Design, synthesis and biological evaluation of novel tripeptidyl epoxyketone derivatives constructed from β-amino acid as proteasome inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.04.011

A series of novel tripeptidyl epoxyketone derivatives constructed from β-amino acid were designed, synthesized and evaluated as proteasome inhibitors. All target compounds were tested for their proteasome inhibitory activities and selected compounds were tested for their anti-proliferation activities against two multiple myeloma (MM) cell lines RPMI 8226 and NCI-H929. Among them, eleven compounds exhibited proteasome inhibitory rates of more than 50% at the concentration of 1 μg/mL and nine compounds showed anti-proliferation activities with IC₅₀ values at low micromolar level. Compound 20h displayed the most potent proteasome inhibitory activities (IC₅₀: 0.11 ± 0.01 μM) and anti-proliferation activities with IC₅₀ values at 0.23 ± 0.01 and 0.17 ± 0.02 μM against two tested cell lines. Additionally, the poly-ubiquitin accumulation in the western blot analysis supported that proteasome inhibition in a cellular system was induced by compound 20h. All these experimental results confirmed that β-amino acid can be introduced as a building block for the development of proteasome inhibitors.

Full text of DOI:10.1016/j.bmc.2014.04.011

Bioorganic & Medicinal Chemistry 22 (2014) 2955–2965
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of novel tripeptidyl
epoxyketone derivatives constructed from b-amino acid
as proteasome inhibitors
a,
Jiankang Zhang a, , Jiayi Cao b, , Lei Xu b, Yubo Zhou b, Tao Liu a, Jia Li b, , Yongzhou Hu
⇑
⇑
a ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
b National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 February 2014
Revised 4 April 2014
Accepted 4 April 2014
Available online 13 April 2014
A series of novel tripeptidyl epoxyketone derivatives constructed from b-amino acid were designed, syn-
thesized and evaluated as proteasome inhibitors. All target compounds were tested for their proteasome
inhibitory activities and selected compounds were tested for their anti-proliferation activities against
two multiple myeloma (MM) cell lines RPMI 8226 and NCI-H929. Among them, eleven compounds exhib-
ited proteasome inhibitory rates of more than 50% at the concentration of 1
showed anti-proliferation activities with IC50 values at low micromolar level. Compound 20h displayed
the most potent proteasome inhibitory activities (IC50: 0.11 0.01 M) and anti-proliferation activities
with IC50 values at 0.23 0.01 and 0.17 0.02 M against two tested cell lines. Additionally, the poly-
lg/mL and nine compounds
Keywords:
l
Proteasome inhibitors
b-Amino acid
Epoxyketone
Anti-cancer
Drug design
SARs
l
ubiquitin accumulation in the western blot analysis supported that proteasome inhibition in a cellular
system was induced by compound 20h. All these experimental results confirmed that b-amino acid
can be introduced as a building block for the development of proteasome inhibitors.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
its longer and irreversible inhibition of the proteasome.14 Herein,
development of carfilzomib analogue with epoxyketone fragment
The functionally active proteasome is a proteolytic complex that
is responsible for digestion of about 80–90% intracellular proteins
including misfolded and abnormal proteins as well as various
regulatory proteins associated with cell growth, differentiation,
and apoptosis into short peptides and amino acids.1–5 The promi-
nent roles of proteasome in protein degradation has made it a
promising anti-cancer drug target.6–8 To date, various proteasome
inhibitors have been identified and developed, and a few of them
are extensively evaluated in clinical trials with possibilities for clin-
ical applications.9
The approval of proteasome inhibitors bortezomib and
carfilzomib (Fig. 1) for the treatment of multiple myeloma (MM)
validates the close correlation between this proteolytic particle
and tumorigenesis, maintenance and progression.10,11 Compared
to bortezomib, the peptidyl epoxyketone compound carfilzomib
causes fewer side effects, especially low rates of peripheral neu-
ropathy.12,13 In addition, carfilzomib was proved to be able to
overcome bortezomib resistance in some cancer models due to
may lead to proteasome inhibitors with enhanced safety profiles.
To best of our knowledge, most proteasome inhibitors
approved or under clinical trials are short peptides sharing a sim-
ilar
a-peptide skeleton. It is reported that adding an extra back-
bone carbon by introducing a b-amino acid building block could
improve the biological activity and enzymatic stability of the
compound.15 Besides, Zhu and colleagues reported some b-amino
acid contained dipeptidyl boronic acid compounds with similar
proteasome inhibitory activities and even better pharmacokinetic
profiles in comparison with bortezomib, which validated the
remarkable value of this building block.16,17 In this manuscript,
33 tripeptidyl epoxyketone proteasome inhibitors (Fig. 2) con-
structed from b-amino acid (i.e. 3-amino-3-phenylpropionic acid
in this paper) were synthesized and evaluated, and structure–
activity relationships (SARs) were discussed in detail.
2. Results and discussion
2.1. Chemistry
⇑
Corresponding authors. Tel.: +86 21 5080 1552; fax: +86 21 5080 0721 (J.L.);
tel./fax: +86 571 8820 8460 (Y.H.).
The epoxyketone fragment 7 was synthesized following the
method described in the literature with modifications,18,19 and
E-mail addresses: jli@simm.ac.cn (J. Li), huyz@zju.edu.cn (Y. Hu).
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2014.04.011
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

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J. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 2955–2965
Figure 1. Structures of approved proteasome inhibitors bortezomib and carfilzomib.
to reaction with corresponding acid to obtain target compounds
10a–e, 13a–e, 15a–e, 18a–n, 20f, 20h, 22f and 22h.
2.2. Proteasome inhibitory activities
The synthesized target compounds were evaluated for their
20S proteasome chymotrypsin-like inhibitory activities in vitro.
Bortezomib and carfilzomib were employed as the positive con-
trols. The results are summarized in Tables 1–3.
As shown in Table 1, compounds (18a–e) with b-amino
acid-Phe-Leu skeleton exhibited the best proteasome inhibitory
activities, and four compounds (18b–e) showed inhibitory rates
Figure 2. Epoxyketone tripeptidyl compounds constructed from b-amino acid. R:
various substitutions at terminal positions of the tripeptides EK: epoxyketone.
of more than 80% at the concentration of 1
ues of 0.34 0.03, 0.44 0.04, 0.38 0.03, and 0.34 0.04
l
g/mL, with IC50 val-
M,
l
the synthetic routes are summarized in Scheme 1. Reaction of
N-Boc-protected amino acid 1 with N,O-dimethylhydroxylamine
hydrochloride gave Weinreb amide 2, which was then treated
with isopropenylmagnesium bromide at 0 °C to form the desired
respectively. However, compounds (10a–e, 13a–e and 15a–e)
with b-amino acid in the middle of the tripeptidyl skeleton
(Leu-b-amino acid-Phe, Leu-b-amino acid-Leu and Phe-b-amino
acid-Leu) displayed weak proteasome inhibitory activities. There-
fore, the peptidyl skeleton played important roles in maintaining
the activities of this series of compounds.
a
,b-unsaturated ketone 3.18 Subsequent reduction of
3
with
sodium borohydride and cerium chloride afforded allylic alcohol
4, which was oxidized into epoxide
5 in the presence of
In order to intensively evaluate the influences of substituents
at the end of carboxamide in compounds 18a–e on proteasome
inhibitory activity, compounds 18f–n with various substituents
at the end of carboxamide were synthesized. According to the
data of inhibitory activities of compounds 18a–n (Tables 1 and
2), compounds 18b–h with phenyl or pyridyl moieties at the
end of carboxamide showed more potent activities than that of
compounds with five-membered aromatic heterocyclic rings
(18j–k) and aliphatic heterocyclic rings (18l–n) at the end of car-
boxamide. In addition, various replacements on the phenyl ring
(18c–g) only have a slight impact on activity.
mCPBA.18,19 The epoxide 5 was not stable enough and was
thereby oxidized into epoxyketone 6 directly with Dess–Martin
reagent.18 Afterwards, deprotection of 6 with trifluoroacetic acid
resulted in compound 7.
The synthetic routes for tripeptidyl epoxyketone derivatives
are summarized in Schemes 2 and 3. The racemic and enantio-
meric N-Boc-protected b-amino acids were purchased from com-
mercial sources. Reaction of epoxyketone 7a or 7b with
corresponding N-Boc-protected amino acid furnished dipeptides
8, 11 or 16, which were deprotected and treated with various
N-Boc-protected amino acids again to afford tripeptides 9, 12,
14, 17, 19 or 21. Finally, the Boc-protecting group of the tripep-
tides were removed and the generated products were subjected
Further study of the impact of stereo configuration of the
b-amino acid on activity was performed with selected two stereo
isomers (20f vs 22f; 20h vs 22h) of compound 18f and 18h. As
Scheme 1. Synthesis of epoxyketone fragments (7a and 7b). Reagents and conditions: (I) HOBt, EDCI, N,O-dimethylhydroxylamine hydrochloride, diisopropylethylamine,
DCM, 0 °C; (II) isopropenylmagnesium bromide, THF, 0 °C; (III) NaBH4, CeCl3Á7H2O, MeOH, THF, 0 °C–rt; (IV) mCPBA, DCM, 0 °C; (V) Dess–Martin periodinane, DCM, 0 °C–rt;
(VI) trifluoroacetic acid, DCM, 0 °C–rt.

J. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 2955–2965
2957
Scheme 2. Synthesis of tripeptidyl target compounds (10a–e, 13a–e, 15a–e, and 18a–n). Reagents and conditions: (I) Boc-3-amino-3-phenylpropionic acid, HOBt, EDCI,
diisopropylethylamine, DCM, rt; (II) trifluoroacetic acid, DCM, 0 °C–rt; (III) Boc-Leu, HOBt, EDCI, diisopropylethylamine, DCM, rt; (IV) RCOOH, HOBt, EDCI, diisopropyleth-
ylamine, DCM, rt; (V) Boc-Phe, HOBt, EDCI, diisopropylethylamine, DCM, rt.
shown in Table 3, the R-isomers 20f (84.44 1.93% inhibition at
g/mL) and 20h (86.08 1.48% inhibition at 1 g/mL) exhibited
much more potent inhibitory activities than that of corresponding
S-isomers 22f (25.63 1.49% inhibition at 1 g/mL) and 22h
(11.76 6.86% inhibition at 1 g/mL). The results suggested that
and two R-configuration compounds 20f and 20h exhibited the
most potent cytotoxic activities (Table 4).
1
l
l
l
2.4. Western blot analysis
l
the stereo configuration of the b-amino acid was also a critical
factor for the activities of these compounds.
Subsequently, the proteasome inhibitory effect of selected com-
pound 20h was tested in a cellular system by Western blot analysis.
Carfilzomib and compound 15e were employed as the positive and
negative control, respectively. As illustrated inFigure 3, poly-ubiqui-
tin is clearly accumulated in RPMI 8226 cells treated withcompound
20h, which indicated that the compound induced proteasomal dys-
function in the cells. Additionally, cleavage of poly (ADP-ribose)
polymerase (PARP), a critical marker of apoptosis,20 was observed,
suggesting that the apoptotic pathway was activated by 20h. On
the contrary, the negative compound 15e was unable to accumulate
poly-ubiquitin and induce PARP cleavage, which was mainly attrib-
uted to its poor proteasome inhibitory activity.
