Development of Novel Epoxyketone-Based Proteasome Inhibitors as a Strategy to Overcome Cancer Resistance to Carfilzomib and Bortezomib

Article abstract of DOI:10.1021/acs.jmedchem.8b01943

Over the past 15 years, proteasome inhibitors (PIs), namely bortezomib, carfilzomib (Cfz) and ixazomib, have significantly improved the overall survival and quality-of-life for multiple myeloma (MM) patients. However, a significant portion of MM patients do not respond to PI therapies. Drug resistance is present either de novo or acquired after prolonged therapy through mechanisms that remain poorly defined. The lack of a clear understanding of clinical PI resistance has hampered the development of next-generation PI drugs to treat MM patients who no longer respond to currently available therapies. Here, we designed and synthesized novel epoxyketone-based PIs by structural modifications at the P1′ site. We show that a Cfz analog, 9, harboring a hydroxyl substituent at its P1′ position was highly cytotoxic against cancer cell lines displaying de novo or acquired resistance to Cfz. These results suggest that peptide epoxyketones incorporating P1′-targeting moieties may have the potential to bypass resistance mechanisms associated with Cfz and to provide additional clinical options for patients resistant to Cfz.

Full text of DOI:10.1021/acs.jmedchem.8b01943

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Article
Development of novel epoxyketone-based proteasome inhibitors as a
strategy to overcome cancer resistance to carfilzomib and bortezomib
Min Jae Lee, Deepak Bhattarai, Zi Soo Yoo, Zachary Miller, Ji Eun
Park, Sukyeong Lee, Wooin Lee, James J. Driscoll, and Kyung Bo Kim
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01943 • Publication Date (Web): 09 Apr 2019
Downloaded from http://pubs.acs.org on April 9, 2019
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Development of novel epoxyketone-based proteasome inhibitors as a strategy
to overcome cancer resistance to carfilzomib and bortezomib
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Min Jae Lee,1,# Deepak Bhattarai,1,# Zi Soo Yoo, Zach Miller, Ji Eun Park, Sukyeong Lee,
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1,*
Wooin Lee, James J. Driscoll, Kyung Bo Kim
1Department of Pharmaceutical Sciences, University of Kentucky, Lexington, KY 40536;
2College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National
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University, Seoul, Korea 08826; Verna and Marrs McLean Department of Biochemistry and
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Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA; Department of
Internal Medicine, Division of Hematology and Oncology and University of Cincinnati Cancer
Institute, Cincinnati, OH 45267
Corresponding Author
*
Phone: 1-859-257-5301. E-mail: kbkim2@uky.edu
KEYWORDS
Proteasome inhibitor, carfilzomib, multiple myeloma, epoxyketones, drug resistance
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Abstract
Over the past 15 years, proteasome inhibitors (PIs), namely bortezomib, carfilzomib (Cfz) and
ixazomib, have significantly improved the overall survival and quality-of-life for multiple
myeloma (MM) patients. However, a significant portion of MM patients do not respond to PI
therapies. Drug resistance is present either de novo or acquired after prolonged therapy through
mechanisms that remain poorly defined. The lack of a clear understanding of clinical PI resistance
has hampered the development of next-generation PI drugs to treat MM patients who no longer
respond to currently-available therapies. Here, we designed and synthesized novel epoxyketone-
based PIs by structural modifications at the P1′ site. We show that a Cfz analog, 9, harboring a
hydroxyl substituent at its P1′ position was highly cytotoxic against cancer cell lines displaying de
novo or acquired resistance to Cfz. These results suggest that peptide epoxyketones incorporating
P1′-targeting moieties may have the potential to bypass resistance mechanisms associated with Cfz
and to provide additional clinical options for patients resistant to Cfz.
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Introduction
Since the FDA approval of bortezomib (Btz) in 2003, proteasome inhibitors (PIs) (Fig 1A) have
become critical components of many MM treatment regimens. Compared to the pre-PI era, the use
of PIs, either as single agents or in combination with an immunomodulatory agent, has
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significantly improved the 5-year overall survival rate for MM patients. Despite these remarkable
successes, MM remains nearly universally incurable due to de novo or acquired drug resistance.
Since 2015, the FDA has approved three non-PI drugs, daratumumab (a monoclonal antibody
(mAb) targeting CD38), elotuzumab (mAb targeting SLAMF7) and panobinostat (HDAC
inhibitor). Although these non-PI drugs offer additional options for MM patients relapsed on
current PI-based therapies, their response rates vary widely. Results from recent clinical trials show
that responses to these non-PI drugs are rather transient with a median duration of ~7-20 months
before relapsing.2-4 Thus, there is an urgent unmet medical need for new agents which can yield
durable responses for MM patients who have exhausted current treatment options.
A
S3
S1
S1
S3
Cl
S1
S3
O
O
N
N
H
N
H
N
O
O
O
H
N
N
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H
N
OH
H
H
N
O
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OH
N
B
N
B
H
H
O
OH
Cl
O
OH
S2
S2
S4
Bortezomib
Carfilzomib
S1
Ixazomib
B
S3
Cleavage site
O
P3
O
P1
O
H
H
N
H
N
N
N
N
N
Under-explored by
existing proteasome
inhibitors
H
H
H
O
P4
O
P2
O
P1'
S4
S2
S1′
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Figure 1. A. Structures of proteasome inhibitors in clinical use. The gray-colored markings denote the functional
groups that are proposed to form favorable interactions with the specificity pockets (S1-4) of a proteasome catalytic
subunit. B. Schematic representation of a prototypical proteasome substrate or substrate-like inhibitor bound to a
proteasome catalytic subunit showing the unprimed residues (P1, P2, P3, and P4) N-terminal to the cleavage site of
the proteasome catalytic subunit (shown as red arrows) and the primed P1′ residue C-terminal to the cleavage site.
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The ubiquitin-proteasome system (UPS) is responsible for maintaining protein homeostasis via
controlled degradation of numerous intracellular proteins. The UPS furthermore plays a critical
role in regulating cellular signaling pathways and several key processes described as the original
5
hallmarks of cancer by Hanahan and Weinberg (Fig 2). The fundamental importance of the UPS
to cancer cells is further reflected by the recent inclusion of the proteotoxic stress as an additional
hallmark of cancer.6
Ub
Ub
Ub
Ub
Ub
Ub
Ub
Ub
E1
E2
E3
Cell proliferation
DNA repair
Proteotoxic stress
Oxidative stress
Angiogenesis
2
0S
9S
2
6S
Metabolic stress
Evading apoptosis
1
Figure 2. The Ubiquitin-Proteasome System (UPS) in the hallmarks of cancer.
The UPS presumably remains essential for cancer cells regardless of resistance to existing PI
drugs. The UPS components upstream of the proteasome (e.g., deubiquitinases, ubiquitin E3
ligases) are being explored as potential anti-cancer therapeutic targets,7-11 but clinical success is
yet to be achieved. Alternatively, it remains to be seen whether the proteasome itself can be
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subsequently re-targeted to achieve further therapeutic gains for MM patients relapsed on existing
PI drugs. Although there are several new PIs in commercial development pipelines (e.g.,
delanzomib, oprozomib, marizomib), early clinical data indicates that those PIs may not be
beneficial for patients with acquired resistance to existing PIs due to issues such as low response
rates in high-risk populations and prevalent cross-resistance with existing PIs.12-17
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Until now, nearly all PIs have been developed through optimization of amino acid side chains (P1,
P2, P3) which interact with the conventional substrate binding pockets (S1, S2, S3, etc.) of
proteasome catalytic subunits (Fig 1B) located N-terminal to the cleavage site. This binding
configuration is further stabilized by the formation of an anti-parallel β-sheet configuration via
hydrogen bonding interactions between the inhibitor’s peptide backbone and the conserved
backbone residues such as Thr21, Gly47, and Ala49 of proteasome catalytic subunits.18-20 When
combined with a C-terminal warhead which targets the catalytic Thr1 residue, this strategy
typically yields potent inhibitors including the three FDA-approved PIs: Btz, Ixz (peptide boronic
acids) and Cfz (a peptide epoxyketone). However, this strategy might have unintentionally
contributed to an increased incidence of cross-resistance among them. A potential strategy to
circumvent PI cross-resistance is to identify and exploit a structural niche not utilized by existing
PIs. In that regard, previously unexplored are P1′ binding sites which lie on the C-terminal side of
the proteasome catalytic subunit’s cleavage site (Fig 1B).
