPMI-1640 medium supplemented with 0.1% FBS.
Inhibitors were serially diluted in RPMI-1640 medium as 10-fold stocks,
added to cells giving final concentrations and incubated for 0 h – 13 h at
37°C and 5% CO2 (Figure S5 – S8). Substrate was added to give a final
concentration of 30 µM and substrate conversion was detected by
fluorescence measurement over 100 min (assay validation) or 60 min
(inhibition assays) using a Tecan M1000 microplate reader. Rate of
fluorescence change over time was calculated, rates for negative (no-cell)
control was subtracted and normalized to positive control (no inhibitor).
Dose-response curves were fitted to the equation “Y=100/(1+10((LogIC50-
X)*HillSlope))” using GraphPad Prism 7.02 (SI chapter 2b, Figure S2 – S4).
(S)-3-((S)-2-((S)-2-(3-(2-(4-(2,8-diethyl-5,5-difluoro-1,3,7,9-
tetramethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-
yl)phenoxy)acetamido)propanamido)-4-methylpentanamido)-4-
methylpen-tan-amido)-5-methyl-2-oxo-N-(3-phenoxyphenyl)hexan-
amide
(34)
(3S)-3-((S)-2-((S)-2-Amino-4-methylpen-tanamido)-4-
methylpentanamido)-2-hydroxy-5-methyl-N-(3-
phenoxyphenyl)hexanamide (207 mg, 0.373 mmol, 1.5 equiv), 3-(2-(4-
(2,8-Diethyl-5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ4,5λ4-dipyrrolo[1,2-
c:2',1'-f][1,3,2]diaza-borinin10-yl)phenoxy)acetamido)propanoic
acid
(130 mg, 0.249 mmol, 1.0, equiv), HATU (104 mg, 0.274 mmol, 1.1 equiv),
and DIPEA (123 μL, 93 mg, 0.721 mmol, 2.9 equiv) in 10 mL of DMF were
reacted according to GP iii. The crude coupling product was directly taken
up in 10 mL of DMSO und oxidized with IBX (139 mg, 0.498 mmol, 2.0
Time-dependent determination of cell viability. For the
determination of time-dependent cytotoxic activity 5.000 cells per well
were plated in a white 384 well-plate in RPMI-1640 medium supplemented
with 0.1% FBS. Real-Time Glo MT cell viability assay (Promega) and
inhibitor were added to the final concentration and luminescence was
determined using a Tecan M1000 microplate reader at the time points
indicated (SI chapter 2b, Figure S9 – S17, Table S2).
equiv). Column chromatography of the residue on silica (cyclohexane
/
AcOEt 1:2) afforded 34 (75 mg, 0.0707 mmol, 28%) as a purple
amorphous solid. Rf = 0.29 (cyclohexane / AcOEt = 1:5). HPLC (360 nm,
VWD): tR = 10.16 min (98.54%). 1H-NMR (500 MHz, DMSO-d6): d = 10.63
(s, 1H), 8.31 – 8.26 (m, 1H), 8.14 – 8.08 (m, 1H), 8.03 (d, J = 8.0 Hz, 1H),
7.90 – 7.81 (m, 1H), 7.59 – 7.56 (m, 1H), 7.55 – 7.51 (m, 1H), 7.39 (t, J =
8.0 Hz, 2H), 7.35 – 7.30 (m, 1H), 7.27 – 7.23 (m, 2H), 7.17 – 7.11 (m, 3H),
7.01 (d, J = 8.0 Hz, 2H), 6.79 – 6.74 (m, 1H), 5.03 – 4.92 (m, 1H), 4.52 (s,
2H), 4.40 – 4.26 (m, 2H), 3.39 – 3.33 (m, 2H), 2.43 (s, 6H), 2.38 – 2.32 (m,
2H), 2.28 (q, J = 7.5 Hz, 4H), 1.73 – 1.65 (m, 1H), 1.61 – 1.49 (m, 4H),
1.47 – 1.37 (m, 4H), 1.30 (s, 6H), 0.93 (t, J = 7.5 Hz, 6H), 0.91 – 0.85 (m,
5H), 0.85 – 0.77 (m, 13H). 13C-NMR (126 MHz, DMSO-d6): d = 196.8,
172.3, 172.2, 171.8, 171.7, 170.5, 170.4, 167.1, 159.7, 159.5, 158.0,
156.9, 156.3, 152.9, 140.4, 139.0, 138.1, 132.4, 130.3, 130.0, 129.3,
127.4, 123.5, 118.7, 115.4, 115.2, 114.4, 110.4, 67.0, 52.1, 51.0, 50.5,
41.1, 40.8, 40.7, 40.5, 38.1, 35.2, 35.0, 24.4, 24.2, 24.0, 23.0, 23.0, 22.8,
21.7, 21.6, 21.0, 20.8, 16.4, 14.5, 12.2, 11.5. 19F NMR (471 MHz, DMSO-
d6): d = -143.02 (dd, J = 66.5, 28.6 Hz). 11B NMR (160 MHz, DMSO-d6): d
= 3.68 (t, J = 33.8 Hz). ESI-MS: m/z = 1040.57 [M-F]+.