2.3. Tumor cell growth inhibition
Based on the evaluation of proteasome chymotrypsin-like
inhibitory activities, selected compounds (18b–h, 20f, and 20h)
were further tested for their cytotoxic activities in vitro against
two MM cell lines (RPMI 8226 and NCI-H929) by MTS assay with
bortezomib and carfilzomib employed as the positive controls. All
the tested compounds showed potent cytotoxic activities against
two MM cell lines, with IC50 values in low micromolar range,

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J. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 2955–2965
Scheme 3. Synthesis of tripeptidyl target compounds (20f, 20h, 22f, and 22h). Reagents and conditions: (I) trifluoroacetic acid, DCM, 0 °C–rt; (II) Boc-(R)-3-amino-3-
phenylpropionic acid, HOBt, EDCI, diisopropylethylamine, DCM, rt; (III) RCOOH, HOBt, EDCI, diisopropylethylamine, DCM, rt; (IV) Boc-(S)-3-amino-3-phenylpropionic acid,
HOBt, EDCI, diisopropylethylamine, DCM, rt.
Table 1
20S proteasome chymotrypsin-like inhibitory activities of tripeptidyl target compounds (10a–e, 13a–e, 15a–e, and 18a–e)
a
a
Compound
R
Inhibitory rate at
g/mLa (%)
IC50
(l
M)
Compound
R
Inhibitory rate at
g/mLa (%)
IC50 (lM)
1
l
1 l
10a
10b
10c
10d
10e
Pyrazin-2-yl
Pyridin-3-yl
Phenyl
4-Fluorophenyl
4-Methoxyphenyl
8.70 0.36
3.16 0.39
1.56 0.32
1.43 0.19
0.39 0.03
NTb
NTb
NTb
NTb
NTb
13a
13b
13c
13d
13e
Pyrazin-2-yl
Pyridin-3-yl
Phenyl
4-Fluorophenyl
4-Methoxyphenyl
9.01 0.01
0.84 0.22
6.42 1.96
5.46 0.92
0.56 0.71
NTb
NTb
NTb
NTb
NTb
15a
15b
15c
15d
15e
Bortezomib
Pyrazin-2-yl
Pyridin-3-yl
Phenyl
4-Fluorophenyl
4-Methoxyphenyl
—
1.51 0.10
0.48 0.04
0.30 0.87
0.37 0.33
0.30 0.69
—
NTb
NTb
NTb
NTb
NTb
18a
18b
18c
18d
18e
Carfilzomib
Pyrazin-2-yl
Pyridin-3-yl
Phenyl
4-Fluorophenyl
4-Methoxyphenyl
—
48.61 1.16
85.76 4.61
88.55 7.10
87.72 3.56
84.12 5.55
—
NTb
0.34 0.03
0.44 0.04
0.38 0.03
0.34 0.04
8.60 1.40 (nM)
9.77 0.89 (nM)
a
The inhibitory rate and IC50 values are an average of three independent determinations.
NT = not tested.
b
inhibitory effects. This study validated the potential of developing
b-amino acid constructed peptides as potent proteasome inhibi-
tors, which might be helpful in further studies.
3. Conclusion
A series of novel tripeptidyl epoxyketone derivatives were syn-
thesized and evaluated for their proteasome inhibitory activities.
Among which, eleven compounds displayed potent proteasome
4. Experimental procedures
4.1. Chemistry
inhibitory activities, with IC50 values lower than 1 lM, which were
consistent with the cytotoxic activities against two MM cell lines
RPMI 8226 and NCI-H929. In addition, the western blot analysis
of selected compound 20h suggested that proteasome inhibition
caused an accumulation of poly-ubiquitin and cleavage of PARP,
which resulted in cell apoptosis, thus explaining the cell growth
Melting points were determined on a Büchi B-540 apparatus
(Büchi Labortechnik, Flawil, Switzerland) and are uncorrected. 1H
and 13C NMR spectra were recorded on Brüker 500 MHz spectrom-

J. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 2955–2965
2959
Table 2
Table 4
20S proteasome chymotrypsin-like inhibitory activities of tripeptidyl target com-
In vitro cytotoxic activities of selected compounds (18b-h, 20f, and 20h) against two
pounds (18f–n)
MM cell lines
Compound
Cytotoxicity (IC50, l
M)a
RPMI 8226
NCI-H929
18b
18c
18d
18e
18f
18g
18h
20f
4.73 0.81
1.68 0.08
1.55 0.12
1.16 0.04
0.32 0.08
0.41 0.09
0.42 0.01
0.34 0.01
0.23 0.01
8.70 0.27
2.69 0.07
2.37 0.13
1.35 0.01
0.96 0.06
0.61 0.03
0.70 0.04
0.23 0.01
0.17 0.02
a
Compound
R
Inhibitory rate at
g/mLa (%)
IC50
(l
M)
20h
1
l
Bortezomib
Carfilzomib
7.96 0.11 (nM)
13.19 0.56 (nM)
10.85 0.51 (nM)
21.32 0.83 (nM)
18f
4-
76.05 0.06
0.22 0.01
(Trifluoromethyl)phenyl
4-Cyanophenyl
2-Naphthyl
Biphenyl-4-yl
2-Furyl
a
The IC50 values are an average of three independent determinations.
18g
18h
18i
18j
18k
18l
18m
18n
Bortezomib
72.00 2.64
74.83 0.25
47.28 1.59
42.27 0.47
62.46 3.92
69.55 19.22
48.48 2.41
39.97 4.23
—
0.36 0.02
0.19 0.01
NTb
NTb
2-Thienyl
0.95 0.07
0.63 0.03
NTb
4-Tetrahydropyranyl
1-Acetylpiperidine-4-yl
N-Morpholinomethyl
—
NTb
8.96 0.56
(nM)
a
The inhibitory rate and IC50 values are an average of three independent
determinations.
b
NT = not tested.
Table 3
20S proteasome chymotrypsin-like inhibitory activities of tripeptidyl target com-
pounds constructed from enantiomeric b-amino acid (20f, 20h, 22f, and 22h)
Figure 3. Immunoblotting analysis of poly-ubiquitin, PARP, and cPARP in RPMI
8226 cells treated with carfilzomib, DMSO (control), or selected b-amino acid
derivatives (15e and 20h).
singlet (s), broad singlet (brs), doublet (d), triplet (t), quartet (q)
and multiplet (m). Mass spectral data were obtained by Esquire-
LC-00075 spectrometer (Brüker Bioscience). Reagents and solvents
were purchased from common commercial suppliers and were
used without further purification unless stated otherwise. Column
chromatography was performed using silica gel (300–400 mesh).
All yields are unoptimized and generally represent the result of a
single experiment.
a
Compound
R
Inhibitory rate
at 1
g/mLa (%)
IC50
(
lM)
l
20f
20h
22f
22h
4-(Trifluoromethyl)phenyl 84.44 1.93
2-Naphthyl 86.08 1.48
4-(Trifluoromethyl)phenyl 25.63 1.49
2-Naphthyl
—
0.17 0.01
0.11 0.01
NTb
11.76 6.86
—
NTb
4.1.1. General procedure for the synthesis of
ketone (3)
a,b-unsaturated
Bortezomib
7.14 0.36 (nM)
To a suspension of Boc-protected amino acid 1 (10.0 mmol) in
DCM (45.0 mL), HOBt (10.0 mmol) and EDCI (15.0 mmol) were
added at 0 °C. The reaction mixture was kept at the same tempera-
ture and stirred for 30 min. Then N,O-dimethylhydroxylamine
hydrochloride (10.0 mmol) and diisopropylethylamine (25.0 mmol)
were added. After stirring at 0 °C for 3 h, the resulting mixture was
washed with aqueous NaHCO3 solution (2 Â 20 mL), brine
(1 Â 20 mL), and dried over Na2SO4. The solvent was evaporated
under reduced pressure and the residue obtained was used in the
next step without further purification.
a
The inhibitory rate and IC50 values are an average of three independent
determinations.
b
NT = not tested.
eter (Brüker Bioscience, Billerica, MA, USA) with CDCl3 or DMSO-d6
as solvent. Chemical shifts (d) were reported in partsper million
(ppm) relative to internal TMS, and coupling constants (J) were
reported in Hertz (Hz). Splitting patterns were designated as

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J. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 2955–2965
To a solution of isopropenylmagnesium bromide (15 mmol,
7.5 Hz, CH2), 1.50 (s, 3H, CH3), 1.36 (s, 9H, CH3); ESI-MS:
m/z = 306 [M+H]+.
0.5 M in THF) was added a solution of Weinreb amide 2 (10 mmol)
in dry THF (50 mL) dropwise at 0 °C under an atmosphere of nitro-
gen. The reaction mixture was stirred at the same temperature
overnight and then poured into 100 mL of saturated NH4Cl solu-
tion. The solution was acidified to pH 2.0 with 6 N HCl, and the
mixture was extracted with EtOAc (3 Â 30 mL). The combined
organic layers were washed with aqueous NaHCO3 (1 Â 30 mL),
brine (1 Â 30 mL), and dried over Na2SO4. The solvent was evapo-
rated in vacuo and the crude product was purified by flash column
chromatography (ethyl acetate–petroleum ether = 1:20) to give
compound 3.
4.1.2.2. tert-Butyl [(1S)-3-methyl-1-{[(2R)-2-methyloxiran-2-
yl]carbonyl}butyl]carbamate (6b).
Colorless oil; yield: 52%
(3 steps); 1H NMR (500 MHz, CDCl3): d = 4.84 (d, 1H, J = 8.5 Hz,
NH), 4.31 (m, 1H, CH), 3.28 (d, 1H, J = 4.5 Hz, OCH2), 2.87 (d, 1H,
J = 4.5 Hz, OCH2), 1.71 (m, 1H, CH), 1.51 (s, 3H, CH3), 1.46 (m, 1H,
CH2), 1.40 (s, 9H, CH3), 1.16 (m, 1H, CH2), 0.94 (dd, 6H, J = 16.5,
6.5 Hz, CH3); ESI-MS: m/z = 272 [M+H]+.
4.1.3. General procedure for the synthesis of deprotected
epoxyketone (7)
4.1.1.1. S-tert-Butyl 4-methyl-3-oxo-1-phenylpent-4-en-2-ylcar-
To a solution of epoxyketone fragment 6 (5.0 mmol) in DCM
(20.0 mL) was added trifluoroacetic acid (5.0 mL) dropwise at
0 °C. The reaction mixture was then warmed to room temperature
and stirred for 1 h. The volatiles were evaporated and the crude 7
salt was put in the next step without further purification.
bamate (3a).
White solid; yield: 75% (2 steps); mp: 92–93 °C;
1H NMR (500 MHz, CDCl3): d = 7.26 (m, 3H, Ar-H), 7.06 (m, 2H, Ar-
H), 6.01 (d, 1H, J = 1.5 Hz, @CH2), 5.85 (d, 1H, J = 1.5 Hz, @CH2), 5.27
(m, 2H, NH+CH), 3.02 (m, 2H, CH2), 1.86 (s, 3H, CH3), 1.41 (s, 9H,
CH3); ESI-MS: m/z = 290 [M+H]+.
4.1.4. General procedure for the synthesis of dipeptidyl
intermediates 8, 11, and 16
4.1.1.2. tert-Butyl [(1S)-3-methyl-1-(2-methylpropyl)-2-oxobut-
3-en-1-yl]carbamate (3b).