Here, we have developed peptide epoxyketones bearing a P1′-targeting moiety. The anticancer
efficacy of these compounds was superior to their non-P1′-targeting parent compounds when
evaluated against models of intrinsic and acquired Cfz resistance. The identified lead compound,
9
, an analog of Cfz containing a hydroxyl group at the P1′ position, displayed potent proteasome
inhibitory activity and cytotoxicity in both Cfz-sensitive and Cfz-resistant cancer cell lines.
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RESULTS
Chemistry
The final compounds (5, 7-12) were prepared as shown in Scheme 1. TBDPS (t-butyldiphenylsilyl)
and methyl-protected alkenes 1a and 1b were synthesized in three steps following a known method
starting from Boc-serine methyl ester.21 Boc-serine methyl ester was treated with dimethyl
methylphosphonate in the presence of n-butyllithium (n-BuLi) to yield a phosphoryl intermediate,
which upon combination of Wittig-Horner and Baylis–Hillman-type one-pot reactions furnished
the hydroxymethyl-substituted enone in a good yield. Protection of a hydroxyl group with TBDPS
or a methyl group was carried out in TBDPS-Cl/DIEA or methyl iodide/silver oxide, respectively.
Hydrogen peroxide was used to prepare epoxide 2a and 2b from the respective enones.
Intermediates 3 and 4 were prepared using a conventional amide coupling reaction. Deprotection
of TBDPS from the intermediate 4 in the presence of TBAF (t-butylammonium fluoride) yielded
the final compound 5 as shown in Scheme 1A. For Cfz P1′ derivatives, the left-hand peptide
backbone of Cfz 6 was separately prepared in three steps from Boc-serine using a series of amide
coupling reactions in good yields. Another amide coupling reaction between benzyl deprotected 6
and Boc deprotected 2a or 2b gave the final compounds 7 and 8. Deprotection of TBDPS from 7
yielded the final compound 9. Compounds 10, 11 and 12 were prepared from compound 9 using
the respective alkylating agents in the presence of organic base DIEA as shown in Scheme 1B.
UK101 and UK102 were used from in-house compound library.
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Scheme 1. Synthesis scheme of 5 (UK101-OH) and 9 (Cfz-OH)
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b
O
a
OCH3
A
B
BocHN
BocHN
BocHN
O
O
OR1
O
OR
O
H
N
O
1a (R1 = TBDPS)
2a (R = TBDPS)
2b (R = CH3)
1b (R1 = CH3)
N
d
H
O
O
O
O
OTBDPS
4
O
O
O
e
H
H2N
N
c
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O
O
H
N
O
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N
H
O
OH
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O
O
O
O
H
H
H
N
H
O
N
N
N
a
b
N
N
N
N
N
O
OH
H
H
H
BocHN
O
O
O
O
O
O
OR
O
6
7
8
(R = TBDPS)
(R = CH3)
c
7
O
O
H
O
O
H
O
H
H
N
O
N
N
d
N
N
N
N
N
N
H
N
H
H
H
O
O
O
O
OR
O
O
O
O
OH
O
S
10 (R = MOM) 12 (R =
1 (R = MEM)
CH3)
9
1
O
Reagents and conditions. A. (a) i) CH
3
PO(OCH
3
)
2
, n-BuLi, THF, -78 °C, 2 h, Boc-serine methylester, THF, -78 °C,
CO , H O, rt., 4 h, 59%; iii) TBDMS-Cl, Imidazole, DCM, rt., 30
O, MeCN, rt, 24 h, 51% (b) benzonitrile, H , DIEA, MeOH, 0 °C, 3 h, 35-41%; (c)
Heptanoic acid, HBTU, HOBt, DIEA, DCM, rt, 18 h, 82%; (d) i) H , Pd/C, methanol, 1 h, ii) Boc-deprotected 2a,
3
h, 55%; ii) Aqueous solution of Formaldehyde, K
2
3
2
min, 71% or iodomethane, Ag
2
2 2
O
2
HBTU, HOBt, DIEA, DCM, rt, 18 h, 91%; (e) TBAF, DCM, 2 h, 70%. B. (a) i) Phenylalanine benzyl ester
hydrochloride, HBTU, HOBt, DIEA, DCM, rt, 18 h, 81%; ii) TFA, DCM, rt, 1 h then evaporation and drying, Boc-
homoPhe-OH, HBTU, HOBt, DIEA, DCM, rt, 18 h, 79%; iii) TFA, DCM, rt, 1 h then evaporate and dried, Morpholin-
4
2
-yl-acetic acid hydrochloride, HBTU, HOBt, DIEA, DCM, rt, 18 h, 65%; (b) i) H /Pd, C, methanol, 1 h; ii) 2a or 2b,
HBTU, HOBt, DIEA, DCM, rt, 18 h, 45% and 48% respectively; (c) TBAF, DCM, 2 h, 64%; (d) MOM-Cl or MEM-Cl
or methanesulfonyl chloride, DIEA, DCM, overnight, 60-64%.
Initial screening for PIs that can overcome de novo resistance to Cfz.
We recently noted that H727 cells derived from human lung adenocarcinoma are intrinsically
resistant to Cfz (IC of 611 nM, Supple. S1) compared to a panel of cancer cell lines (IC ’s in
5
0
50
the low nM range). Our mechanistic investigation via chemical and genetic modulation of
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proteasome catalytic subunits suggests that proteasome catalytic subunit composition contributes
2
1
to the de novo resistance of H727 cells to Cfz. Therefore, we envisioned that H727 cells can
serve as an initial screening system in identifying PIs that can overcome de novo resistance to Cfz
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(currently there is no MM model with de novo resistance to Cfz). We hypothesized that alternative
PIs with distinct structural features may interact differentially with proteasomes in H727 cells to
induce proteasome inhibition and consequent cell death. As a control, we used another lung cancer
cell, H23, which are highly sensitive to Cfz (IC of 18 nM). We compared the cytotoxic effects
5
0
of PIs with differing pharmacophores including P1′-targeted peptide epoxyketones (UK101 and
2
2
UK102 previously developed in our laboratory) in H727 and H23 cells (Figure 3).
A
O
O
H
O
O
H
O
O
N
N
N
N
H
H
O
O
O
O
OH
Si
O
O
UK101
UK102
B
IC (µM)
5
0
Fold difference
H23
H727
(
H727/H23)
Carfilzomib
Bortezomib
MG-132
0.018 ±0.004
0.010 ±0.001
0.48 ± 0.01
3.37 ± 0.19
3.40 ± 0.18
5.23 ± 0.22
0.611 ±0.047
0.025 ±0.006
33.3
2.5
2
0.98 ± 0.03
5.21 ± 0.11
4.49 ± 0.26
> 100
UK101
1.5
1.3
> 19
UK102
Lactacystin
Figure 3. A. Structures of UK101 and UK102. B. Comparison of the cytotoxic potency (72-hour IC50 values) of H727
and H23 cells to carfilzomib, bortezomib, MG-132, UK101, UK102, and lactacystin. Data are reported as the mean ±
SD.
Btz (a peptide boronic acid) and MG-132 (a peptide aldehyde) displayed a comparable cytotoxicity
in H727 and H23 cells. UK101 and UK102 were also not cross-resistant to Cfz, displaying similar
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IC values in H727 cells and H23 cells. On the other hand, lactacystin (a lactone) was not as
5
0
effective in H727 cells as in H23 cells. These results suggest that proteasome inhibition using
alternative compounds can still be a viable therapeutic option even in the presence of resistance to
Cfz.
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0
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0
The anticancer efficacy of PIs with P1′ moieties (UK101 & UK102) is not attenuated by
acquired Cfz resistance in cancer cells.
As a next step to examine the effectiveness of P1′-targeting epoxyketones in cancer cells, we
examined whether these compounds can overcome acquired Cfz resistance, a major clinical
2
3
challenge facing Cfz-based therapies. We utilized a Cfz-resistant subline of RPMI8226 (derived
from human MM cells) which had been adapted to escalating concentrations of Cfz over several
months.
A
IC50 (nM)
Parental
7.4
RPMI8226
Parental CfzR
Cfz-resistant 269.8
1
1
25
00
kDa
250
7
5
5
0
P-gp
150
25
0
β-actin
1
10
100
1000
Carfilzomib(nM)
B
IC (µM)
5
0
RPMI8266
(parental)
Fold difference
(CfzR/parental)
RPMI8266/CfzR
Carfilzomib
Epoxomicin
UK101
0.007 ±0.001
0.011 ±0.005
0.269 ±0.055
0.541 ±0.058
3.72 ± 0.10
36.5
49.2
1.7
2.14 ± 0.08
2.08 ± 0.11
UK102
3.41 ± 0.09
1.6
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Figure 4. A. Effects of carfilzomib (Cfz) on the viability of an RPMI8266 cell line with acquired Cfz resistance
(RPMI8266/CfzR) in comparison to the parental cell line. Cell viability was measured by MTS assay after 72 h drug
treatment (Left). Immunoblotting analysis showing a marked increase of P-gp expression in RPMI8226 Cfz-resistant
cells in comparison to parental cells (Right). B. Comparison of the sensitivity (72-hour IC50 values) of RPMI8226
parental and Cfz-resistant cells to Cfz, epoxomicin, UK101, and UK102. Data are reported as the mean ± SD. For
epoxomicin and carfilzomib, the SD values were obtained from three independent experiments. For UK101 and
UK102, the SD values were from non-linear regression analysis using three replicates.