Danio rerio embryo toxicity assay. Fish keeping protocols are
approved by the Darmstadt administrative authority and documented. All
fish were treated humanely. Inhibitors 7, 9, 27 as well as bortezomib and
carfilzomib were prepared at 25 µM, 10 µM, 5 µM and 1 µM inhibitor in E3
medium. For better solubility of the compounds in the media, the wells
contain 1 % v/v DMSO. Embryos were decollated into separate wells
containing a total volume of 200 µL and the inhibitor at the given
concentration.
Danio rerio escape response assay. 24 hours past fertilization
embryos were dechorionated and transferred into 96-well plates
containing 198 µL of E3 media at 26°C. Embryos were treated at 24 h,
48 h or 72 h with bortezomib or compound 27 by adding 2 µL 100-fold
concentrated proteasome inhibitor in DMSO to give a final concentration
of 25 µM or 50 µM. Incubation times were 24 h, 48 h and 72 h. Survival of
the embryos was determined by visual analysis of heart-beat. For
recording the touch evoked escape response, embryos were transferred
to a microscopy slide with cavity that is placed on a SL-300 LED Soft Light
(Dörr, Neu-Ulm, Germany) under a MotionBlitz EoSens mini1-1 MC1370
high speed camera (Mikrotron, Unterschleißheim, Germany, Figure S18).
An escape response was triggered by a touch stimulus at the trunk of
embryos using a small sewing needle, which is slightly blunted. For each
embryo, a maximum of 50 stimuli was applied. Two (in case where only 2
of 4 embryos survived) or three embryos per incubation time and
concentration were assayed. No escape response was determined if only
one embryo was alive at 96 hpf. Movement was recorded at 96 hpf at 500
frames per second for 3 seconds using the MotionBlitzDirector 2 software.
Determination of the amplitude α defined by the body angle spanned
between the head, yolk and tail using a MATLAB-based software tool and
plotted using GraphPad Prism 7.02. Statistical analysis was performed
using the two-sided, unpaired t-test (Parametric test, α = 0.05, 95% CI)
under the Null hypothesis (H0) that there is no difference in means after
treatment and the alternate hypothesis that there is a difference (H1) using
GraphPad Prism 7.02. These data were used to obtain the amplitude and
length of the C-bend, total number of movements and response duration.
Detailed statistical parameters including 95% confidence intervals and R2
values are given in thesupporting information (Tables S4, S5; Figures S19
– S23).
Biology
Proteasome inhibition assay. Bortezomib (Selleckchem) and
Carfilzomib (MedChemExpress) were purchased and used without further
purification. The 20S Proteasome Assay Kit for Drug Discovery was used
according to the supplier’s protocol using Suc-LLVY-AMC (β5), Bz-VGR-
AMC (β2) or Z-LLE-AMC (β1) as substrates (Enzo Life Sciences). Single
point measurements of residual activity of proteasome activity were
performed at a final concentration of 100 nM (n = 3, β5c, β5i) or 1 µM (n =
1, β1c, β2c, β5c, β5i, SI chapter 2a, Table S1). For key compounds 7, 9,
27 and carfilzomib 3 in this study half-maximal inhibitory values were
determined using inhibitor concentrations spanning four orders of
magnitude (β5c, β5i; 5 nM to 10 µM Figure S1). Inhibitors were prepared
in 2-fold concentration (cfinal = 100 nM) and added to proteasomes (mfinal
=
200 ng/well). After 30 min incubation, substrate was added (cfinal = 100
µM), incubated for 60 minutes and fluorescence was read using a Tecan
M1000 microplate reader. Dose-response curves were fitted to the
equation Y=100/(1+10((LogIC50-X)*HillSlope)) using GraphPad Prism 7.02. IC50
values were determined from technical duplicates as mean value of three
independent experiments.