Colorless oil; yield: 72% (2 steps);
To a suspension of Boc-protected b-amino acid or Phe (2.0 mmol)
in DCM (8.0 mL), HOBt (2.0 mmol) and EDCI (3.0 mmol) were added.
After stirring at room temperature for 30 min, corresponding amine
7a or 7b (2.0 mmol) and diisopropylethylamine (4.0 mmol) were
added. The reaction mixture was stirred for another 3 h and washed
with aqueous NaHCO3 solution (2 Â 10 mL), brine (1 Â 10 mL), and
dried over Na2SO4. The solvent was evaporated in vacuo and the
obtained residue was purified by flash column chromatography
(ethyl acetate–petroleum ether = 1:6–1:4).
1H NMR (500 MHz, CDCl3): d = 6.07 (s, 1H, @CH2), 5.87 (s, 1H,
@CH2), 5.14 (d, 1H, J = 8.0 Hz, NH), 5.05 (m, 1H, CH), 1.89 (s, 3H,
CH3), 1.72 (m, 1H, CH), 1.46 (m, 1H, CH2), 1.42 (s, 9H, CH3), 1.32
(m, 1H, CH2), 0.98 (d, 3H, J = 6.5 Hz, CH3), 0.90 (d, 3H, J = 6.5 Hz,
CH3); ESI-MS: m/z = 256 [M+H]+.
4.1.2. General procedure for the synthesis of Boc-protected
epoxyketone (6)
To a solution of compound 3 (5.0 mmol) in MeOH (10.0 mL) and
THF (10.0 mL) was added CeCl3Á7H2O (7.0 mmol) under an atmo-
sphere of nitrogen at 0 °C. Then NaBH4 (7.0 mmol) was added.
The reaction mixture was kept at 0 °C for 2 h and quenched with
glacial acetic acid (4.0 mL). The volatiles were evaporated under
reduced pressure, and the residue was diluted with H2O
(20.0 mL) and extracted with EtOAc (3 Â 15 mL). The combined
organic layers were washed with H2O (1 Â 30 mL), brine
(1 Â 30 mL), and dried over Na2SO4. The solvent was evaporated
in vacuo and the crude product 4 was used in the next step without
further purification.
4.1.4.1. tert-Butyl 3-[S-1-(R-2-methyloxiran-2-yl)-1-oxo-3-phen
ylpropan-2-ylamino]-3-oxo-1-phenylpropylcarbamate
(8).
White solid; yield: 91%; mp: 144–146 °C; 1H NMR
(500 MHz, CDCl3): d = 7.29 (m, 8H, Ar-H), 6.99 (m, 2H, Ar-H), 6.08
and 5.99 (br s, 50:50, 1H, NH), 5.90 (m, 1H, CH), 4.99 (br s, 1H,
NH), 4.85 (m, 1H, CH), 2.93 and 2.82 (m, 50:50, 1H, CH2), 2.68
(m, 4H, CH2 + OCH2), 2.60 and 2.47 (d, 50:50, 1H, J = 4.5 Hz,
OCH2), 1.43 (s, 3H, CH3), 1.41 (s, 9H, CH3); ESI-MS: m/z = 453
[M+H]+.
To a solution of alcohol 4 (5.0 mmol) in DCM (20.0 mL) was
added mCPBA (5.5 mmol) at 0 °C. The reaction mixture was stirred
at the same temperature for 3 h, and then warmed to room tem-
perature. The solution was washed with aqueous NaHCO3
(2 Â 30 mL), brine (1 Â 30 mL), and dried over Na2SO4. The solvent
was evaporated under reduced pressure and the crude product 5
was put in the next step without further purification.
To a solution of abovementioned compound 5 (5.0 mmol) in
DCM (20.0 mL) was added Dess–Martin periodinane (7.5 mmol)
in DCM (15.0 mL) at 0 °C under an atmosphere of nitrogen. The
reaction was stirred at 0 °C overnight. The resulting mixture was
poured into aqueous NaHCO3 solution (30 mL), and filtrated. The
layers were separated, and the aqueous layer was extracted with
EtOAc (3 Â 30 mL). The combined organic layers were washed with
H2O (2 Â 20 mL), brine (1 Â 20 mL), and dried over Na2SO4. The
solvent was evaporated under reduced pressure. Purification by
flash column chromatography (ethyl acetate–petroleum
ether = 1:15–1:10) gave epoxyketone 6.
4.1.4.2. tert-Butyl 3-[S-4-methyl-1-(R-2-methyloxiran-2-yl)-1-o
xopentan-2-ylamino]-3-oxo-1-phenylpropylcarbamate
(11).
White solid; yield: 92%; mp: 62–64 °C; 1H NMR
(500 MHz, CDCl3): d = 7.27 (m, 5H, Ar-H), 6.19 and 5.80 (m,
60:40, 1H, NH), 5.79 (m, 1H, NH), 5.06 (m, 1H, CH), 4.55 (m, 1H,
CH), 3.25 and 3.20 (d, 60:40, 1H, J = 5.0 Hz, OCH2), 2.89 and 2.87
(d, 60:40, 1H, J = 5.0 Hz, OCH2), 2.70 (m, 2H, CH2), 1.49 and 1.48
(s, 60:40, 3H, CH3), 1.43 and 1.41 (s, 60:40, 9H, CH3), 1.38 (m,
1H, CH2), 1.11 (m, 2H, CH + CH2), 0.89 and 0.81 (m, 60:40, 6H,
CH3); ESI-MS: m/z = 419 [M+H]+.
4.1.4.3. tert-Butyl (S)-1-{S-4-methyl-1-[R-2-methyloxiran-2-yl]-
1-oxopentan-2-ylamino}-1-oxo-3-phenylpropan-2-ylcarbamate
(16).
White solid; yield: 87%; mp: 151–153 °C; 1H NMR
(500 MHz, CDCl3): d = 7.20 (m, 5H, Ar-H), 6.18 (d, 1H, J = 6.5 Hz,
NH), 4.95 (d, 1H, J = 6.5 Hz, NH), 4.57 (m, 1H, CH), 4.32 (m, 1H,
CH), 3.24 (d, 1H, J = 4.5 Hz, OCH2), 3.03 (m, 2H, CH2), 2.88 (d, 1H,
J = 4.5 Hz, OCH2), 1.63 (m, 1H, CH), 1.49 (s, 3H, CH3), 1.46 (m, 1H,
CH2), 1.43 (s, 9H, CH3), 1.17 (m, 1H, CH2), 0.92 (d, 3H, J = 6.5 Hz,
CH3), 0.87 (d, 3H, J = 6.5 Hz, CH3); ESI-MS: m/z = 419 [M+H]+.
4.1.2.1. tert-Butyl S-1-(R-2-methyloxiran-2-yl)-1-oxo-3-phenyl-
propan-2-ylcarbamate (6a).
White solid; yield: 39% (3
steps); mp: 86–87 °C; 1H NMR (500 MHz, CDCl3): d = 7.28 (m, 3H,
Ar-H), 7.16 (m, 2H, Ar-H), 4.93 (d, 1H, J = 8.0 Hz, NH), 4.58 (m,
1H, CH), 3.28 (d, 1H, J = 5.0 Hz, OCH2), 3.10 (dd, 1H, J = 13.5,
5.0 Hz, CH2), 2.90 (d, 1H, J = 5.0 Hz, OCH2), 2.73 (dd, 1H, J = 13.5,
4.1.5. General procedure for the synthesis of tripeptidyl interme
diates 9, 12, 14, 17, 19, and 21
To a solution of dipeptidyl fragment 8, 11, or 16 (5.0 mmol) in
DCM (20.0 mL) was added trifluoroacetic acid (5.0 mL) dropwise

J. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 2955–2965
2961
at 0 °C. The reaction mixture was then warmed to room tempera-
ture and stirred for 1 h. The volatiles were evaporated and the
crude amine salt was put in the next step without further
purification.
5.91 (m, 2H, NH), 4.99 (br s, 1H, CH), 4.48 (m, 2H, CH), 3.17 (d,
1H, J = 5.0 Hz, OCH2), 2.87 (m, 3H, OCH2 + CH2), 2.69 (m, 2H,
CH2), 1.49 (s, 3H, CH3), 1.41 (m, 11H, CH2 + CH3), 1.12 (m, 1H,
CH), 0.86 (m, 6H, CH3); ESI-MS: m/z = 566 [M+H]+.
To a suspension of corresponding Boc-protected amino acid
(2.0 mmol) in DCM (8.0 mL), HOBt (2.0 mmol) and EDCI (3.0 mmol)
were added. After stirring at room temperature for 30 min, afore-
mentioned amine salt (2.0 mmol) and diisopropylethylamine
(4.0 mmol) were added. The reaction mixture was stirred for
another 3 h and was washed with aqueous NaHCO3 solution
(2 Â 10 mL), brine (1 Â 10 mL), and dried over Na2SO4. The solvent
was evaporated in vacuo and the obtained residue was purified by
flash column chromatography (ethyl acetate–petroleum
ether = 1:3–2:1).
4.1.5.6. tert-Butyl (S)-3-{S-1-[S-4-methyl-1-(R-2-methyloxiran-
2-yl)-1-oxopentan-2-ylamino]-1-oxo-3-phenylpropan-2-ylami-
no}-3-oxo-1-phenylpropylcarbamate (21).
White solid;
yield: 73%; mp: 135–137 °C; 1H NMR (500 MHz, CDCl3): d = 7.26
(m, 8H, Ar-H), 7.01 (d, 2H, J = 7.5 Hz, Ar-H), 6.24 (br s, 1H, NH),
5.98 (m, 2H, NH), 5.00 (br s, 1H, CH), 4.51 (m, 1H, CH), 4.46 (m,
1H, CH), 3.15 (d, 1H, J = 5.0 Hz, OCH2), 2.87 (m, 2H, OCH2 + CH2),
2.63 (m, 3H, CH2), 1.48 (s, 3H, CH3), 1.42 (m, 11H, CH2 + CH3),
1.11 (m, 1H, CH), 0.86 (dd, 6H, J = 13.5, 6.0 Hz, CH3); ESI-MS:
m/z = 566 [M+H]+.
4.1.5.1. tert-Butyl (S)-4-methyl-1-{3-[S-1-(R-2-methyloxiran-2-
yl)-1-oxo-3-phenylpropan-2-ylamino]-3-oxo-1-phenylpropyla-
4.1.6. General procedure for the synthesis of target compounds
10a–e, 13a–e, 15a–e, 18a–n, 20f, 20h, 22f, and 22h
mino}-1-oxopentan-2-ylcarbamate (9).
White solid; yield:
80%; mp: 158–160 °C; 1H NMR (500 MHz, CDCl3): d = 7.67 and
7.62 (d, 50:50, 1H, J = 8.0 Hz, NH), 7.25 (m, 8H, Ar-H), 7.01 (m,
2H, Ar-H), 6.48 and 5.89 (d, 50:50, 1H, J = 7.0 Hz, NH), 5.38 and
5.27 (m, 50:50, 1H, NH), 5.02 and 4.90 (m, 50:50, 1H, CH), 4.87
(m, 1H, CH), 4.15 (br s, 1H, CH), 2.93 (m, 1H, CH2), 2.66 (m, 4H,
CH2 + OCH2), 2.55 and 2.40 (d, 50:50, 1H, J = 4.5 Hz, OCH2), 1.69
(m, 2H, CH2), 1.45 (m, 13H, CH + CH3), 0.95 (m, 6H, CH3); ESI-MS:
m/z = 566 [M+H]+.