0
1
2
3
4
5
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8
9
0
1
2
3
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8
9
0
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0
1
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9
0
As shown in Figure 4A, the Cfz-resistant RPMI8226 cells displayed a marked (greater than 35-
fold) increase in IC values and elevated expression of the efflux transporter P-glycoprotein (P-
5
0
gp), similar to the previous reports.24, 25 The use of the P-gp inhibitor reversin 121 almost
completely restored Cfz sensitivity (Supple. S2), suggesting the predominant contribution of P-gp
in the current model of acquired Cfz resistance. While both Cfz and epoxomicin (also reported as
26
a P-gp substrate ) displayed a marked increase in IC values, UK101 and UK102 showed
5
0
minimal changes in IC values in Cfz-resistant RPMI 8226 cells compared to the parental control
5
0
(Fig 4B).
The anticancer efficacy of UK101 and UK102 is not impaired by acquired Btz resistance in
primary MM samples.
We next investigated whether UK101 and UK102 can be effective in primary MM samples from
patients who do not respond to Btz (We were not able to obtain MM samples from patients who
are resistant to Cfz, but it was previously reported that patients with MM refractory to or relapsed
27
on Btz are often cross-resistant to Cfz. ). Using primary MM samples from 14 different donors (6
from PI-naïve patients and 8 from patients relapsed on Btz therapy), their sensitivity to Cfz was
assessed. The results indicated varying degrees of sensitivity to Cfz, but the MM samples from the
patients who relapsed on Btz tended to be less responsive to Cfz than those from patients from the
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PI-naïve group (Fig 5A). Due to the limited quantities of primary MM samples from 5 patients
(#5-#8 and #14), we utilized only 9 primary MM samples (4 Btz-resistant and 5 PI-naïve) to
examine the effect of UK101 or UK102. When 9 primary MM samples treated with UK101 or
UK102 for 48 h, the results indicated that cytotoxic effects of UK101 or UK102 were similar.
Although the current sample size was small, the lack of apparent cross-resistance between Cfz/Btz
and UK101/UK102 was encouraging.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
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0
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0
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2
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8
9
0
Prior Btz therapy
PI naive
1
00
80
A
6
4
0
0
(
Cfz)
20
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
1
#
#
#
#
#
#
#
#
#
1
1
1
1
#
#
#
#
#
1
00
80
B
6
0
(
UK101)
40
2
0
0
1
2
3
4
9
0
1
2
3
1
#
#
#
#
#
1
1
1
#
#
#
#
100
8
6
4
2
0
0
0
0
0
C
(UK102)
1
2
3
4
9
0
1
2
3
1
#
#
#
#
#
1
1
1
#
#
#
#
Figure 5. Effects of carfilzomib (Cfz), UK101, and UK102 on cell viability of primary MM samples from 14 different
donors, 6 from Btz/Cfz-naïve patients and 8 from patients relapsed on Btz therapy. Primary MM cells were treated
with Cfz (A, 50 nM), UK101 (B, 10 μM) or UK102 (C, 10 μM ) for 48 h. Cell viability was measured using an ATP-
based luminescent assay.
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Introduction of hydrophilic residues at the P1′ position of peptide epoxyketones improves
the proteasome inhibitory potency.
To improve the potency of UK101 and UK102 and obtain structure-activity relationship (SAR)
information, we first utilized docking models of UK101 bound to β1i and β5, known targets of
UK101. UK101 was initially identified as a selective inhibitor of the β1i subunit at nanomolar
0
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8
concentration ranges. Later, UK101 was also found to inhibit the β5 subunit (chymotrypsin-like
2
9
(CT-L) activity) at micromolar concentration ranges. Docking structures shown in Fig 6 were
generated by Autodock Vina using the previously reported X-ray structures from mouse 20S
3
0
proteasomes complexed with ONX 0914 and were used as a guide to our optimization effort at
the P1′ position. As shown in Fig 6A, the P1-P3 residues of UK101 were predicted to occupy the
S1-S3 pockets located deep inside of the active sites of the β1i and β5 subunits.
However, unlike Cfz bound to β5, docking models indicated that UK101 occupies an additional
binding pocket (S1′) of the β5 as well as β1i subunits via its P1′ group (highlighted in purple
circles, Fig 6A) which is not occupied by Cfz or Btz.31 Although the P1′ group (t-
butyldimethylsilyl, TBDMS) of UK101 points toward the surface of β1i, it is predicted to
contribute to a favorable hydrogen bonding interaction with the hydroxyl side chain of Ser168
within the S1′ pocket (bottom-left, Fig. 6A). In the case of UK101 bound to β5, the amino acid
residue changes within the S1′ pocket (especially, β1i-Ser168 → β5-Tyr169) are predicted to
abolish this additional hydrogen bonding interaction with the TBDMS (top-left, Fig. 6A).
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A
B
O
H
O
O
O
H
N
O
N
N
H
N
H
O
O
O
O
OH
Si
UK101
5 (UK101-OH)
P1’
1
1
1
1
1
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1
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0
1
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8
9
0
IC50 (µM)
UK101
Carfilzomib
UK101
> 10
UK101-OH
1.7 ± 0.1
CT-L
β1i
0.14 ± 0.01 0.036 ± 0.01
H23
3.37 ± 0.19
5.21 ± 0.11
0.58 ± 0.02
0.69 ± 0.01
H727
RPMI8266
2.14 ± 0.08 0.31 ± 0.01
/CfzR
UK101-OH
β1i/β5
UK101
Figure 6. A. Predicted docking models of UK101 and carfilzomib (Cfz) bound to β5 or β1i. The location of UK101’s
TBDMS group positioned within putative P1′ pockets is highlighted using a purple-colored circle. β5 (PDB ID:
3UNB) and β1i (PDB ID: 3UNF) from mammalian 20S proteasomes were used as templates. In cartoon presentation,
β1i (gray, PDB ID: 3UNF) was superposed to β5 (yellow, PDB ID: 3UNB) and only different amino acid residues are
shown in stick model. B. Comparison of UK101 and 5 (UK101-OH) in terms of their potency (IC50 values) against
proteasome chymotrypsin-like activity (in RPMI8226 cell lysate), LMP2 catalytic activity (in 20S purified human
immunoproteasome), and against H23, H727, and Cfz-resistant RPMI8226 cells as measured by MTS cell viability
assay. Data are reported as the mean ± SD. Docking model of UK101-OH bound to β1i (PDB ID: 3UNF): The P1′-
OH of UK101-OH (5) is perfectly positioned to form hydrogen bonds with Ser168 and Ser21.
The resulting change in amino acid residues of the S1′ pocket is also predicted to force the large
hydrophobic TBDMS group to be surrounded by the polar amino acids (Asp17, Thr21, Ser130)
within the S1′ pocket, a plausible contributing factor towards UK101’s low potency against the β5
3
1
subunit. Based on this prediction, we hypothesized that the introduction of small polar groups at
the P1′ position could provide energetically-favorable hydrogen bonding interactions with the
polar amino acid residues within the S1′ pockets of β1i and β5, potentially improving potency
against both subunits. To test this, we replaced the bulky hydrophobic P1′ substituent (TBDMS)
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5
6
of UK101 with a hydroxyl group (Scheme 1A) and assessed its impact. This simple P1′ substitution
increased in vitro 20S proteasome inhibitory potency by ~4-10 fold (Fig 6B). This experimental
result is consistent with the docking models of UK101-OH (5) bound to β1i (PDB ID: 3UNF), in
which the P1′ hydroxyl group of UK101-OH (5) is perfectly positioned to form hydrogen bonds
with the oxygen of Ser168 carbonyl backbone and the hydroxyl side chain of Ser21. Consequently,
the P1′ hydroxyl group also considerably improved the cytotoxic activity of compound 5 in cancer
cell lines, including our models of both de novo and acquired Cfz resistance. Based on this result,
we suspect that the P1′ residue of peptide epoxyketones may play an important role in overcoming
the resistance of cancer cells to Cfz.
