Synthesis, bioactivity, docking and molecular dynamics studies of furan-based peptides as 20s proteasome inhibitors

Article abstract of DOI:10.1002/cmdc.201402484

Proteasome inhibitors are promising compounds for a number of therapies, including cardiovascular and eye diseases, diabetes, and cancers. We previously reported a series of furanbased peptidic inhibitors with moderate potencies against the proteasome b5 subunit, hypothesizing that the C-terminal furyl ketone motif could form a covalent bond with the catalytic residue, threonine 1. In this context, we describe further optimizations of the furan-based peptides, and a series of dipeptidic and tripeptidic inhibitors were designed and synthesized, aiming at improved potency and better solubility. Most of the tripeptidic inhibitors demonstrated improved potency and selectivity as b5 subunit inhibitors in both enzymatic and cellular assays, and good antineoplastic activities in various tumor cell lines were also observed. However, no inhibitory effects were observed for the dipeptidic compounds, which led us to presume that a noncovalent binding mode is adopted. Docking studies and molecular dynamics simulations were carried out to verify this presumption, with results showing that the distance between the furyl ketone motif and Thr1 is slightly too long to form covalent bond.

Full text of DOI:10.1002/cmdc.201402484

DOI: 10.1002/cmdc.201402484
Full Papers
Synthesis, Bioactivity, Docking and Molecular Dynamics
Studies of Furan-Based Peptides as 20S Proteasome
Inhibitors
[
a]
[b]
[a]
[a]
[a]
[a]
[a]
Qi Sun, Bo Xu, Yan Niu,* Fengrong Xu, Lei Liang, Chao Wang, Jiapei Yu,
[a]
[a]
[b]
[a, b]
Gang Yan, Wei Wang, Hongwei Jin,* and Ping Xu*
Proteasome inhibitors are promising compounds for a number
of therapies, including cardiovascular and eye diseases, diabe-
tes, and cancers. We previously reported a series of furan-
based peptidic inhibitors with moderate potencies against the
proteasome b5 subunit, hypothesizing that the C-terminal furyl
ketone motif could form a covalent bond with the catalytic
residue, threonine 1. In this context, we describe further opti-
mizations of the furan-based peptides, and a series of dipepti-
dic and tripeptidic inhibitors were designed and synthesized,
aiming at improved potency and better solubility. Most of the
tripeptidic inhibitors demonstrated improved potency and se-
lectivity as b5 subunit inhibitors in both enzymatic and cellular
assays, and good antineoplastic activities in various tumor cell
lines were also observed. However, no inhibitory effects were
observed for the dipeptidic compounds, which led us to pre-
sume that a noncovalent binding mode is adopted. Docking
studies and molecular dynamics simulations were carried out
to verify this presumption, with results showing that the dis-
tance between the furyl ketone motif and Thr1 is slightly too
long to form covalent bond.
Introduction
In eukaryotic cells, the major
pathway of intracellular protein
degradation is the ubiquitin pro-
teasome system, which is essen-
tial for many key cellular func-
tions, such as cell cycling, signal
transduction, antigen processing
for appropriate immune re-
[
1]
sponses, and apoptosis. Protea-
some inhibitors have been ex-
plored extensively as potential
antitumor, anti-inflammatory, an-
tiviral, and immunosuppressive
agents. Much research has indi-
cated that tumor cells are more
sensitive to proteasome inhibi-
Figure 1. a) Clinically approved proteasome inhibitors. b) Compound 12. c) Designed dipeptidic (n=0) and tripep-
tors than normal cells, and there- tidic (n=1) inhibitors.
[
a] Dr. Q. Sun, Dr. Y. Niu, F. Xu,
fore, the proteasome is regarded as one of the most promising
antitumor drug targets. The 26S proteasome is a large multica-
talytic proteinase complex, consisting of one 20S catalytic core
and two 19S regulatory particles. Crystal structure information
shows that the active sites of the 20S proteasome are located
on the b1, b2, and b5 subunits, which have caspase-like (C-L),
trypsin-like (T-L), and chymotrypsin-like (ChT-L) activities, re-
spectively. Studies have shown that inhibition of the b5 subu-
Dr. L. Liang, Dr. C. Wang, J. Yu, G. Yan, W. Wang, Prof. P. Xu
Department of Medicinal Chemistry, School of Pharmaceutical Sciences
Peking University Health Science Center
3
8 Xueyuan Road, Beijing 100191 (China)
E-mail: yanniu480@126.com
pingxu@bjmu.edu.cn
[b] Dr. B. Xu, Dr. H. Jin, Prof. P. Xu
State Key Laboratory of Natural and Biomimetic Drugs
School of Pharmaceutical Sciences
Peking University Health Science Center
[2]
nit alone is sufficient to achieve therapeutic effects. Success-
3
8 Xueyuan Road, Beijing, 100191 (China)
ful examples include bortezomib, carfilzomib, and marizomib
E-mail: jinhw@bjmu.edu.cn
(
Figure 1a), which were approved by the US Food and Drug
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402484.