To a solution of tripeptidyl fragment 9, 12, 14, 17, 19, or 21
(5.0 mmol) in DCM (20.0 mL) was added trifluoroacetic acid
(5.0 mL) dropwise at 0 °C. The reaction mixture was then warmed
to room temperature and stirred for 1 h. The volatiles were evapo-
rated and the crude amine salt was put in the next step without
further purification.
To a suspension of corresponding acid (2.0 mmol) in DCM
(8.0 mL), HOBt (2.0 mmol) and EDCI (3.0 mmol) were added. After
stirring at room temperature for 30 min, aforementioned amine
salt (2.0 mmol) and diisopropylethylamine (4.0 mmol) were added.
The reaction mixture was stirred for another 3 h and was washed
with aqueous NaHCO3 solution (2 Â 10 mL), brine (1 Â 10 mL),
and dried over Na2SO4. The solvent was evaporated in vacuo and
the obtained residue was purified by flash column chromatography
(ethyl acetate–petroleum ether = 1:3–10:1).
4.1.5.2. tert-Butyl (S)-4-methyl-1-{3-[S-4-methyl-1-(R-2-methyl
oxiran-2-yl)-1-oxopentan-2-ylamino]-3-oxo-1-phenylpropylam
ino}-1-oxopentan-2-ylcarbamate (12).
White solid; yield:
88%; mp: 185–187 °C; 1H NMR (500 MHz, CDCl3): d = 7.99 and
7.50 (d, 50:50, 1H, J = 8.5 Hz, NH), 7.28 (m, 5H, Ar-H), 6.64 and
5.90 (d, 50:50, 1H, J = 6.5 Hz, NH), 5.50 and 5.26 (m, 50:50, 1H,
NH), 4.95 (m, 1H, CH), 4.53 (m, 1H, CH), 4.19 (m, 1H, CH), 3.27
and 3.25 (d, 50:50, 1H, J = 5.0 Hz, OCH2), 2.87 (m, 1H, OCH2), 2.68
(m, 2H, CH2), 1.71 (m, 2H, CH2), 1.48 (m, 14H, CH2 + CH3), 1.22
(m, 2H, CH2), 0.95 (m, 6H, CH3), 0.87 (m, 6H, CH3); ESI-MS: m/
z = 532 [M+H]+.
4.1.6.1.
xo-3-phenylpropan-2-ylamino]-3-oxo-1-phenylpropylamino}-1
-oxopentan-2-yl}pyrazine-2-carboxamide (10a). White
N-{S-4-Methyl-1-{3-[S-1-(R-2-methyloxiran-2-yl)-1-o
solid; yield: 75%; 1H NMR (500 MHz, CDCl3): d = 9.36 (s, 1H, pyra-
zine-H), 8.75 and 8.73 (d, 50:50, 1H, J = 2.0 Hz, pyrazine-H), 8.53
and 8.46 (d, 50:50, 1H, J = 2.0 Hz, pyrazine-H), 8.24 and 8.17 (d,
50:50, 1H, J = 7.5 Hz, NH), 7.86 and 7.78 (d, 50:50, 1H, J = 8.0 Hz,
NH), 7.25 (m, 8H, Ar-H), 7.00 (m, 2H, Ar-H), 6.44 and 5.93 (d,
50:50, 1H, J = 7.5 Hz, NH), 5.39 and 5.29 (m, 50:50, 1H, CH), 4.85
and 4.79 (q, 50:50, 1H, J = 7.0 Hz, CH), 4.68 (m, 1H, CH), 2.91 (m,
1H, CH2), 2.65 (m, 4H, CH2 + OCH2), 2.52 and 2.40 (d, 50:50, 1H,
J = 4.5 Hz, OCH2), 1.80 (m, 3H, CH + CH2), 1.43 and 1.40 (s, 50:50,
3H, CH3), 0.98 (m, 6H, CH3); ESI-MS: m/z = 572 [M+H]+.
4.1.5.3. tert-Butyl (S)-1-{3-[S-4-methyl-1-(R-2-methyloxiran-2-
yl)-1-oxopentan-2-ylamino]-3-oxo-1-phenylpropylamino}-1-ox
o-3-phenylpropan-2-ylcarbamate (14).
White solid; yield:
82%; mp: 167–169 °C; 1H NMR (500 MHz, CDCl3): d = 7.69 and
6.96 (d, 50:50, 1H, J = 8.5 Hz, NH), 7.23 (m, 10H, Ar-H), 6.36 and
5.80 (br s, 50:50, 1H, NH), 5.15 (m, 2H, NH + CH), 4.50 (m, 2H,
CH + CH), 3.23 and 3.17 (d, 50:50, 1H, J = 5.0 Hz, OCH2), 3.06 (m,
2H, CH2), 2.85 and 2.83 (d, 50:50, 1H, J = 5.0 Hz, OCH2), 2.63 (m,
2H, CH2), 1.65 (m, 1H, CH), 1.50 and 1.46 (s, 50:50, 3H, CH3),
1.40 (m, 11H, CH2 + CH3), 0.86 (m, 6H, CH3); ESI-MS: m/z = 566
[M+H]+.
4.1.6.2.
xo-3-phenylpropan-2-ylamino]-3-oxo-1-phenylpropylamino}-1
-oxopentan-2-yl}nicotinamide (10b). White solid; yield:
N-{S-4-Methyl-1-{3-[S-1-(R-2-methyloxiran-2-yl)-1-o
4.1.5.4. tert-Butyl 3-{S-1-[S-4-methyl-1-(R-2-methyloxiran-2-
yl)-1-oxopentan-2-ylamino]-1-oxo-3-phenylpropan-2-ylami-
56%; 1H NMR (500 MHz, CDCl3): d = 9.13 and 9.10 (s, 50:50, 1H,
pyridine-H), 8.69 (s, 1H, pyridine-H), 8.21 (m, 2H, pyridine-
H + NH), 7.75 (d, 1H, J = 2.5 Hz, pyridine-H), 7.41 and 7.37 (m,
50:50, 1H, NH), 7.26 (m, 8H, Ar-H), 7.02 (m, 2H, Ar-H), 6.83 and
6.41 (br s, 50:50, 1H, NH), 5.43 and 5.27 (q, 50:50, 1H, J = 6.5 Hz,
CH), 4.78 (m, 2H, CH + CH), 2.76 (m, 5H, CH2 + OCH2), 2.43 and
2.39 (d, 50:50, 1H, J = 4.5 Hz, OCH2), 1.74 (m, 3H, CH + CH2), 1.42
and 1.38 (s, 50:50, 3H, CH3), 0.95 (m, 6H, CH3); ESI-MS:
m/z = 571 [M+H]+.
no}-3-oxo-1-phenylpropylcarbamate (17).
White solid;
yield: 73%; mp: 95–97 °C; 1H NMR (500 MHz, CDCl3): d = 7.28
(m, 8H, Ar-H), 7.12 (m, 1H, Ar-H), 7.02 (m, 1H, Ar-H), 6.13 (m,
1H, NH), 5.86 (m, 2H, NH), 4.98 (m, 1H, CH), 4.48 (m, 2H, CH),
3.19 and 3.16 (d, 45:55, 1H, J = 5.0 Hz, OCH2), 2.87 (m, 5H, OCH2 + -
CH2), 1.58 (s, 9H, CH3), 1.49 and 1.48 (s, 3H, CH3), 1.42 (m, 3H,
CH + CH2), 0.87 (m, 6H, CH3); ESI-MS: m/z = 566 [M+H]+.
4.1.5.5. tert-Butyl (R)-3-{S-1-[S-4-methyl-1-(R-2-methyloxiran-
4.1.6.3.
N-{S-4-Methyl-1-{3-[S-1-(R-2-methyloxiran-2-yl)-1-o
2-yl)-1-oxopentan-2-ylamino]-1-oxo-3-phenylpropan-2-ylamin
xo-3-phenylpropan-2-ylamino]-3-oxo-1-phenylpropylamino}-1
o}-3-oxo-1-phenylpropylcarbamate
(19).
White
solid;
-oxopentan-2-yl}benzamide (10c).
White solid; yield: 49%;
yield: 65%; mp: 139–141 °C; 1H NMR (500 MHz, CDCl3): d = 7.28
(m, 8H, Ar-H), 7.11 (d, 2H, J = 7.5 Hz, Ar-H), 6.23 (br s, 1H, NH),
1H NMR (500 MHz, CDCl3): d = 7.81 (m, 2H, Ar-H), 7.45 (m, 3H,
Ar-H), 7.32 (m, 8H, Ar-H), 7.26 (m, 8H, Ar-H), 7.03 (m, 2H, Ar-H),

2962
J. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 2955–2965
6.85 (m, 1H, NH), 6.69 and 6.62 (d, 40:60, 1H, J = 7.5 Hz, NH), 6.02
and 5.95 (d, 40:60, 1H, J = 8.0 Hz, NH), 5.42 and 5.28 (m, 40:60, 1H,
CH), 4.77 (m, 2H, CH + CH), 2.89 (m, 1H, CH2), 2.74 (m, 4H, CH2 + -
OCH2), 2.53 and 2.42 (d, 60:40, 1H, J = 4.5 Hz, OCH2), 1.71 (m, 3H,
CH + CH2), 1.44 and 1.41 (s, 60:40, 3H, CH3), 0.99 (m, 6H, CH3);
ESI-MS: m/z = 570 [M+H]+.
J = 6.5 Hz, CH), 4.50 (m, 1H, CH), 3.26 and 3.24 (d, 50:50, 1H,
J = 5.0 Hz, OCH2), 2.85 and 2.82 (d, 50:50, 1H, J = 5.0 Hz, OCH2),
2.71 (m, 2H, CH2), 1.74 (m, 2H, CH2), 1.47 (m, 5H, CH2 + CH3),
1.19 (m, 2H, CH2), 0.99 (m, 6H, CH3), 0.86 (m, 6H, CH3); ESI-MS:
m/z = 536 [M+H]+.
4.1.6.9.
ethyloxiran-2-yl)-1-oxopentan-2-ylamino]-3-oxo-1-phenylprop
ylamino}-1-oxopentan-2-yl}benzamide (13d). White solid;
4-Fluoro-N-{S-4-methyl-1-{3-[S-4-methyl-1-(R-2-m
4.1.6.4. 4-Fluoro-N-{S-4-methyl-1-{3-[S-1-(R-2-methyloxiran-2-
yl)-1-oxo-3-phenylpropan-2-ylamino]-3-oxo-1-phenylpropyla-
mino}-1-oxopentan-2-yl}benzamide (10d).