0
1
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9
0
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0
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9
0
Development of 9 (Cfz-OH) with an improved potency in the models of de novo and acquired
Cfz resistance
In our efforts to further optimize 5, we considered that the P1-P4 groups of Cfz have already been
thoroughly optimized for the S1-S4 pockets of β5 and β5i (as well as β1i, to a lesser extent, based
on the largely β1i/β5 superimposed model, Fig 6A). We thus decided to attach several different
P1′ moieties to Cfz (Fig 7). Our hypothesis was that Cfz analogs bearing a P1′-targeting group
could overcome cross-resistance to Cfz. We also expected that a Cfz analog containing a polar P1′
moiety may have an improved inhibitory potency against both β5 and β1i compared to Cfz, due to
additional P1′:S1′ interactions (Fig 1B). We prepared Cfz analogs having a series of P1′ moieties
varying from bulky hydrophobic to small hydrophilic residues (Scheme 1B). We subsequently
measured their activity against cell lysates to measure CT-L (β5/β5i) inhibition and against 20S
purified immunoproteasomes to measure β1i inhibition. As predicted by in Silico docking analysis,
9
and 12 having small polar moieties were most potent in vitro with IC values similar to that of
50
Cfz. When these two compounds were tested using Cfz-resistant H727 cells, 12 was less potent
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than Cfz against H727. On the other hand, 9 (Cfz-OH, a Cfz analog with a hydroxyl group at the
P1′ position) demonstrated an improved potency by ~10-fold relative to Cfz. When tested against
RPMI8226 with acquired Cfz resistance, 9 demonstrated an almost 3-fold improvement in potency
against Cfz-resistant RPMI8226 cells as compared to Cfz (Fig 7).
1
1
1
1
1
1
1
1
1
1
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0
Ph
R =
Si
7
8
Ph
CH3
O
O
H
H
O
O
N
N
N
N
N
H
9 (Cfz-OH)
10
H
H
O
O
O
O
OCH3
R
O
OCH3 11
12
O
Cfz-O-R
S CH3
O
IC (nM)
5
0
Proteasome
inhibitors
RPMI
266/CfzR
CT-L
LMP2
H23
H727
8
Carfilzomib
2.7 ± 2.2
111.6 ± 11.1
>1000
2940 ± 22.3
18.3 ± 6.6
610.2 ± 46.9 269.8 ± 54.8
7
8
(Cfz-TBDPS)
(Cfz-CH3)
-
> 10,000
> 10,000
-
-
-
-
-
9 (Cfz-OH)
0 (Cfz-MOM)
11 (Cfz-MEM)
2 (Cfz-Sulfone)
2.1 ± 0.9
> 1000
743.5 ± 10.7
29.4 ± 2.5
61.0 ± 10.7
106.2 ± 28.9
1
-
-
-
-
-
-
-
-
> 1000
-
-
1
8.0 ± 4.9
310.7 ± 12.3
834.4± 41.6
Figure 7. The potency (IC50 values) for compounds with various substitutions at the P1′site against proteasome
chymotrypsin-like activity (RPMI8226 cell lysate), LMP2 activity (purified human 20S immunoproteasome), and cell
viability of H23, H727, and Cfz-resistant RPMI8226 cells. Data reported as the mean ± SD (carfilzomib, n = 3
independent experiments) or from a single experiment (3 replicates, 7, 9, and 12).
Compound 9 (Cfz-OH) has improved metabolic stability compared to its close structural
analog, Cfz
32
Cfz is rapidly metabolized in vivo with plasma half-lives of less than 1 hr, likely contributing to
3
3
its lack of efficacy against solid cancers especially those with poorly developed vasculature. The
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metabolic inactivation of Cfz is reported to be mediated mainly by microsomal epoxide hydrolase
(
mEH) and peptidases.34 In the case of mEH, the active site harbors two conserved tyrosine
residues which may contribute to substrate specificity and orientation of substrates within the
active site.35, 36 The prototypical substrates of mEH include planar hydrophobic compounds such
0
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3
7
as various epoxides of polycyclic aromatic hydrocarbons and steroids. It is thus expected that the
epoxide ring of Cfz occupies the active site of mEH with a position suitable for hydrolysis to yield
Cfz-diol (Fig 8A). We hypothesized that the addition of a hydroxyl group adjacent to the epoxide
ring of Cfz (compound 9) may hinder the hydrolysis of the epoxide ring by inhibiting access to the
active site of mEH. When the metabolic stability of 9 was examined in rat liver homogenates, 9
was indeed more stable than Cfz (Fig 8B).
A
B
50
O
O
OH
OH
H
N
H
O
N
Cfz
9 (Cfz-OH)
N
N
N
H
mEH
mEH
4
3
2
1
0
0
0
0
0
H
N
O
O
O
O
H
O
carfilzomib-diol
Carfilzomib
(
Inactive)
O
H
O
H
N
O
N
N
H
N
N
H
O
X
H
O
O
O
O
5
10
20
9
Time (min)
Figure 8. A. Schematic depicting the rapid metabolism of Cfz by microsomal epoxide hydrolase (mEH) to the inactive
diol. B. Quantification of the remaining levels of Cfz or 9 following the incubation with rat liver homogenate
containing active mEH and peptidase activities for 5, 10, and 20 minutes respectively. Data presented as mean ± SD.
DISCUSSION
In expanding the clinical utility of existing PI drugs, cancer resistance, either de novo or acquired,
remains a major obstacle. Currently, MM patients who do not respond to PI therapy or relapse
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after prolonged PI have very dismal outcomes with median survival of less than one year. To date,
several mechanisms of PI resistance have been reported, including mutations/amplification of
proteasome catalytic subunits, dysregulations of unfolded protein response and autophagy,
overexpression of MARCKS proteins or efflux transporters, increased formation of aggresomes,
and others.38-41 However, clinically relevant mechanisms of PI resistance remain to be uncovered,
making it difficult to achieve an eventual goal of developing therapeutic options for patients
refractory to current PI drugs.
1
1
1
1
1
1
1
1
1
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0
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0
Several reports indicate that the proteasome remains indispensable to the survival of cancer cells
despite their resistance to PI drugs. The proteasome plays fundamental roles in various cellular
functions and to date there appears no pathway/machinery which can fully compensate for the loss
of proteasome function. Several reports showed that Btz-resistant MM cells remain susceptible to
the disruption of proteasome pathways.22, 42-44 For instance, an inhibitor targeting the non-catalytic
α7 proteasome subunit was effective against bortezomib-resistant THP-1 cells harboring a mutated
4
5
PSMB5 gene encoding the catalytic 5 subunit. These in vitro results require further validation
in clinical settings, but they support that proteasome inhibition may offer therapeutic gains even
in patients with MM relapsed/refractory to currently used PIs in clinic.
In this study, we used two established cancer cell lines with de novo or acquired resistance to Cfz
and identified a novel PI compound with promising efficacy against both cell lines. Among the
tested compounds, we found that epoxyketones with previously underexplored P1′ substituents
can overcome both de novo and to a lesser degree, acquired Cfz resistance in cell line models.
Compared to the boronic acid-based PIs, peptide epoxyketones feature a much improved
selectivity in their interactions with the proteasome by forming an 1,4-oxazepane adduct with the
N-terminal catalytic threonine residue of the proteasome.46 Recent extensive reviews of the
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literatures that describe the efficacy and toxicity profiles of carfilzomib-based regimens in clinic
further supports the safety and tolerability of the peptide epoxyketone family.47, 48 Peptide
epoxyketones with an improved metabolic stability and an ability to bypass resistance mechanisms
may offer a more promising route for further drug development efforts. Future studies will address
the in vivo efficacy and metabolic stability of epoxyketones with previously underexplored P1′
substituents that can overcome both de novo and acquired Cfz resistance.
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CONCLUSION
By exploring the chemical space at the P1′ position of peptide epoxyketones, we identified novel
PIs effective against cell line models of de novo and acquired resistance to Cfz as well as primary
MM cells. Our lead compound closely mimics the FDA-approved Cfz except for the addition of a
hydroxyl group at the P1′ position. Our lead peptide epoxyketone with an S1′-occupying
component displayed an improved metabolic stability over Cfz. This could provide potentially
important advantages in expanding the utility of PIs to the many solid cancers in need of improved
therapeutics. Further investigations regarding the contribution of P1′ substituents to metabolic
stability and activity against PI-resistant patient samples are ongoing.
EXPERIMENTAL SECTION
General: All chemical reagents starting materials and solvents were purchased from commercial
vendors and used without further purification. TLC was performed on precoated plates and spots
were detected by using UV or staining solution of phosphomolybdic acid hydrate (PMA) or iodine.