Administration (FDA) in 2003, 2012, and 2014, respectively, for
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the treatment of multiple myeloma. Several second-generation
[
3]
proteasome inhibitors are currently in clinical trials. We previ-
ously reported two series of furan-based b5 subunit inhibitors,
among which nine compounds were tripeptide derivatives and
[
4]
six were statine peptidomimetics.
The unique potent compound we identified was peptide de-
rivative 12 (Figure 1b), with IC50 values of 7.85 mm against the
b5 subunit, and 34.2 and 30.07 mm toward HepG2 and HL-60
cell growth inhibition, respectively. The C-terminal furyl ketone
group of compound 12 was initially hypothesized to form a co-
valent bond with Thr1, and the peptide backbone to adopt an
antiparallel b-sheet conformation typical for peptide-based in-
hibitors.
Scheme 1. Synthesis of compound 3. Reagents and conditions: a) HOBt, EDCl,
DMF, NMM, RT, 12 h, 91%; b) nBuLi, furan, THF, 08C, 30 min, then À788C,
2
2 2
.5 h, 90%; c) 50% TFA/CH Cl , 08C, 2 h.
As di- and tripeptides were reported to be able to cross in-
[
5,6]
testinal epithelial barriers,
a series of di- and tripeptidic in-
Scheme 2. In the synthesis of intermediates 6a,b, we found
that under the conditions of methyl iodide and potassium car-
bonate as base, the monomethylated byproducts 5a,b were
generated together with the double-methylated product at
nearly 30%. Therefore, to improve the yield of double-methy-
lated product 6a,b, the Boc- or Cbz-protected tyrosine groups
were first mono-methylated with methyl iodide under the rela-
tively mild basic conditions of sodium bicarbonate to give
5a,b almost quantitatively, followed by further etherification
with methyl iodide and potassium carbonate to give 6a,b. The
N-protecting group of 6a was then removed with 50% TFA/di-
chloromethane to give 4d, whereas 6a,b were hydrolyzed to
produce the carboxylic acids 7a,b (Scheme 3).
hibitors were thus designed in this study, and their structures
1
are depicted in Figure 1c. The R isopropyl group has been re-
tained, and commercially available substituted N-termini such
as tert-butoxycarbonyl (Boc) and benzyloxycarbonyl (Cbz)
4
groups at R were adopted owing to the results of our previ-
ous structure–activity relationship (SAR) study. Given that the
b5 subunit preferentially cleaves peptides with hydrophobic
2
3
residues, isopropyl was selected as the R or R group in the in-
2
itial design. Bulky residues were also introduced at the R and
3
R positions, and these were expected to reinforce hydropho-
bic interactions and to improve the specificity of these inhibi-
tors for the b5 subunit. To improve solubility and to increase
the number of hydrogen bonds, hydroxy- and methoxy-substi-
tuted phenylalanine residues were also introduced. In summa-
ry, with the aim of improving affinity, specificity, and solubility,
a series of di- and tripeptides with a furyl ketone group as the
C terminus were designed and synthesized.
The target tripeptide compounds 10a–m were synthesized
as shown in Scheme 4. The N-protected l-amino acids (7a–f;
Both enzyme- and cell-based assays were carried out to eval-
uate the potencies of these new compounds against the three
proteasome subunits. The potent inhibitors discovered were
further evaluated for their antitumor effects in assays with
eleven available tumor cell lines. Docking and molecular dy-
namics (MD) simulations were also used to explore the binding
modes of these molecules with the specific proteasome subu-
nits.
Scheme 2. Synthesis of compounds 4a–c. Reagents and conditions: a) SOCl
CH OH, RT, 5 h, 81–89%.