White solid;
yield: 96%; 1H NMR (500 MHz, CDCl3): d = 8.11 and 7.83 (d,
50:50, 1H, J = 9.0 Hz, NH), 7.78 (m, 2H, Ar-H), 7.23 (m, 6H, Ar-
H + NH), 7.02 (m, 2H, Ar-H), 6.96 and 6.27 (m, 50:50, 1H, NH),
5.46 and 5.23 (m, 50:50, 1H, CH), 4.77 and 4.69 (m, 50:50, 1H,
CH), 4.50 (m, 1H, CH), 3.26 and 3.24 (d, 50:50, 1H, J = 5.0 Hz,
OCH2), 2.85 and 2.83 (d, 50:50, 1H, J = 5.0 Hz, OCH2), 2.70 (m, 2H,
CH2), 1.73 (m, 2H, CH2), 1.47 (m, 5H, CH2 + CH3), 1.25 (m, 2H,
CH2), 0.96 (m, 6H, CH3), 0.84 (m, 6H, CH3); ESI-MS: m/z = 554
[M+H]+.
yield: 56%; 1H NMR (500 MHz, CDCl3): d = 7.82 (m, 2H, Ar-H),
7.24 (m, 8H, Ar-H), 7.26 (m, 8H, Ar-H), 7.07 (m, 5H, Ar-H + NH),
6.89 and 6.73 (d, 50:50, 1H, J = 7.0 Hz, NH), 6.57 and 5.95 (d,
50:50, 1H, J = 7.0 Hz, NH), 5.41 and 5.26 (q, 50:50, 1H, J = 7.0 Hz,
CH), 4.79 (m, 2H, CH + CH), 2.91 (m, 1H, CH2), 2.68 (m, 4H, CH2 + -
OCH2), 2.50 and 2.42 (d, 50:50, 1H, J = 5.0 Hz, OCH2), 1.70 (m, 3H,
CH + CH2), 1.44 and 1.41 (s, 50:50, 3H, CH3), 0.98 (m, 6H, CH3);
ESI-MS: m/z = 588 [M+H]+.
4.1.6.5.
an-2-yl)-1-oxo-3-phenylpropan-2-ylamino]-3-oxo-1-phenylpro
pylamino}-1-oxopentan-2-yl}benzamide (10e). White
4-Methoxy-N-{S-4-methyl-1-{3-[S-1-(R-2-methyloxir
4.1.6.10. 4-Methoxy-N-{S-4-methyl-1-{3-[S-4-methyl-1-(R-2-m
ethyloxiran-2-yl)-1-oxopentan-2-ylamino]-3-oxo-1-phenylprop
ylamino}-1-oxopentan-2-yl}benzamide (13e).
White solid;
solid; yield: 46%; 1H NMR (500 MHz, CDCl3): d = 7.78 (m, 3H, Ar-
H + NH), 7.28 (m, 8H, Ar-H), 7.08 (m, 2H, Ar-H), 6.90 (t, 2H,
J = 9.0 Hz, Ar-H), 6.78 and 6.74 (d, 50:50, 1H, J = 6.5 Hz, NH), 6.66
and 6.05 (br s, 50:50, 1H, NH), 5.42 and 5.27 (q, 50:50, 1H,
J = 6.5 Hz, CH), 4.77 (m, 2H, CH + CH), 3.84 and 3.83 (s, 50:50, 3H,
CH3), 2.89 (m, 1H, CH2), 2.71 (m, 4H, CH2 + OCH2), 2.51 and 2.42
(d, 50:50, 1H, J = 4.5 Hz, OCH2), 1.75 (m, 3H, CH + CH2), 1.44 and
1.41 (s, 50:50, 3H, CH3), 0.97 (m, 6H, CH3); ESI-MS: m/z = 600
[M+H]+.
yield: 69%; 1H NMR (500 MHz, CDCl3): d = 8.06 and 7.31 (d,
50:50, 1H, J = 8.5 Hz, NH), 7.77 (m, 2H, Ar-H), 7.26 (m, 5H, Ar-H),
6.94 and 6.08 (d, 50:50, 1H, J = 8.0 Hz, NH), 6.91 (m, 2H, Ar-H),
6.75 (m, 1H, NH), 5.49 and 5.21 (q, 50:50, 1H, J = 7.0 Hz, CH),
4.78 and 4.67 (q, 50:50, 1H, J = 8.0 Hz, CH), 4.50 (m, 1H, CH), 3.84
and 3.83 (s, 3H, CH3), 3.25 (m, 1H, OCH2), 2.77 (m, 3H, OCH2 + CH2),
1.75 (m, 2H, CH2), 1.48 (m, 5H, CH2 + CH3), 1.22 (m, 2H, CH2), 0.96
(m, 6H, CH3), 0.85 (m, 6H, CH3); ESI-MS: m/z = 566 [M+H]+.
4.1.6.11. N-{S-1-{3-[S-4-Methyl-1-(R-2-methyloxiran-2-yl)-1-o
4.1.6.6. N-{S-4-Methyl-1-{3-[S-4-methyl-1-(R-2-methyloxiran-2
xopentan-2-ylamino]-3-oxo-1-phenylpropylamino}-1-oxo-3-p
-yl)-1-oxopentan-2-ylamino]-3-oxo-1-phenylpropylamino}-1-o
henylpropan-2-yl}pyrazine-2-carboxamide
(15a).
White
xopentan-2-yl}pyrazine-2-carboxamide (13a).
White solid;
solid; yield: 79%; 1H NMR (500 MHz, CDCl3): d = 9.33 (s, 1H, pyra-
zine-H), 8.74 and 8.72 (d, 50:50, 1H, J = 2.5 Hz, pyrazine-H), 8.53
and 8.50 (d, 50:50, 1H, J = 2.5 Hz, pyrazine-H), 8.36 (m, 1H, NH),
7.77 and 7.29 (d, 50:50, 1H, J = 9.0 Hz, NH), 7.23 (m, 9H, Ar-H),
6.93 (m, 1H, Ar-H), 6.46 and 5.82 (d, 50:50, 1H, J = 8.0 Hz, NH),
5.39 and 5.15 (q, 50:50, 1H, J = 6.0 Hz, CH), 4.87 (m, 1H, CH), 4.45
(m, 1H, CH), 3.21 (m, 3H, CH2 + OCH2), 2.82 (m, 1H, OCH2), 2.67
(m, 2H, CH2), 1.46 and 1.44 (s, 50:50, 3H, CH3), 1.39 (m, 2H, CH2),
1.18 (m, 1H, CH), 0.83 (m, 6H, CH3); ESI-MS: m/z = 572 [M+H]+.
yield: 73%; 1H NMR (500 MHz, CDCl3): d = 9.37 (m, 1H, pyrazine-
H), 8.77 and 8.75 (d, 60:40, 1H, J = 2.5 Hz, pyrazine-H), 8.56 and
8.53 (m, 60:40, 1H, pyrazine-H), 8.20 (m, 1H, NH), 8.12 and 7.62
(d, 60:40, 1H, J = 8.0 Hz, NH), 7.25 (m, 5H, Ar-H), 6.70 and 5.93
(d, 60:40, 1H, J = 8.0 Hz, NH), 5.49 and 5.28 (q, 60:40, 1H,
J = 6.0 Hz, CH), 4.74 and 4.66 (q, 40:60, 1H, J = 7.5 Hz, CH), 4.51
(m, 1H, CH), 3.24 and 3.22 (d, 60:40, 1H, J = 5.0 Hz, OCH2), 2.86
(m, 1H, OCH2), 2.70 (m, 2H, CH2), 1.77 (m, 2H, CH2), 1.47 (m, 5H,
CH2 + CH3), 1.25 (m, 2H, CH2), 1.01 (m, 6H, CH3), 0.88 (m, 6H,
CH3); ESI-MS: m/z = 538 [M+H]+.
4.1.6.12. N-{S-1-{3-[S-4-Methyl-1-(R-2-methyloxiran-2-yl)-1-o
xopentan-2-ylamino]-3-oxo-1-phenylpropylamino}-1-oxo-3-p
4.1.6.7. N-{S-4-Methyl-1-{3-[S-4-methyl-1-(R-2-methyloxiran-2
-yl)-1-oxopentan-2-ylamino]-3-oxo-1-phenylpropylamino}-1-o
henylpropan-2-yl}nicotinamide (15b).
White solid; yield:
92%; 1H NMR (500 MHz, CDCl3): d = 8.96 and 8.93 (d, 50:50, 1H,
J = 1.5 Hz, pyridine-H), 8.68 (m, 1H, pyridine-H), 8.04 (m, 1H, pyr-
idine-H), 7.84 and 7.52 (d, 50:50, 1H, J = 9.0 Hz, NH), 7.51 and 7.42
(d, 50:50, 1H, J = 7.0 Hz, NH), 7.34 (m, 1H, pyridine-H), 7.24 (m, 9H,
Ar-H), 7.00 (m, 1H, Ar-H), 6.67 and 6.07 (d, 50:50, 1H, J = 8.0 Hz,
NH), 5.41 and 5.18 (q, 50:50, 1H, J = 7.0 Hz, CH), 4.94 (m, 1H,
CH), 4.46 (m, 1H, CH), 3.21 (m, 3H, CH2 + OCH2), 2.83 and 2.80
(d, 50:50, 1H, J = 5.0 Hz, OCH2), 2.73 (m, 2H, CH2), 1.45 and 1.42
(s, 50:50, 3H, CH3), 1.39 (m, 2H, CH2), 1.22 (m, 1H, CH), 0.84 (m,
6H, CH3); ESI-MS: m/z = 571 [M+H]+.
xopentan-2-yl}nicotinamide (13b).
White solid; yield: 78%;
1H NMR (500 MHz, CDCl3): d = 9.02 (d, 1H, J = 11.0 Hz, pyridine-
H), 8.70 (m, 1H, pyridine-H), 8.10 (m, 1H, pyridine-H), 8.06 and
7.85 (d, 50:50, 1H, J = 8.0 Hz, NH), 7.49 and 7.30 (d, 50:50, 1H,
J = 7.5 Hz, NH), 7.40 (m, 1H, pyridine-H), 7.25 (m, 5H, Ar-H), 6.95
and 6.26 (d, 50:50, 1H, J = 7.5 Hz, NH), 5.48 and 5.21 (q, 50:50,
1H, J = 7.0 Hz, CH), 4.72 (m, 1H, CH), 4.49 (m, 1H, CH), 3.24 (m,
1H, OCH2), 2.84 (m, 1H, OCH2), 2.70 (m, 2H, CH2), 1.74 (m, 2H,
CH2), 1.46 (m, 5H, CH2 + CH3), 1.24 (m, 2H, CH2), 0.98 (m, 6H,
CH3), 0.83 (m, 6H, CH3); ESI-MS: m/z = 537 [M+H]+.
4.1.6.13. N-{S-1-{3-[S-4-Methyl-1-(R-2-methyloxiran-2-yl)-1-o
4.1.6.8. N-{S-4-Methyl-1-{3-[S-4-methyl-1-(R-2-methyloxiran-2
xopentan-2-ylamino]-3-oxo-1-phenylpropylamino}-1-oxo-3-p
-yl)-1-oxopentan-2-ylamino]-3-oxo-1-phenylpropylamino}-1-o
xopentan-2-yl}benzamide (13c).
NMR (500 MHz, CDCl3): d = 8.11 (m, 1H, NH), 7.80 (m, 2H, Ar-H),
7.45 (m, 3H, Ar-H), 7.30 (m, 5H, Ar-H), 7.03 and 6.94 (d, 50:50,
1H, J = 7.5 Hz, NH), 7.00 and 6.22 (br s, 50:50, 1H, NH), 5.47 and
5.23 (q, 50:50, 1H, J = 5.5 Hz, CH), 4.82 and 4.71 (q, 50:50, 1H,
henylpropan-2-yl}benzamide (15c).