Silica gel column chromatography was performed on 60 Å silica gel, 230–400 mesh (Merck).
Proton and carbon NMR spectra were recorded on a Varian spectrometer at 400 MHz. Chemical
shifts are reported in ppm units with tetramethylsilane as a reference standard. Multiplicities of
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NMR signals were reported using different abbreviations like singlet (s), doublet (d), triplet (t),
quartet (q) and multiplet (m). Chemical shifts were reported in parts per million (ppm) and
coupling constant (J) is reported in Hz. Mass spectra were recorded on an Agilent Technologies
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120 Quadrupole LC/MS. The mobile phases used were A: H O containing 0.1% formic acid and
2
B: acetonitrile (CH CN) containing 0.1% formic acid. The linear gradient of B/A from 5:95 to
3
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0:10 over 18 min and then 100:0 over 7 min was used in an Agilent Eclipse XBD-C18 column
(5-μm particle size), 4.6 mm dia.  150 mm with a flow rate of 0.4 mL/min. The purity of all final
compound used in bioassay was determined in an Agilent Technologies 1200 series equipped with
a UV detector set at 254 nm using same column, gradient and flow rate to be ≥ 95%. Melting
points were determined on Thermo Scientific 1001D apparatus and were uncorrected.
Synthesis
General procedure for the deprotection of benzyl group: Benzyl ester was dissolved in
methanol and 5 mol% Pd/C was added. The solution was stirred under hydrogen gas environment
for 30 min. Deprotected carboxylic acid was filtered through celite and solvent was concentrated.
Deprotected carboxylic acid was used in the next step without further purification.
General procedure for the deprotection of Boc group: To the solution of Boc-protected amine
in dichloromethane (DCM), excess of TFA was added and the reaction solution was stirred at room
temperature for 1 h. Solvent was evaporated under reduced pressure and the crude TFA salt of
amine was used in next step without further purification.
(S)-tert-Butyl
2-((tert-butyldiphenylsilyloxy)methyl)-6-methyl-3-oxohept-1-en-4-
ylcarbamate (1a)
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A solution of n-BuLi (25.46 mL, 40.76 mmol) in hexane was added dropwise to the stirred solution
of dimethyl methylphosphonate (4.43 mL, 40.76 mmol) in THF at –78 °C. The reaction mixture
was stirred at the same temperature for 2 h. A solution of N-(tert-Butoxycarbonyl)-L-leucine
methyl ester (2.5 g, 10.19 mmol) in THF was then added to the reaction mixture and the reaction
solution was further stirred for 3 h at same temperature. Reaction temperature was gradually
increased up to room temperature and the reaction was quenched with the addition of water. The
product was extracted with ethyl acetate 3 times and the collected organic layer was dried over
sodium sulfate. It was then filtered, concentrated under reduced pressure and purified through flash
column chromatography using 50% ethyl acetate in hexane to yield pure product ((S)-tert-butyl 1-
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(dimethoxyphosphoryl)-5-methyl-2-oxohexan-3-ylcarbamate) as a colorless sticky oil (1.88 g,
1
5
5% yield). H NMR (400 MHz, CDCl ) δ 5.18 (d, J = 8.6 Hz, 1H), 4.40 - 4.26 (m, 1H), 3.79 (d,
3
J = 4.5 Hz, 3H), 3.76 (d, J = 4.4 Hz, 3H), 3.32 (dd, J = 14.2, 22.6 Hz, 1H), 3.08 (dd, J = 14.2, 22.0
Hz, 1H), 1.76 - 1.61 (m, 2H), 1.43 (s, 10H), 0.99 - 0.86 (m, 6H).
To the stirred solution of (S)-tert-butyl 1-(dimethoxyphosphoryl)-5-methyl-2-oxohexan-3-
ylcarbamate (1.88 g, 5.57 mmol) and formaldehyde (1.35 ml, 16.71 mmol) in 30 mL water, aq.
solution of potassium carbonate (0.92 g, 6.68 mmol) was added drop-wise at room temperature.
Reaction mixture was further stirred for 4 h at same temperature. The crude product was extracted
with ethyl acetate 3 times and the collected organic layer was dried over sodium sulfate. It was
then filtered, concentrated under reduced pressure and purified by flash column chromatography
using 30% ethyl acetate in hexane to yield pure product ((S)-tert-butyl 2-(hydroxymethyl)-6-
1
methyl-3-oxohept-1-en-4-ylcarbamate) as colorless oil (0.89 g, 59% yield). H NMR (400 MHz,
CDCl ) δ 6.22 (s, 1H), 6.17 - 6.06 (m, 1H), 5.03 (d, J = 12.6 Hz, 2H), 4.34 (t, J = 10.2 Hz, 2H),
3
1.73 (s, 1H), 1.42 (d, J = 3.4 Hz, 12H), 0.99 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.5 Hz, 3H).
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To the stirred solution of (S)-tert-butyl 2-(hydroxymethyl)-6-methyl-3-oxohept-1-en-4-
ylcarbamate (0.9 g, 3.30 mmol) and imidazole (0.67 g, 9.92 mmol) in DCM at room temperature,
TBDPS-Cl (2.53 mL, 9.92 mmol) was added drop-wise and the reaction solution was stirred at
same temperature for 30 min. Solvent was removed under reduced pressure and residue was
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purified by flash column chromatography using 5% ethyl acetate in hexane to yield pure product
1
(
1a) as colorless thick oil (1.2 g, 71% yield). H NMR (400 MHz, CDCl ) δ 7.66 - 7.58 (m, 4H),
3
7
.44 - 7.31 (m, 6H), 6.33 (s, 1H), 6.24 (s, 1H), 5.10 - 4.93 (m, 2H), 4.50 - 4.27 (m, 2H), 1.68 (t, J
7.6 Hz, 1H), 1.24 (ddd, J = 4.5, 10.0, 14.0 Hz, 2H), 1.05 (d, J = 2.2 Hz, 9H), 0.97 (d, J = 6.5 Hz,
H), 0.87 (d, J = 6.6 Hz, 3H).
=
3
(S)-tert-Butyl 2-(methoxymethyl)-6-methyl-3-oxohept-1-en-4-ylcarbamate (1b)
To the solution of (S)-tert-butyl 2-(hydroxymethyl)-6-methyl-3-oxohept-1-en-4-ylcarbamate (100
mg, 0.36 mmol) and iodomethane (0.23 µL, 3.68 mmol) in acetonitrile, silver oxide (427 mg, 1.84
mmol) was added and the reaction mixture was stirred at room temperature for 24 h. The solvent
was removed under reduced pressure and residue was purified by flash column chromatography
using 10% ethyl acetate in hexane. Pure product was collected as colorless sticky solid (54 mg,
1
5
1% yield). H NMR (400 MHz, CDCl ) δ 6.23 (s, 1H), 6.09 (s, 1H), 5.09 (d, J = 9.0 Hz, 1H),
3
5.00 (td, J = 3.5, 9.5 Hz, 1H), 4.09 (t, J = 1.5 Hz, 2H), 3.36 (s, 3H), 1.80 - 1.62 (m, 1H), 1.39 (m,
1H), 0.97 (d, J = 6.5 Hz, 3H), 0.87 (d, J = 6.7 Hz, 3H).
1
tert-Butyl
(S)-1-((S)-2-((tert-butyldiphenylsilyloxy)methyl)oxiran-2-yl)-4-methyl-1-
oxopentan-2-ylcarbamate (2a) (General procedure for epoxidation)
The mixture of 1a (1.2 g, 2.35 mmol), benzonitrile (2.4 mL, 23.5 mmol) and hydrogen peroxide
(3.99 mL, 58.75 mmol) in methanol was stirred at 0 °C and DIEA (4.10 mL, 23.5 mmol) was
added drop-wise. The reaction mass was stirred at the same temperature for 3 h. Methanol was
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removed under reduced pressure and the reaction mixture was extracted with ethyl acetate 3 times.
The collected organic layer was dried under sodium sulfate, filtered and concentrated. Crude
product was purified by flash column chromatography using 10% ethyl acetate in hexane. Pure
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required product was collected as colorless sticky solid (500 mg, 41% yield). H NMR (400 MHz,
CDCl ) δ 7.68 (m, 4H), 7.46 – 7.36 (m, 6H), 5.08 (dd, J = 7.2, 23.4 Hz, 2H), 4.47 (d,J = 10.5 Hz,
3
1
H), 3.49 (d, J = 11.5 Hz, 1H), 3.17 (d, J = 4.9 Hz, 1H), 2.98 (d, J = 4.9 Hz, 1H), 1.23 - 1.11 (m,
H), 1.00 (d, J = 1.9 Hz, 10H), 0.93 (d, J = 5.6 Hz, 6H).