Cell lines. MV4-11 (ACC 102), Jurkat (ACC 282) and THP-1 (ACC 16)
cells were obtained from DSMZ and maintained at 37°C and 5% CO2 at
densities between 0.2 – 1 · 106 cells / ml in RMPI-1640 medium
Fluorescence characterization of BODIPY-labeled ketoamide
proteasome inhibitors. Absorption and fluorescence emission spectra
have been determined in acetonitrile at 10 µM using a Tecan M1000 Pro
microplate reader (Figure S24).
supplemented with 10% FBS, L-glutamine (2 mM) and penicillin
streptomycin.
/
Cell viability endpoint assay. For the determination of cytotoxicity of
proteasome inhibitors 50.000 cells per well were plated in 90 µl RPMI-
1640 medium supplemented with 0.1% FBS and incubated overnight at
37°C and 5% CO2. Inhibitors were serially diluted in RPMI-1640 medium
as 10-fold stocks and added to cells giving the final concentration (1 nM to
10 µM, Figure S2 – S4) and incubated for 72 h at 37°C and 5% CO2. Cell
viability was determined using the Celltiter Blue assay (Promega) by
adding 20 µl of reagent, incubation for 120 minutes at 37°C and 5% CO2
and fluorescent read-out using a Tecan M1000 microplate reader. Dose-
Imaging of embryos treated with BODIPY-conjugated PI. To
analyze the location, where fluorescent proteasome inhibitors are mounted
and metabolized within the zebrafish embryo, dechorionated embryos
were decollated into 96-well-plates containing concentrations of 25 µM
inhibitor in E3 media. 96 hours post fertilization and 72 hours post the start
of embryo´s incubation in the presence of fluorescent inhibitor, the
embryos were placed under an AxioScope A1 fluorescence microscope
equipped with a Nuance Fx multispectral imaging system. Untreated
embryos were investigated using the same exposure time to evaluate the
response curves were fitted to the equation “Y=Bottom
+ (Top-
14
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10.1002/cmdc.201900472
ChemMedChem
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Author Contributions
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The manuscript was written through contributions of all authors. All authors
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contributed equally.
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Notes
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The authors declare no competing financial interest.
The authors declare no conflict of interest.
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§Present address: Center for Systems Biology Dresden, Dresden,
Germany; Max Planck Institute of Molecular Cell Biology and Genetics,
Dresden, Germany.
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Acknowledgements
This work was partially funded by the DFG (GRK1657, project
2E). We thank Marietheres Kleuter, Yannick Kristiansen and Anke
Imrich for technical assistance and Dr. Tobias Meckel and Prof.
Dr. Michael Groll for helpful discussion.
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Keywords: 20S Proteasome • α-Ketoamides • Cancer • Drug
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[33]
Discovery • Ubiquitin
Abbreviations: CP: 20S proteasome core particle; yCP: yeast
proteasome core particle; cCP: constitutive proteasome core
particle; iCP: immunoproteasome core particle; PI: proteasome
inhibitor; MOE: Molecular Operating Environment.
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15
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FULL PAPER
Entry for the Table of Contents
Bridging the gap. Identification of novel covalent reversible binding P1’-site extended α-ketoamide inhibitors for proteasome’s β5
subunit. The development was guided by a new in-cell proteasome inhibition assay and the lead compound was profiled in a Danio
rerio embryo escape response assay showing superior properties in comparison to Bortezomib. BODIPY conjugated activity-based
probes give insight into in-cell localization and distribution in zebrafish embryos.
16
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