White solid; yield: 88%;
White solid; yield: 75%; 1H
1H NMR (500 MHz, CDCl3): d = 7.78 and 7.47 (d, 50:50, 1H,
J = 8.5 Hz, NH), 7.73 (m, 2H, Ar-H), 7.37 (m, 3H, Ar-H), 7.25 (m,
9H, Ar-H), 6.99 (m, 2H, Ar-H + NH), 6.55 and 5.95 (d, 50:50, 1H,
J = 8.0 Hz, NH), 5.39 and 5.17 (q, 50:50, 1H, J = 7.5 Hz, CH), 4.91
(m, 1H, CH), 4.46 (m, 1H, CH), 3.23 (m, 3H, CH2 + OCH2), 2.79 (m,

J. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 2955–2965
2963
1H, OCH2), 2.59 (m, 2H, CH2), 1.45 and 1.43 (s, 50:50, 3H, CH3), 1.39
(m, 2H, CH2), 1.23 (m, 1H, CH), 0.84 (m, 6H, CH3); ESI-MS: m/
z = 570 [M+H]+.
CH), 4.46 (m, 1H, CH), 3.16 and 3.13 (d, 50:50, 1H, J = 5.0 Hz,
OCH2), 2.86 (m, 5H, OCH2 + CH2), 1.48 and 1.47 (s, 3H, CH3), 1.42
(m, 2H, CH2), 1.08 (m, 1H, CH), 0.86 (m, 6H, CH3); ESI-MS: m/
z = 570 [M+H]+.
4.1.6.14. 4-Fluoro-N-{S-1-{3-[S-4-methyl-1-(R-2-methyloxiran-
2-yl)-1-oxopentan-2-ylamino]-3-oxo-1-phenylpropylamino}-1-
1.1.6.19. 4-Fluoro-N-{3-{S-1-[S-4-methyl-1-(R-2-methyloxiran-2
oxo-3-phenylpropan-2-yl}benzamide (15d).
White solid;
-yl)-1-oxopentan-2-ylamino]-1-oxo-3-phenylpropan-2-ylamino
yield: 68%; 1H NMR (500 MHz, CDCl3): d = 7.81 and 7.50 (d,
50:50, 1H, J = 8.5 Hz, NH), 7.73 (m, 2H, Ar-H), 7.22 (m, 13H, Ar-
H + NH), 6.61 and 6.00 (d, 50:50, 1H, J = 8.0 Hz, NH), 5.39 and
5.18 (q, 50:50, 1H, J = 6.5 Hz, CH), 4.91 (m, 1H, CH), 4.46 (m, 1H,
CH), 3.20 (m, 3H, CH2 + OCH2), 2.80 (m, 1H, OCH2), 2.60 (m, 2H,
CH2), 1.45 and 1.43 (s, 50:50, 3H, CH3), 1.38 (m, 2H, CH2), 1.19
(m, 1H, CH), 0.83 (m, 6H, CH3); ESI-MS: m/z = 588 [M+H]+.
}-3-oxo-1-phenylpropyl}benzamide (18d).
yield: 66%; IR (KBr): 3291, 3065, 2959, 1722, 1643, 1543, 738,
700 cmÀ1 1H NMR (500 MHz, CDCl3): d = 8.58 and 8.23 (d, 55:45,
1H, J = 7.5 Hz, NH), 7.88 (m, 2H, Ar-H), 7.29 (m, 4H, Ar-H), 7.23
(m, 4H, Ar-H), 7.10 (m, 3H, Ar-H), 6.92 (m, 1H, Ar-H), 6.33 (m,
1H, NH), 5.93 (m, 1H, NH), 5.50 (m, 1H, CH), 4.54 (m, 1H, CH),
4.46 (m, 1H, CH), 3.15 and 3.12 (d, 45:55, 1H, J = 5.0 Hz, OCH2),
2.87 (m, 5H, OCH2 + CH2), 1.48 and 1.47 (s, 3H, CH3), 1.43 (m, 2H,
CH2), 1.08 (m, 1H, CH), 0.85 (m, 6H, CH3); ESI-MS: m/z = 588
[M+H]+.
White solid;
;
4.1.6.15. 4-Methoxy-N-{S-1-{3-[S-4-methyl-1-(R-2-methyloxir
an-2-yl)-1-oxopentan-2-ylamino]-3-oxo-1-phenylpropylamin
o}-1-oxo-3-phenylpropan-2-yl}benzamide
(15e).
White
solid; yield: 84%; 1H NMR (500 MHz, CDCl3): d = 7.77 and 7.42 (d,
50:50, 1H, J = 9.0 Hz, NH), 7.66 (m, 2H, Ar-H), 7.24 (m, 9H, Ar-
H + NH), 6.87 (m, 4H, Ar-H), 6.62 and 5.96 (d, 50:50, 1H,
J = 7.5 Hz, NH), 5.39 and 5.16 (q, 50:50, 1H, J = 6.0 Hz, CH), 4.93
and 4.86 (q, 50:50, 1H, J = 7.5 Hz, CH), 4.46 (m, 1H, CH), 3.83 and
3.82 (s, 50:50, 3H, CH3), 3.21 (m, 3H, CH2 + OCH2), 2.80 (m, 1H,
OCH2), 2.59 (m, 2H, CH2), 1.47 and 1.45 (s, 50:50, 3H, CH3), 1.42
(m, 2H, CH2), 1.23 (m, 1H, CH), 0.84 (m, 6H, CH3); ESI-MS: m/
z = 600 [M+H]+.
4.1.6.20. 4-Methoxy-N-{3-{S-1-[S-4-methyl-1-(R-2-methyloxir
an-2-yl)-1-oxopentan-2-ylamino]-1-oxo-3-phenylpropan-2-yla
mino}-3-oxo-1-phenylpropyl}benzamide
solid; yield: 79%; IR (KBr): 3292, 3068, 2960, 1725, 1644, 1545,
748, 701 cmÀ1 1H NMR (500 MHz, CDCl3): d = 8.38 and 8.04 (d,
(18e).
White
;
50:50, 1H, J = 7.5 Hz, NH), 7.83 (m, 2H, Ar-H), 7.29 (m, 4H, Ar-H),
7.22 (m, 4H, Ar-H), 7.10 (m, 1H, Ar-H), 6.93 (m, 3H, Ar-H), 6.33
(m, 1H, NH), 5.96 (m, 1H, NH), 5.51 (m, 1H, CH), 4.54 (m, 1H,
CH), 4.47 (m, 1H, CH), 3.85 and 3.84 (s, 3H, CH3), 3.16 and 3.13
(d, 50:50, 1H, J = 5.0 Hz, OCH2), 2.85 (m, 5H, OCH2 + CH2), 1.48
and 1.47 (s, 3H, CH3), 1.42 (m, 2H, CH2), 1.08 (m, 1H, CH), 0.85
(m, 6H, CH3); ESI-MS: m/z = 600 [M+H]+.
4.1.6.16. N-{3-{S-1-[S-4-Methyl-1-(R-2-methyloxiran-2-yl)-1-o
xopentan-2-ylamino]-1-oxo-3-phenylpropan-2-ylamino}-3-oxo
-1-phenylpropyl}pyrazine-2-carboxamide
solid; yield: 92%; IR (KBr): 3294 cm-1, 3063 cm-1, 2959 cm-1
1721 cm-1, 1645 cm-1, 1519 cm-1, 746 cm-1, 700 cm-1 1H NMR
(18a).
White
,
4.1.6.21. N-{3-{S-1-[S-4-Methyl-1-(R-2-methyloxiran-2-yl)-1-o
xopentan-2-ylamino]-1-oxo-3-phenylpropan-2-ylamino}-3-ox
o-1-phenylpropyl}-4-(trifluoromethyl)benzamide
;
(500 MHz, CDCl3): d = 9.44 and 9.06 (d, 55:45, 1H, J = 8.0 Hz, NH),
9.37 and 9.35 (d, 55:45, J = 1.0 Hz, pyrazine-H), 8.74 (m, 1H, pyra-
zine-H), 8.60 and 8.57 (m, 55:45, 1H, pyrazine-H), 7.33 (m, 4H, Ar-
H), 7.28 (m, 1H, Ar-H), 7.18 (m, 3H, Ar-H), 7.11 (m, 1H, Ar-H), 6.95
(m, 1H, Ar-H), 6.27 (m, 1H, NH), 5.89 and 5.84 (d, 55:45, 1H,
J = 8.0 Hz, NH), 5.56 (m, 1H, CH), 4.59 (m, 1H, CH), 4.45 (m, 1H,
CH), 3.15 and 3.13 (d, 45:55, 1H, J = 5.0 Hz, OCH2), 2.85 (m, 5H,
OCH2 + CH2), 1.48 (s, 3H, CH3), 1.41 (m, 2H, CH2), 1.11 (m, 1H,
CH), 0.86 (m, 6H, CH3); ESI-MS: m/z = 572 [M+H]+.
(18f).
White solid; yield: 71À%1; IR (KBr): 3303, 3067, 2962,
1724, 1646, 1544, 743, 702 cm
;
1H NMR (500 MHz, CDCl3):
d = 8.77 and 8.43 (d, 50:50, 1H, J = 7.5 Hz, NH), 7.98 (m, 2H, Ar-
H), 7.70 (m, 2H, Ar-H), 7.28 (m, 8H, Ar-H), 7.11 (m, 1H, Ar-H),
6.93 (m, 1H, Ar-H), 6.34 (m, 1H, NH), 5.87 (d, 1H, J = 8.0 Hz, NH),
5.52 (m, 1H, CH), 4.56 (m, 1H, CH), 4.46 (m, 1H, CH), 3.13 and
3.11 (d, 50:50, 1H, J = 5.0 Hz, OCH2), 2.86 (m, 5H, OCH2 + CH2),
1.48 (s, 3H, CH3), 1.42 (m, 2H, CH2), 1.09 (m, 1H, CH), 0.83 (m,
6H, CH3); ESI-MS: m/z = 638 [M+H]+.
4.1.6.17. N-{3-{S-1-[S-4-Methyl-1-(R-2-methyloxiran-2-yl)-1-o
xopentan-2-ylamino]-1-oxo-3-phenylpropan-2-ylamino}-3-oxo
4.1.6.22. 4-Isocyano-N-{3-{S-1-[S-4-methyl-1-(R-2-methyloxir
-1-phenylpropyl}nicotinamide (18b).
67%; IR (KBr): 3294 cm-1 3063 cm-1
1648 cm-1, 1541 cm-1, 749 cm-1, 700 cm-1
White solid; yield:
2958 cm-1 1719 cm-1
1H NMR (500 MHz,
an-2-yl)-1-oxopentan-2-ylamino]-1-oxo-3-phenylpropan-2-yla
,
,
,
,
mino}-3-oxo-1-phenylpropyl}benzamide
solid; yield: 57%; IR (KBr): 3296, 3064, 2958, 1721, 1645, 1541,
700 cmÀ1 1H NMR (500 MHz, CDCl3): d = 8.87 and 8.54 (d, 50:50,
(18g).