2
tert-Butyl (S)-1-((S)-2-(methoxymethyl)oxiran-2-yl)-4-methyl-1-oxopentan-2-ylcarbamate
2b)
(
Compound 2b was prepared using same general procedure for epoxidation as 2a from (S)-tert-
butyl 2-(methoxymethyl)-6-methyl-3-oxohept-1-en-4-ylcarbamate (1b) to yield colorless sticky
1
solid (20 mg, 35% yield). H NMR (400 MHz, CDCl ) δ 4.89 (dd, J = 7.2, 23.4 Hz, 2H), 4.53 (d,
3
J = 10.5 Hz, 1H), 3.51 (d, J = 11.5 Hz, 1H), 3.15 (d, J = 4.9 Hz, 1H), 2.98 (d, J = 4.9 Hz, 1H), 1.85
- 1.63 (m, 1H), 1.39-1.28 (m, 11H), 0.99 (d, J = 6.5 Hz, 3H), 0.87 (d, J = 6.7 Hz, 3H).
(S)-Benzyl 2-heptanamidopropanoate (3) (General procedure for amide coupling)
To the stirred solution of alanine benzyl ester hydrochloride (1 g, 2.84 mmol) and heptanoic acid
(
0.44 mL, 3.13 mmol) in DCM, HBTU (2.15 g, 5.68 mmol), HOBt (0.77 g, 5.68 mmol), and DIEA
2.48 mL, 14.2 mmol) were added at room temperature and the resulting reaction mixture was
(
stirred at the same temperature overnight. Solvent was removed under reduced pressure and
residue was purified by flash column chromatography using 30% ethyl acetate in Hexane. Pure
1
product was collected as white sticky solid (0.68 g, 82%). H NMR (400 MHz, CDCl ) δ 7.63-
3
7
.31 (m, 5H), 5.95 (s, 1H), 5.16 (t, J = 4.6 Hz, 2H), 4.72 - 4.56 (m, 1H), 2.18 (td, J = 2.8, 8.0 Hz,
H), 1.59 (d, J = 9.0 Hz, 2H), 1.46 - 1.12 (m, 9H), 0.86 (t, J = 8.6 Hz, 3H).
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N-((S)-1-((S)-1-((S)-2-((tert-Butyldiphenylsilyloxy)methyl)oxiran-2-yl)-4-methyl-1-
oxopentan-2-ylamino)-1-oxopropan-2-yl)heptanamide (4)
Benzyl ester was deprotected from compound 3 using general procedure for benzyl deprotection
reaction and Boc group was deprotected from intermediate 2a using general procedure for Boc
deprotection reaction. Thus obtained free carboxylic acid and amines were coupled using general
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procedure for amide coupling reaction to yield intermediate 4 as colorless sticky solid (91%). H
NMR (400 MHz, CDCl ) δ 7.74 - 7.59 (m, 4H), 7.38 (dt, J = 6.7, 8.9 Hz, 6H), 6.71 (d, J = 7.5 Hz,
3
1
1
H), 6.09 (d, J = 7.5 Hz, 1H), 4.65 - 4.52 (m, 2H), 4.48 (d, J = 11.4 Hz, 1H), 3.47 (d, J = 11.5 Hz,
H), 3.20 (d, J = 5.1 Hz, 1H), 2.97 (d, J= 5.1 Hz, 1H), 2.25 - 2.07 (m, 2H), 1.82 - 1.50 (m, 4H),
1.42 - 1.16 (m, 10H), 0.99 (s, 9H), 0.95 (d, J = 6.3 Hz, 3H), 0.91 (d, J = 6.3 Hz, 3H), 0.89 - 0.81
m, 3H).
(
N-((S)-1-((S)-1-((R)-2-(Hydroxymethyl)oxiran-2-yl)-4-methyl-1-oxopentan-2-ylamino)-1-
oxopropan-2-yl)heptanamide (5) (General procedure for TBDPS deprotection)
To the solution of compound 4 (71 mg, 0.12 mmmol) in DCM, TBAF (0.25 mL, 0.25 mmol) was
added at room temperature and the reaction mass was stirred at the same temperature for 30 min.
Solvent was removed under reduced pressure and the resulting residue was purified by flash
column chromatography using 5% methanol in DCM to yield pure product as colorless sticky solid
1
(
30 mg, 70% yield). H NMR (400 MHz, CDCl ) δ 6.97 (d, J = 7.4 Hz, 1H), 6.16 (d, J = 7.6 Hz,
3
1H), 4.65 - 4.44 (m, 2H), 4.19 (dd, J = 4.3, 12.7 Hz, 1H), 3.70 (dd, J = 4.7, 12.7 Hz, 1H), 3.31 (d,
J = 5.0 Hz, 1H), 3.06 (d, J = 5.0 Hz, 1H), 2.54 (d, J = 6.6 Hz, 1H), 2.15 (dt, J = 4.3, 8.8 Hz, 2H),
1
.96 (d, J = 4.6 Hz, 1H), 1.72 - 1.49 (m, 4H), 1.41 - 1.15 (m, 9H), 0.91 (t, J = 7.6 Hz, 5H), 0.87 -
1
3
0.80 (m, 3H). C NMR (101 MHz, CDCl ) δ 207.72, 173.14, 172.51, 62.02, 61.42, 51.01, 49.31,
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8.28, 39.07, 36.56, 31.46, 28.86, 25.55, 25.13, 23.30, 22.43, 21.09, 17.97, 13.98; LCMS (ES+)
+
m/z calcd for C H N O [M+H] 371.2, found: 371.2.
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(S)-Benzyl 2-((S)-4-methyl-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido) pentan-
emido)-3-phenylpropanoate (6)
Intermediate 6 was prepared in three steps starting from Boc-Leu-OH. Dipeptide (S)-benzyl 2-
((S)-2-(tert-butoxycarbonylamino)-4-methylpentanamido)-3-phenylpropanoate was prepared
using the general procedure for amide coupling reaction from Boc-Leu-OH and phenylalanine
1
benzyl ester hydrochloride as white solid (81%). H NMR (400 MHz, CDCl ) δ 7.34 (m, 3H), 7.30
3
- 7.22 (m, 2H), 7.19 (m, 3H), 7.00 (t, J = 4.3 Hz, 2H), 6.45 (d, J = 7.8 Hz, 1H), 5.19 - 5.05 (m,
2
H), 4.91 – 4.83 (m, 1H), 4.77 (s, 1H), 3.10 (t, J = 6.7 Hz, 2H), 1.67 - 1.51 (m, 3H), 1.41 (s, 9H),
.87 (dt, J = 4.0, 2.1 Hz, 6H).
0
The Boc deprotected above prepared dipeptide was further coupled with Boc-L-
homophenylalanine using the general procedure for amide coupling reaction to prepare tripeptide
(S)-benzyl
2-((S)-2-((S)-2-(tert-butoxycarbonylamino)-4-phenylbutanamido)-4-
1
methylpentanamido)-3-phenylpropanoate as white solid (79%). White solid (79%). H NMR (400
MHz, CDCl ) δ 7.33 (dd, J = 4.8, 1.8 Hz, 2H), 7.29 - 7.22 (m, 5H), 7.21 – 7.12 (m, 5H), 7.01 -
3
6
.94 (m, 2H), 6.43 (d, J = 7.5 Hz, 1H), 6.37 (d, J = 8.0 Hz, 1H), 5.20 – 5.00 (m, 2H), 4.97 - 4.77
m, 2H), 4.36 (td, J = 8.7, 4.9 Hz, 1H), 3.99 (d, J = 7.4 Hz, 1H), 3.07 (q, J = 7.9, 7.1 Hz, 2H), 2.62
(t, J = 7.9 Hz, 2H), 2.06 (dt, J = 14.2, 7.0 Hz, 1H), 1.85 (dd, J = 14.1, 7.6 Hz, 1H), 1.57 (s, 3H),
.42 (s, 9H), 0.84 (t, J = 5.9 Hz, 6H).