2
,
3
Results and Discussion
Chemistry
The synthetic routes for the furan-based peptides are outlined
in Schemes 1–5. Furan-based leucine 3 was synthesized from
Boc-protected l-leucine via three steps (Scheme 1). Firstly,
Weinreb amide 1 was obtained through a published coupling
[
7]
approach, followed by treatment with n-butyllithium and
furan at À788C to give compound 2 in high yield (90%). Com-
pound 2 was then deprotected with 50% trifluoroacetic acid
(TFA)/dichloromethane to give 3, which was neutralized in situ
by N-methylmorpholine (NMM) and used in the next step with-
out purification.
Scheme 3. Synthesis of (4-methoxyphenyl)alanine analogues. Reagents and
conditions: a) CH I, NaHCO , DMF, RT, 5 h; b) CH I, K CO , DMF, RT, 5–10 h,
83–85%; c) 1n NaOH, THF, RT, 2 h; d) 50% TFA/CH Cl , 08C, 2 h.
Methyl esters 4a–c were produced by methylation of the di-
3
3
3
2
3
2
verse commercially available amino acids at R , as illustrated in
2
2
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toward T-L and C-L activities due
to their IC50 values being
>
50 mm. Among these active tri-
peptides, 10b was the most
potent, with the lowest IC50
value of 1.64 mm and the best
selectivity for the b5 subunit.
Studies have shown that in-
hibition of the b5 subunit alone
is sufficient to achieve therapeu-
tic effects, especially in the treat-
ment of cancer. Furthermore, it
is known that the inhibition of
all proteasome subunits is gen-
erally cytotoxic toward normal
[8]
cells. The above results sug-
gested that these inhibitors
might have the potential to be
developed as therapeutic agents
Scheme 4. Synthesis of tripeptide derivatives 10a–m. Reagents and conditions: a) HOBt, EDCl, DMF, NMM, RT, 12–
4 h, 72–98%; b) 1n NaOH, THF, RT, 2 h; c) 3, HOBt, EDCl, DMF, NMM, RT, 12–24 h, 51–91%.
2
the syntheses of 7a,b are mentioned above, and 7c–f were
commercially available) were coupled with the corresponding
methyl esters 4a–d under typical peptide-coupling conditions.
The obtained dipeptidic esters 8a–m were hydrolyzed to give
the corresponding acids 9a–m, followed by coupling with 3
and purification by flash chromatography to form compounds
against cancer with decreased toxicity.
Based on these results, SAR studies were focused on the tri-
1
peptide backbones. Maintaining isopropyl as the R group in
this study, loss of activity was observed by introducing isopro-
2
pyl at R (10e versus 10h, 10j, 10m), suggesting that an aro-
matic group at this position is essential for strong binding.
Meanwhile, enhanced potency was achieved by introducing 4-
1
9
1
0a–m. The commercially available N-protected amino acids
n–q were coupled with 3 to give dipeptide compounds
0n–q by the same method (Scheme 5). The structures were
2
hydroxy or 4-methoxy groups on the phenyl ring at R , an
effect that can be observed by comparing 10b, 10i/10l, 10j/
10m, and 10k versus 10a, 10g, 10h, and 10 f. If the R group
is phenyl or 4-hydroxyphenyl, variation from Cbz to Boc at R
1
13
2
confirmed by H NMR, C NMR, and HRMS spectra. All substitu-
ents are summarized in Table 1.
4
generates increased potencies, as observed with 10b and 10h
versus 10j and 10a, for example. Conversely, compounds with
2
4
-methoxyphenyl substitution at the R position show greater
inhibitory potency: 10l and 10m versus 10c and 10d. Unlike
2
3
R , modifications at R showed less importance for recognition
4
of the b5 subunit. Regardless of the group at R , compounds
2
3
with R as 4-hydroxyphenyl and R as 4-methoxyphenyl dem-
onstrated the best activities (10b and 10j), which could be
due to their improved solubility and the p-electron system of
the aromatic moiety forming strong interactions with b5 subu-
nit and more favorable shape fitting into the binding pockets.
Scheme 5. Synthesis of dipeptide derivatives 10n–q. Reagents and condi-
tions: a) 3, HOBt, EDCl, DMF, NMM, RT, 12–24 h, 63–87%.
1
2
3
The optimal substituents for positions R , R , and R on this
furan-based scaffold were found to be isopropyl, 4-hydroxy-
phenyl, and 4-methoxyphenyl, respectively, which are suggest-
ed to be maintained in further optimizations.