White
;
CDCl3): d = 9.30 and 9.20 (s, 50:50, 1H, pyridine-H), 8.89 and 8.83
(d, 50:50, 1H, J = 7.5 Hz, NH), 8.72 (m, 1H, pyridine-H), 8.35 and
8.27 (d, 50:50, 1H, J = 7.5 Hz, pyridine-H), 7.46 (m, 1H, pyridine-
H), 7.31 (m, 4H, Ar-H), 7.15 (m, 5H, Ar-H), 6.95 (m, 1H, Ar-H),
6.57 and 6.10 (d, 50:50, 1H, J = 7.5 Hz, NH), 6.38 (m, 1H, NH),
5.56 (m, 1H, CH), 4.57 (m, 1H, CH), 4.40 (m, 1H, CH), 3.12 and
3.08 (d, 50:50, 1H, J = 5.0 Hz, OCH2), 2.84 (m, 5H, OCH2 + CH2),
1.47 (m, 5H, CH2 + CH3), 1.10 (m, 1H, CH), 0.83 (m, 6H, CH3); ESI-
MS: m/z = 571 [M+H]+.
;
1H, J = 8.0 Hz, NH), 7.99 (dd, 2H, J = 17.0, 8.5 Hz, Ar-H), 7.75 (t,
2H, J = 8.0 Hz, Ar-H), 7.29 (m, 8H, Ar-H), 7.11 (d, 1H, J = 7.0 Hz,
Ar-H), 6.92 (m, 1H, Ar-H), 6.24 and 6.21 (d, 50:50, 1H, J = 7.5 Hz,
NH), 5.78 (m, 1H, NH), 5.52 (m, 1H, CH), 4.51 (m, 2H, CH + CH),
3.13 and 3.10 (d, 50:50, 1H, J = 5.0 Hz, OCH2), 2.89 (m, 5H, OCH2 + -
CH2), 1.49 (s, 3H, CH3), 1.44 (m, 2H, CH2), 1.09 (m, 1H, CH), 0.84 (m,
6H, CH3); ESI-MS: m/z = 595 [M+H]+.
4.1.6.23. N-{3-{S-1-[S-4-Methyl-1-(R-2-methyloxiran-2-yl)-1-o
4.1.6.18. N-{3-{S-1-[S-4-Methyl-1-(R-2-methyloxiran-2-yl)-1-o
xopentan-2-ylamino]-1-oxo-3-phenylpropan-2-ylamino}-3-ox
xopentan-2-ylamino]-1-oxo-3-phenylpropan-2-ylamino}-3-ox
o-1-phenylpropyl}-2-naphthamide (18h).
White
solid;
o-1-phenylpropyl}benzamide (18c).
White solid; yield: 65%;
yield: 54%; IR (KBr): 3289, 3063, 2957, 1721, 1643, 1535, 699 cm
À1
IR (KBr): 3287, 3066À1, 2959, 1724, 1644, 1538, 737, 701 cmÀ1; 1H
NMR (500 MHz, CDCl3): d = 8.53 and 8.17 (d, 50:50, 1H, J = 8.0 Hz,
NH), 7.89 (m, 2H, Ar-H), 7.43 (m, 3H, Ar-H), 7.30 (m, 4H, Ar-H),
7.20 (m, 4H, Ar-H), 7.11 (m, 1H, Ar-H), 6.92 (m, 1H, Ar-H), 6.32
(m, 1H, NH), 5.92 (m, 1H, NH), 5.54 (m, 1H, CH), 4.55 (m, 1H,
;
1H NMR (500 MHz, CDCl3): d = 8.62 and 8.26 (d, 50:50, 1H,
J = 7.5 Hz, NH), 7.90 (m, 4H, Ar-H), 7.55 (m, 2H, Ar-H), 7.35 (m,
5H, Ar-H), 7.23 (m, 5H, Ar-H), 6.96 (m, 1H, Ar-H), 6.32 (m, 1H,
NH), 5.91 (m, 1H, NH), 5.61 (m, 1H, CH), 4.57 (m, 1H, CH), 4.45
(m, 1H, CH), 3.13 (m, 1H, OCH2), 2.91 (m, 5H, OCH2 + CH2), 1.47

2964
J. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 2955–2965
(s, 3H, CH3), 1.40 (m, 2H, CH2), 1.07 (m, 1H, CH), 0.81 (m, 6H, CH3);
(m, 2H, CH2), 1.49 and 1.48 (s, 50:50, 3H, CH3), 1.42 (m, 2H, CH2),
ESI-MS: m/z = 620 [M+H]+.
1.11 (m, 1H, CH), 0.85 (m, 6H, CH3); ESI-MS: m/z = 619 [M+H]+.
4.1.6.24. N-{3-{S-1-[S-4-Methyl-1-(R-2-methyloxiran-2-yl)-1-o
4.1.6.29. (S)-N-[S-4-Methyl-1-(R-2-methyloxiran-2-yl)-1-oxope
xopentan-2-ylamino]-1-oxo-3-phenylpropan-2-ylamino}-3-ox
ntan-2-yl]-2-[3-(2-morpholinoacetamido)-3-phenylpropanami-
o-1-phenylpropyl}biphenyl-4-carboxamide
solid; yield: 47%; IR (KBr): 3287, 3061, 2954, 1721, 1647, 1538,
699 cmÀ1 1H NMR (500 MHz, CDCl3): d = 8.56 and 8.21 (d, 50:50,
1H, J = 7.5 Hz, NH), 7.96 (m, 2H, Ar-H), 7.62 (m, 4H, Ar-H), 7.45
(m, 2H, Ar-H), 7.39 (m, 9H, Ar-H), 7.12 (m, 1H, Ar-H), 6.95 (m,
1H, Ar-H), 6.29 (m, 1H, NH), 5.88 (m, 1H, NH), 5.56 (m, 1H, CH),
4.57 (m, 1H, CH), 4.47 (m, 1H, CH), 3.16 and 3.13 (d, 50:50, 1H,
J = 5.0 Hz, OCH2), 2.90 (m, 5H, OCH2 + CH2), 1.49 and 1.47 (s,
50:50, 3H, CH3), 1.41 (m, 2H, CH2), 1.09 (m, 1H, CH), 0.83 (m, 6H,
CH3); ESI-MS: m/z = 646 [M+H]+.
(18i).
White
do]-3-phenylpropanamide (18n).
White solid; yield: 63%; IR
(KBr): 3285, 3067, 2951, 1721, 1647, 1541, 700 cm À1
;
1H NMR
;
(500 MHz, CDCl3): d = 8.57 and 7.33 (d, 50:50, 1H, J = 7.5 Hz, NH),
7.28 (m, 8H, Ar-H), 7.13 (m, 1H, Ar-H), 7.02 (m, 1H, Ar-H), 6.40
(m, 1H, NH), 6.29 and 6.02 (d, 50:50, 1H, J = 7.5 Hz, NH), 5.33 (m,
1H, CH), 4.57 (m, 1H, CH), 4.49 (m, 1H, CH), 3.76 (m, 4H, CH2),
3.20 and 3.18 (d, 50:50, 1H, J = 5.0 Hz, OCH2), 2.92 (m, 5H, OCH2 + -
CH2), 2.70 (m, 2H, CH2), 2.58 (m, 4H, CH2), 1.48 (s, 3H, CH3), 1.42
(m, 2H, CH2), 1.15 (m, 1H, CH), 0.86 (m, 6H, CH3); ESI-MS: m/
z = 593 [M+H]+.
4.1.6.25. N-{3-{S-1-[S-4-Methyl-1-(R-2-methyloxiran-2-yl)-1-o
4.1.6.30. N-{R-3-{S-1-[S-4-Methyl-1-(R-2-methyloxiran-2-yl)-1-
xopentan-2-ylamino]-1-oxo-3-phenylpropan-2-ylamino}-3-ox
oxopentan-2-ylamino]-1-oxo-3-phenylpropan-2-ylamino}-3-o
o-1-phenylpropyl}furan-2-carboxamide (18j).
White solid;
xo-1-phenylpropyl}-4-(trifluoromethyl)
(20f). White solid; yield: 63%; IR (KBr): 3308, 3065, 2961,
1721, 1645, 1538, 699 cmÀ1 1H NMR (500 MHz, CDCl3): d = 8.42
benzamide
yield: 48%; IR (KBr): 3288, 3067, 2957, 1724, 1645, 1532, 700 cm
À1
;
1H NMR (500 MHz, CDCl3): d = 8.32 and 7.94 (d, 50:50, 1H,
;
J = 8.0 Hz, NH), 7.48 and 7.45 (d, 50:50, 1H, J = 1.5 Hz, furan-H),
7.29 (m, 10H, Ar-H+furan-H), 6.95 (m, 1H, Ar-H), 6.49 (m, 1H,
furan-H), 6.32 (m, 1H, NH), 5.96 (m, 1H, NH), 5.49 (m, 1H, CH),
4.58 (m, 1H, CH), 4.46 (m, 1H, CH), 3.17 and 3.14 (d, 50:50, 1H,
J = 5.0 Hz, OCH2), 2.89 (m, 5H, OCH2 + CH2), 1.48 (s, 3H, CH3),
1.41 (m, 2H, CH2), 1.09 (m, 1H, CH), 0.85 (m, 6H, CH3); ESI-MS:
m/z = 560 [M+H]+.
(d, 1H, J = 7.5 Hz, NH), 7.96 (d, 2H, J = 8.5 Hz, Ar-H), 7.68 (d, 2H,
J = 9.0 Hz, Ar-H), 7.28 (m, 8H, Ar-H), 7.09 (d, 2H, J = 7.0 Hz, Ar-H),
6.31 (d, 1H, J = 6.0 Hz, NH), 5.90 (d, 1H, J = 8.0 Hz, NH), 5.51 (m,
1H, CH), 4.53 (q, 1H, J = 7.0 Hz, CH), 4.45 (m, 1H, CH), 3.13 (d, 1H,
J = 5.0 Hz, OCH2), 2.94 (m, 2H, CH2), 2.85 (d, 1H, J = 5.0 Hz, OCH2),
2.78 (m, 2H, CH2), 1.48 (s, 3H, CH3), 1.42 (m, 2H, CH2), 1.08 (m,
1H, CH), 0.83 (dd, 6H, J = 10.0, 6.0 Hz, CH3) ppm; 13C NMR
(125 MHz, CDCl3): d = 207.82, 170.71, 170.23, 165.28, 140.64,
137.50, 135.99, 129.21, 128.82, 128.70, 127.72, 127.67, 127.21,
126.13, 125.58, 124.81, 122.64, 58.90, 54.44, 52.22, 50.89, 50.21,
41.30, 40.17, 38.36, 25.02, 23.22, 21.26, 16.61; ESI-MS: m/z = 638
[M+H]+.
4.1.6.26. N-{3-{S-1-[S-4-Methyl-1-(R-2-methyloxiran-2-yl)-1-o
xopentan-2-ylamino]-1-oxo-3-phenylpropan-2-ylamino}-3-ox
o-1-phenylpropyl}thiophene-2-carboxamide (18k).
solid; yield: 56%; IR (KBr): 3296, 3064, 2957, 1721, 1644, 1538,
700 cmÀ1 1H NMR (500 MHz, CDCl3): d = 8.37 and 8.04 (d, 50:50,
White
;
1H, J = 8.0 Hz, NH), 7.62 (d, 1H, J = 3.0 Hz, thiophene-H), 7.48 (t,
1H, J = 5.0 Hz, thiophene-H), 7.24 (m, 10H, Ar-H + thiophene-H),
6.94 (m, 1H, Ar-H), 6.29 (m, 1H, NH), 5.89 (m, 1H, NH), 5.49 (m,
1H, CH), 4.53 (m, 2H, CH + CH), 3.16 and 3.13 (d, 50:50, 1H,
J = 4.5 Hz, OCH2), 2.86 (m, 5H, OCH2 + CH2), 1.48 (s, 3H, CH3),
1.43 (m, 2H, CH2), 1.09 (m, 1H, CH), 0.85 (m, 6H, CH3); ESI-MS:
m/z = 576 [M+H]+.