Compound 6 was prepared from Boc deprotected tripeptide (S)-benzyl 2-((S)-2-((S)-2-(tert-
(
1
butoxycarbonylamino)-4-phenylbutanamido)-4-methylpentanamido)-3-phenylpropanoate
and
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morpholin-4-yl-acetic acid using the general procedure for amide coupling reaction. White solid
1
(
65%). H NMR (400 MHz, CDCl ) δ 7.95 (s, 2H), 7.56 (d, J = 8.4 Hz, 1H), 7.34 - 7.27 (m, 2H),
3
7.26 - 7.18 (m, 3H), 7.17 - 7.08 (m, 5H), 6.97 (dd, J = 7.2, 2.3 Hz, 2H), 6.84 (d, J = 7.8 Hz, 1H),
.66 (d, J = 7.7 Hz, 1H), 5.14 - 4.98 (m, 2H), 4.83 (dt, J = 7.6, 6.0 Hz, 1H), 4.44 (td, J = 8.1, 5.8
Hz, 1H), 4.35 (ddd, J = 9.0, 7.8, 5.3 Hz, 1H), 3.74 - 3.63 (m, 4H), 3.12 - 2.97 (m, 2H), 2.94 (d, J
5.9 Hz, 2H), 2.57 (dq, J = 8.8, 3.1 Hz, 2H), 2.46 (q, J = 4.5 Hz, 4H), 2.17 - 2.03 (m, 1H), 1.97 -
.84 (m, 1H), 1.62 - 1.39 (m, 3H), 0.81 (dd, J = 9.5, 6.1 Hz, 6H).
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
=
1
(S)-N-((S)-1-((S)-1-((S)-2-((tert-Butyldiphenylsilyloxy)methyl)oxiran-2-yl)-4-methyl-1-
oxopentan-2-ylamino)-1-oxo-3-phenylpropan-2-yl)-4-methyl-2-((S)-2-(2-
morpholinoacetamido)-4-phenylbutanamido)pentanamide (7)
Compound 7 was prepared from benzyl deprotected 6 and Boc deprotected 2a using the general
1
procedure for amide coupling reaction. White solid (45%); melting point, 87-91 °C; H NMR (400
MHz, CD OD) δ 7.69-7.66 (m, 4H), 7.47 - 7.34 (m, 6H), 7.29 - 7.09 (m, 9H), 7.09 - 6.98 (m, 1H),
3
4
2
.69 - 4.50 (m, 3H), 4.41- 4.33 (m, 2H), 3.69 (m, 4H), 3.35 (d, J = 11.4 Hz, 1H), 3.18 - 3.04 (m,
H), 3.04 - 2.97 (m, 2H), 2.96 - 2.83 (m, 2H), 2.62 - 2.42 (m, 6H), 2.01 - 1.86 (m, 2H), 1.72 - 1.72
(m, 2H), 1.57-1.44 (m, 3H), 1.34 (t, J = 10.5 Hz, 1H), 0.99 (d, J = 0.7 Hz, 9H), 0.94 – 0.78 (m,
1
3
1
1
2H). C NMR (101 MHz, CD OD) δ 135.31, 135.24, 129.58, 129.57, 128.95, 128.08, 128.05,
3
27.91, 127.43, 127.41, 126.23, 125.66, 66.45, 63.45, 61.02, 53.88, 53.31, 52.55, 51.07, 48.20,
47.99, 47.84, 47.77, 47.56, 47.35, 47.13, 46.92, 40.47, 38.21, 37.40, 33.99, 31.66, 25.74, 24.88,
+
2
9
4.39, 22.49, 21.97, 20.56, 20.12; LCMS (ES+) m/z calcd for C H N O Si [M+H] 974.5, found:
5
6
76
5
8
74.5; Purity: ≥ 98% and retention time is 14.21 min by HPLC analysis.
2
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6
(
S)-N-((S)-1-((S)-1-((S)-2-(Methoxymethyl)oxiran-2-yl)-4-methyl-1-oxopentan-2-ylamino)-
1
-oxo-3-phenylpropan-2-yl)-4-methyl-2-((S)-2-(2-morpholinoacetamido)-4-
phenylbutanamido)pentanamide (8)
0
1
2
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6
7
8
9
0
1
2
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8
9
0
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0
1
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7
8
9
0
Compound 8 was prepared from benzyl deprotected 6 and Boc deprotected 2b using the general
1
procedure for amide coupling reaction. White solid (48%); melting point, 129-133 °C; H NMR
(400 MHz, CD OD) δ 7.28 - 7.22 (m, 2H), 7.22 - 7.08 (m, 7H), 7.08 - 7.00 (m, 1H), 4.66 - 4.57
3
(m, 1H), 4.51 (dd, J = 2.8, 10.9 Hz, 1H), 4.37 (ddd, J = 5.8, 9.0, 22.4 Hz, 2H), 4.19 (ddd, J = 0.7,
1
3
1.3, 16.9 Hz, 1H), 3.70 (td, J = 2.1, 4.8, 5.3 Hz, 4H), 3.29 (ddd, J = 1.3, 2.2, 4.1 Hz, 3H), 3.26 –
.11 (m, 2H), 3.09 (dt, J = 1.3, 5.3 Hz, 1H), 3.04 - 2.99 (m, 2H), 2.99 - 2.93 (m, 1H), 2.93 - 2.77
(m, 2H), 2.64 - 2.44 (m, 6H), 2.08 - 1.82 (m, 2H), 1.73 - 1.41 (m, 5H), 1.29 (ddd, J = 4.5, 10.8,
13
1
4.3 Hz, 1H), 0.95 - 0.70 (m, 12H). C NMR (101 MHz, CD OD) δ 205.96, 172.59, 172.24,
3
1
71.83, 170.93, 141.03, 136.75, 128.93, 128.08, 128.05, 127.91, 126.24, 125.67, 71.00, 66.45,
61.28, 61.02, 58.46, 53.84, 53.32, 52.54, 51.76, 50.74, 48.20, 47.99, 47.78, 47.56, 47.35, 47.14,
4
6.93, 40.46, 37.89, 37.35, 34.02, 31.65, 24.76, 24.37, 22.37, 21.96, 20.56, 20.05.; LCMS (ES+)
+
m/z calcd for C H N O [M+H] 750.4, found: 750.4; Purity: ≥ 96% and retention time is 10.14
4
1
60
5
8
min by HPLC analysis.
(S)-N-((S)-1-((S)-1-((R)-2-(Hydroxymethyl)oxiran-2-yl)-4-methyl-1-oxopentan-2-ylamino)-
1
-oxo-3-phenylpropan-2-yl)-4-methyl-2-((S)-2-(2-morpholinoacetamido)-4-
phenylbutanamido)pentanamide (9)
Compound 9 was prepared from compound 7 using the general procedure for TBDPS deprotection
1
reaction. White solid (64%); melting point, 118-122 °C; H NMR (400 MHz, CD OD) δ 7.30 -
3
7.21 (m, 2H), 7.21 - 7.09 (m, 7H), 7.09 - 7.01 (m, 1H), 4.61 (dd, J = 5.4, 8.7 Hz, 1H), 4.52 (dd, J
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=
2.9, 11.0 Hz, 1H), 4.36 (ddd, J = 5.6, 8.8, 19.5 Hz, 2H), 4.26 (d, J = 12.6 Hz, 1H), 3.79 – 3.66
(m, 4H), 3.45 (d, J = 12.6 Hz, 1H), 3.14 – 3.05 (m, 2H), 3.00 (dd, J = 5.0, 10.1 Hz, 3H), 2.86 (dd,
J = 8.7, 14.0 Hz, 1H), 2.63 - 2.42 (m, 6H), 2.06 - 1.83 (m, 2H), 1.72 - 1.42 (m, 5H), 1.29 (ddd, J
1
1
1
1
1
1
1
1
1
1
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2
2
2
2
2
2
2
2
2
3
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3
4
4
4
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4
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5
5
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0
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2
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5
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9
0
1
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9
0
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9
0
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7
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9
0
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2
3
4
5
6
7
8
9
0
13
=
3.9, 10.9, 14.5 Hz, 1H), 0.97 - 0.77 (m, 12H). C NMR (101 MHz, CD OD) δ 206.57, 172.59,
3
1
6
4
72.26, 171.80, 170.95, 141.03, 136.74, 128.92, 128.07, 128.05, 127.90, 126.23, 125.66, 66.45,
2.40, 61.02, 60.28, 53.85, 53.31, 52.55, 51.76, 50.74, 48.19, 48.05, 47.98, 47.84, 47.76, 47.55,
7.34, 47.12, 46.91, 40.43, 37.82, 37.35, 33.98, 31.64, 24.76, 24.37, 22.36, 21.94, 20.53, 20.02;
+
LCMS (ES+) m/z calcd for C H N O [M+H] 736.4, found: 736.4; Purity: ≥ 95% and retention
4
0
58
5
8
time is 13.18 min by HPLC analysis.