4.1.6.31. N-{R-3-{S-1-[S-4-Methyl-1-(R-2-methyloxiran-2-yl)-1-
oxopentan-2-ylamino]-1-oxo-3-phenylpropan-2-ylamino}-3-
oxo-1-phenylpropyl}-2-naphthamide (20h).
White solid;
yield: 54%; IR (KBr): 3292, 3061, 2958, 1720, 1643, 1533, 699 cm
À1
;
1H NMR (500 MHz, CDCl3): d = 8.37 (s, 1H, Ar-H), 8.28 (d, 1H,
J = 7.5 Hz, NH), 7.88 (m, 4H, Ar-H), 7.54 (m, 2H, Ar-H), 7.37 (d,
2H, J = 7.5 Hz, Ar-H), 7.30 (t, 2H, J = 7.5 Hz, Ar-H), 7.22 (m, 4H,
Ar-H), 7.10 (d, 2H, J = 7.0 Hz, Ar-H), 6.38 (d, 1H, J = 6.0 Hz, NH),
5.95 (d, 1H, J = 7.0 Hz, NH), 5.57 (m, 1H, CH), 4.57 (q, 1H,
J = 7.0 Hz, CH), 4.45 (m, 1H, CH), 3.13 (d, 1H, J = 5.0 Hz, OCH2),
2.95 (m, 2H, CH2), 2.84 (m, 3H, OCH2 + CH2), 1.47 (s, 3H, CH3),
1.40 (m, 2H, CH2), 1.05 (m, 1H, CH), 0.81 (dd, 6H, J = 15.0, 6.0 Hz,
CH3) ppm; 13C NMR (125 MHz, CDCl3): d = 207.83, 170.73,
170.30, 166.76, 140.97, 136.12, 134.85, 132.67, 131.40, 129.24,
129.10, 128.78, 128.66, 128.39, 127.82, 127.72, 127.65, 127.56,
127.14, 126.66, 126.25, 123.76, 58.90, 54.42, 52.24, 50.91, 50.15,
41.62, 40.15, 38.30, 25.01, 23.21, 21.29, 16.62; ESI-MS: m/z = 620
[M+H]+.
4.1.6.27. N-{3-{S-1-[S-4-Methyl-1-(R-2-methyloxiran-2-yl)-1-o
xopentan-2-ylamino]-1-oxo-3-phenylpropan-2-ylamino}-3-ox
o-1-phenylpropyl}-tetrahydro-2H-pyran-4-carboxamide
(18l).
White solid; yield: 66%; IR (KBr): 3293, 3064, 2957,
1721, 1647, 1541, 700 cmÀ1
;
1H NMR (500 MHz, CDCl3): d = 7.71
and 7.40 (d, 50:50, 1H, J = 7.5 Hz, NH), 7.29 (m, 8H, Ar-H), 7.11
(m, 1H, Ar-H), 6.93 (m, 1H, Ar-H), 6.21 (d, 1H, J = 7.0 Hz, NH),
5.84 (m, 1H, NH), 5.34 (m, 1H, CH), 4.49 (m, 2H, CH + CH), 4.01
(m, 2H, CH2), 3.42 (m, 2H, CH2), 3.17 and 3.14 (d, 50:50, 1H,
J = 5.0 Hz, OCH2), 2.88 (m, 5H, OCH2 + CH2), 2.42 (m, 1H, CH),
1.81 (m, 4H, CH2), 1.49 and 1.48 (s, 50:50, 3H, CH3), 1.44 (m, 2H,
CH2), 1.15 (m, 1H, CH), 0.87 (m, 6H, CH3); ESI-MS: m/z = 578
[M+H]+.
4.1.6.32. N-{S-3-{S-1-[S-4-Methyl-1-(R-2-methyloxiran-2-yl)-1-o
xopentan-2-ylamino]-1-oxo-3-phenylpropan-2-ylamino}-3-ox
o-1-phenylpropyl}-4-(trifluoromethyl)
(22f). White solid; yield: 68%; IR (KBr): 3307, 3064, 2961,
1721, 1645, 1539, 699 cmÀ1 1H NMR (500 MHz, CDCl3): d = 8.77
benzamide
4.1.6.28. 1-Acetyl-N-{3-{S-1-[S-4-methyl-1-(R-2-methyloxiran-2
-yl)-1-oxopentan-2-ylamino]-1-oxo-3-phenylpropan-2-ylamino
}-3-oxo-1-phenylpropyl}piperidine-4-carboxamide
;
(d, 1H, J = 7.5 Hz, NH), 7.99 (d, 2H, J = 8.0 Hz, Ar-H), 7.70 (d, 2H,
J = 8.0 Hz, Ar-H), 7.27 (m, 8H, Ar-H), 6.92 (d, 2H, J = 7.0 Hz, Ar-H),
6.34 (d, 1H, J = 6.0 Hz, NH), 5.93 (d, 1H, J = 8.0 Hz, NH), 5.53 (br s,
1H, CH), 4.57 (m, 1H, CH), 4.45 (m, 1H, CH), 3.10 (d, 1H,
J = 5.0 Hz, OCH2), 2.91 (m, 3H, OCH2 + CH2), 2.69 (m, 2H, CH2),
1.48 (s, 3H, CH3), 1.42 (m, 2H, CH2), 1.08 (m, 1H, CH), 0.84 (dd,
6H, J = 21.0, 5.5 Hz, CH3) ppm; 13C NMR (125 MHz, CDCl3):
d = 207.81, 170.86, 170.02, 165.09, 140.44, 137.45, 135.77,
(18m).
White solid; yield: 73%; IR (KBr): 3285, 3061, 2958,
1721, 1642, 1525, 700 cmÀ1
;
1H NMR (500 MHz, CDCl3): d = 7.85
and 7.54 (d, 50:50, 1H, J = 7.5 Hz, NH), 7.28 (m, 8H, Ar-H), 7.09
(m, 1H, Ar-H), 6.92 (m, 1H, Ar-H), 6.39 (m, 1H, NH), 6.06 (m, 1H,
NH), 5.32 (m, 1H, CH), 4.50 (m, 3H, CH + CH + CH2), 3.83 (m, 1H,
CH2), 3.15 (m, 1H, OCH2), 3.08 (m, 1H, CH2), 2.84 (m, 6H, OCH2 +
CH2), 2.40 (m, 1H, CH), 2.07 (s, 3H, CH3), 1.89 (m, 2H, CH2), 1.63

J. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 2955–2965
2965
129.26, 128.79, 128.65, 127.76, 127.68, 127.17, 126.06, 125.63,
124.82, 122.65, 58.88, 54.02, 52.19, 50.82, 50.22, 41.37, 40.29,
38.27, 25.05, 23.21, 21.28, 16.59; ESI-MS: m/z = 638 [M+H]+.
4.2.3. Western blot analysis
Poly ADP-ribose polymerase (PARP), and Ubiquitin antibody
were purchased from Cell Signaling Technology (Boston, MA,
USA); b-actin antibody was obtained from Sigma. All antibodies
were used as recommended by the manufacturers.
4.1.6.33. N-{S-3-{S-1-[S-4-Methyl-1-(R-2-methyloxiran-2-yl)-1-
oxopentan-2-ylamino]-1-oxo-3-phenylpropan-2-ylamino}-3-ox
For experiments, cells were grown in 6-well plates incubated
with Compounds for 12 h. Cells were rinsed twice with ice-cold
PBS and lysed with 1Â SDS loading buffer. Samples were electro-
phoresed on 10% SDS–polyacrylamide gels, and transferred to
PVDF membranes. The membranes were blocked for 1 h with 5%
w/v milk, incubated with indicated antibodies for 2 h, washed 3
times with PBS, incubated with the anti-rabbit or anti-mouse sec-
ondary antibody for 1 h, washed 3 times with PBS, and detected
with the ECL Kit.
o-1-phenylpropyl}-2-naphthamide
(22h).
White
solid;
yield: 46%; IR (KBr): 3283, 3062, 2957, 1722, 1643, 1536, 699 cm
À1
;
1H NMR (500 MHz, CDCl3): d = 8.64 (d, 1H, J = 7.0 Hz, NH),
8.42 (s, 1H, Ar-H), 7.90 (m, 4H, Ar-H), 7.55 (m, 2H, Ar-H), 7.34
(m, 4H, Ar-H), 7.18 (m, 4H, Ar-H), 6.94 (d, 2H, J = 7.0 Hz, Ar-H),
6.41 (d, 1H, J = 6.0 Hz, NH), 6.02 (d, 1H, J = 7.5 Hz, NH), 5.61 (m,
1H, CH), 4.61(q, 1H, J = 6.0 Hz, CH), 4.48 (m, 1H, CH), 3.12 (d, 1H,
J = 4.5 Hz, OCH2), 2.91 (m, 2H, CH2), 2.82 (d, 1H, J = 5.0 Hz, OCH2),
2.73 (m, 2H, CH2), 1.47 (s, 3H, CH3), 1.40 (m, 2H, CH2), 1.08 (m,
1H, CH), 0.82 (dd, 6H, J = 25.0, 6.0 Hz, CH3) ppm; 13C NMR
(125 MHz, CDCl3): d = 207.85, 170.90, 170.17, 166.92, 140.77,
135.91, 134.87, 132.70, 131.34, 129.29, 129.13, 128.76, 128.62,
128.44, 127.72, 127.68, 127.63, 127.43, 127.10, 126.66, 126.19,
123.73, 58.88, 54.08, 52.21, 50.77, 50.16, 44.35, 40.28, 38.20,
25.04, 23.22, 21.29, 16.61; ESI-MS: m/z = 620 [M+H]+.
Acknowledgements
The authors thank the National Natural Science Foundation of
China (81125023, 91029716) and the National Science and Tech-
nology Major Projects for Major New Drugs Innovation and Devel-
opment (2012ZX09301-001-004) for financial support. We also
thank Jianyang Pan (Pharmaceutical Informatics Institute, Zhe-
jiang University) for performing NMR and Mass spectrometry for
structure elucidation.
4.2. Biological evaluation
4.2.1. 20S proteasome chymotrypsin-like inhibition assay
Chymotrypsin-like enzyme activity assay was carried out in
References and notes
50
fied human proteasome (25
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15 min, and then added with 39 L synthesized substrate Suc-
Leu-Leu-Val-Tyr-AMC (50 M) (GL Biochem Ltd., Shanghai, P.R.
l
L volume. One microliter compound was added into 10
lL puri-
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3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium (MTS) was purchased from
Promega (Madison, WI, USA). Human multiple myeloma (MM) cell
lines NCI-H929, RPMI 8226, were gift from Professor Jian Hou
(Chang zheng hospital, Shanghai, P.R. China), and were grown in
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A 100 l
L NCI-H929 or RPMI 8226 cells (104/well) were seeded
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regression model (curvefit) based on sigmoidal dose response
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sion 5.02, Graphpad Software.