(S)-N-((S)-1-((S)-1-((S)-2-((Methoxymethoxy)methyl)oxiran-2-yl)-4-methyl-1-oxopentan-2-
ylamino)-1-oxo-3-phenylpropan-2-yl)-4-methyl-2-((S)-2-(2-morpholinoacetamido)-4-
phenylbutanamido)pentanamide (10).
To the solution of compound 9 (25 mg, 0.033 mmol) in DCM, MOM-Cl (13 µL, 0.165 mmol) and
DIEA (29 µL, 0.165 mmol) were added and the reaction mixture was stirred at room temperature
for 24 h. Solvent was removed under reduced pressure and residue was purified by flash column
chromatography using 5% methanol in DCM to yield 16 mg (60%) white solid as pure compound.
1
Melting point, 107-110 °C; H NMR (400 MHz, CD OD) δ 7.29 - 7.20 (m, 2H), 7.20 - 7.09 (m,
3
7
4
H), 7.09 - 6.99 (m, 1H), 4.64 - 4.58 (m, 1H), 4.58 - 4.55 (m, 2H), 4.51 (dd, J = 2.8, 10.9 Hz, 1H),
.42 - 4.31 (m, 3H), 3.70 (td, J = 2.1, 4.4 Hz, 4H), 3.32 (d, J = 11.4 Hz, 1H), 3.29 (p, J = 1.6 Hz,
3H), 3.11 (dd, J = 5.2, 14.3 Hz, 2H), 3.00 (dd, J = 5.3, 7.7 Hz, 3H), 2.91 – 2.76 (m, 2H), 2.65 –
2
–
.42 (m, 6H), 2.10 - 1.80 (m, 2H), 1.61 - 1.39 (m, 4H), 1.29 (ddd, J = 4.5, 10.9, 13.8 Hz, 2H), 0.97
1
3
0.75 (m, 12H). C NMR (101 MHz, CD OD) δ 209.84, 176.54, 176.20, 175.78, 174.88, 144.97,
3
140.69, 132.87, 132.02, 131.99, 131.85, 130.18, 129.60, 100.10, 70.38, 69.96, 65.09, 64.95, 58.11,
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4
7.77, 57.25, 56.48, 55.69, 54.64, 52.14, 51.97, 51.92, 51.78, 51.71, 51.50, 51.28, 51.07, 50.86,
4.38, 41.79, 41.29, 37.95, 35.59, 28.68, 28.31, 26.32, 25.90, 24.48, 23.98; LCMS (ES+) m/z calcd
+
for C H N O [M+H] 780.4, found: 780.4; Purity: ≥ 97% and retention time is 14.60 min by
4
2
62
5
9
0
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9
0
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8
9
0
HPLC analysis.
(S)-N-((S)-1-((S)-1-((S)-2-(((2-Methoxyethoxy)methoxy)methyl)oxiran-2-yl)-4-methyl-1-
oxopentan-2-ylamino)-1-oxo-3-phenylpropan-2-yl)-4-methyl-2-((S)-2-(2-
morpholinoacetamido)-4-phenylbutanamido)pentanamide (11)
To the solution of compound 9 (25 mg, 0.033 mmol) in DCM, MEM-Cl (19 µL, 0.165 mmol) and
DIEA (29 µL, 0.165 mmol) were added and the reaction mass was stirred at room temperature for
24 h. Solvent was removed under reduced pressure and residue was purified by flash column
chromatography using 5% methanol in DCM to yield 18 mg (64%) white solid as pure compound.
1
Melting point. 85-88 °C; H NMR (400 MHz, CD OD) δ 7.28 - 7.20 (m, 2H), 7.20 - 7.10 (m, 7H),
3
7.08 - 7.00 (m, 1H), 4.68 - 4.59 (m, 3H), 4.51 (dd, J = 2.8, 10.9 Hz, 1H), 4.42 - 4.30 (m, 3H), 3.70
(
td, J = 2.1, 4.7 Hz, 4H), 3.66 - 3.57 (m, 2H), 3.57 - 3.49 (m, 2H), 3.37 (dd, J = 0.7, 11.4 Hz, 1H),
.33 (dd, J = 0.8, 3.7 Hz, 1H), 3.29 (ddt, J = 1.3, 2.3, 3.3 Hz, 2H), 3.17 - 3.05 (m, 2H), 3.05 – 2.95
m, 3H), 2.86 (dd, J = 8.7, 14.0 Hz, 1H), 2.64 - 2.43 (m, 6H), 2.06 - 1.94 (m, 1H), 1.94 - 1.83 (m,
3
(
1
3
1
H), 1.72 - 1.63 (m, 1H), 1.60 - 1.42 (m, 4H), 1.34 - 1.23 (m, 2H), 0.95 - 0.77 (m, 12H). C NMR
(101 CD OD) δ 205.91, 172.59, 172.24, 171.82, 170.93, 141.04, 136.74, 128.93, 128.08, 128.06,
3
1
27.91, 126.24, 125.67, 95.20, 71.44, 66.52, 66.45, 66.07, 61.13, 61.03, 57.62, 53.83, 53.32, 52.54,
51.76, 50.69, 40.46, 37.91, 37.36, 34.02, 31.66, 24.75, 24.38, 22.38, 21.97, 20.56, 20.08; LCMS
+
(ES+) m/z calcd for C H N O [M+H] 824.4, found: 824.4; Purity: ≥ 95% and retention time
4
4
66
5
10
is 12.66 min by HPLC analysis.
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(
(S)-2-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-1-morpholino-2,5,8,11-tetraoxo-4-
phenethyl-3,6,9,12-tetraazahexadecanecarbonyl)oxiran-2-yl)methyl methanesulfonate (12)
A mixture of compound 9 (25 mg, 0.033 mmol), methanesulfonyl chloride (13 µL, 0.165 mmol)
and DIEA (29 µL, 0.165 mmol) in DCM was stirred at room temperature for 24 h. Solvent was
removed under reduced pressure and residue was purified by flash column chromatography using
1
1
1
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1
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1
1
1
2
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2
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5
5
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0
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8
9
0
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4
5
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7
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9
0
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
% methanol in DCM to yield 20 mg (64%) pure compound as white solid. Melting point, 87-90
1
°
C; H NMR (400 MHz, CD OD) δ 7.25 (dd, J = 6.6, 8.5 Hz, 2H), 7.21 – 7.11 (m, 7H), 7.11 –
3
7
1
.01 (m, 1H), 5.04 (d, J = 11.4 Hz, 1H), 4.61 (dd, J = 5.4, 8.8 Hz, 1H), 4.47 (dd, J = 3.0, 10.8 Hz,
H), 4.36 (ddd, J = 5.5, 8.8, 21.9 Hz, 2H), 4.03 (d, J = 11.6 Hz, 1H), 3.70 (td, J = 2.1, 4.5 Hz, 4H),
3.15 – 3.05 (m, 5H), 3.02 (d, J = 4.9 Hz, 2H), 2.86 (dd, J = 8.8, 14.0 Hz, 1H), 2.65 – 2.45 (m, 6H),
2
–
.06 – 1.95 (m, 1H), 1.95 – 1.84 (m, 1H), 1.68 (dd, J = 3.6, 6.7 Hz, 1H), 1.62 – 1.40 (m, 4H), 1.39
1
3
1.22 (m, 1H), 0.96 – 0.81 (m, 12H). C NMR (101 MHz, CD OD) δ 208.65, 176.56, 176.24,
3
175.85, 174.88, 144.97, 140.64, 132.86, 132.02, 132.01, 131.87, 130.21, 129.62, 72.69, 70.38,
6
5
4.94, 63.58, 57.75, 57.25, 56.52, 55.72, 54.25, 52.77, 52.14, 51.93, 51.71, 51.50, 51.29, 51.07,
0.86, 44.34, 41.77, 41.25, 39.76, 37.94, 35.60, 28.66, 28.31, 26.24, 25.90, 24.47, 23.97; LCMS
+
(ES+) m/z calcd for C H N O S [M+H] 814.4, found: 814.4; Purity: ≥ 96% and retention time
41 60
5
10
is 14.24 min by HPLC analysis.
Biological assay
Enzyme Kinetics Assays
Purified human 20S immunoproteasome (Boston Biochem) or RPMI 8226 cell lysates were diluted
in 20S proteasome assay buffer (20 mM Tris-HCl, 0.5 mM EDTA, 0.035% SDS, pH 8.0) and
incubated with various concentrations of each inhibitor for 1 h in a 96-well plate. The fluorogenic
substrates Suc-Leu-Leu-Val-Tyr-AMC (Bachem) or Ac-Pro-Ala-Leu-AMC (Boston Biochem)
2
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