Biological evaluations
To determine whether these compounds are cell permeable
and can also selectively inhibit ChT-L activity at the cellular
The newly synthesized furan-based compounds 10a–q were
tested with enzyme- and cell-based assays for their protea-
some inhibitory properties. All compounds were first evaluated
against three subunits (b1, b2, and b5) of purified human 20S
constitutive proteasome. All tripeptide compounds, with the
exception of 10e and 10h, showed promising inhibitory po-
tencies toward the ChT-L activity of the proteasome, inhibiting
TM
level, cell-based (Proteasome-Glo , Promega) assays were car-
[9]
ried out with HL-60 cells, and the results are summarized in
Table 1. The results show that seven of the eleven potent com-
pounds identified in the enzyme-based assay have micromolar
IC50 values in addition to selectivity for ChT-L activity due to
their significantly diminished inhibitory effects on T-L and C-L
À1
>
40% of the enzyme activity at 10 mm, whereas no inhibitory
activities at 25 mgmL (inhibition: <30%). Compound 10b
effects were observed among the dipeptides. Continuous
assays were then performed to determine the IC50 values, as
listed in Table 1. However, none showed inhibitory effects
maintained the lowest IC50 value (3.18 mm) among all com-
pounds tested, which is consistent with results of the enzyme
assays.
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Table 1. Structures and proteasome ChT-L inhibitory activities of 10a–q as determined by enzyme- and cell-based assays.
[
a]
[a]
Enzymatic assay
Cell-based assay
2
3
4
[b]
[c]
Compd
R
R
R
n
Inhib. [%]
IC50 [mm]
Inhib. [%]
IC50 [mm]
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0a
0b
0c
0d
0e
0 f
0g
0h
0i
0j
0k
0l
0m
0n
0o
0p
0q
Ph
(4-OH)
C
C
6
6
H
H
4
(4-OCH
3
)
)
Boc
Boc
Boc
Boc
Cbz
Cbz
Cbz
Cbz
Cbz
Cbz
Cbz
Cbz
Cbz
Boc
Boc
Cbz
Cbz
–
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
–
73Æ1.7
6.82Æ0.20
1.64Æ0.44
9.42Æ0.56
8.15Æ0.11
77Æ1.5
17.09Æ0.09
3.18Æ0.05
21.4Æ0.05
18.83Æ0.14
C
6
H
4
4
(4-OCH
3
89.7Æ1.3
60.6Æ0.4
69.7Æ0.2
36.4Æ0.3
58.5Æ0.1
47.5Æ1.6
14.9Æ0.8
67.1Æ0.5
84.8Æ2.9
73.4Æ0.3
82.2Æ4.1
60.5Æ1.6
7.7Æ0.3
16.5Æ2.7
5.7Æ1.2
13.6Æ0.4
–
85.4Æ0.2
66.5Æ0.6
67Æ0.3
C
C
6
6
H
H
4
(4-OCH
(4-OCH
iPr
3
)
)
Ph
4
3
C
C
6
6
H
H
4
(4-OCH
3
)
)
4
(4-OCH
3
>10
38.6Æ1.1
54.7Æ0.9
38.1Æ0.8
13.9Æ0.8
18.2Æ1
>50
Ph
Ph
Ph
iPr
Ph
7.56Æ0.25
8.39Æ0.83
27.54Æ0.16
>50
>50
>50
>50
25.36Æ0.06
14.60Æ0.12
>50
ND
ND
ND
ND
C
C
6
6
H
H
4
(4-OCH
3
)
)
>10
C
C
6
H
H
4
(4-OH)
(4-OH)
Ph
6.42Æ0.16
3.27Æ0.21
5.85Æ0.13
2.62Æ0.04
6.01Æ0.58
6
4
4
(4-OCH
3
16.1Æ0.5
83.1Æ2
C
C
C
6
6
6
H
H
H
4
4
4
(4-OCH
(4-OCH
(4-OCH
Ph
3
3
3
)
)
)
iPr
Ph
89.6Æ0.1
59.3Æ0.6
C
6
H
4
(4-OCH
3
)
[
d]
–
–
–
–
–
>10
ND
ND
ND
ND
C
C
6
H
H
4
(4-OH)
Ph
>10
>10
>10
37.3Æ3.1 nm
6
4
(4-OH)
–
[
e]
MG132
–
0.28Æ0.06
[
a] Values represent the meanÆSD of three independent experiments, each based on four biological replicates. [b] Percent inhibition at 10 mm. [c] Percent
À1
inhibition at 25 mgmL . [d] ND: not determined. [e] N-(benzyloxycarbonyl)-leucinyl-leucinyl-leucinal: a potent proteasome inhibitor, used herein as refer-
ence compound.
Encouraged by the above results, all active compounds were
tested for their inhibitory activities toward the proliferation of
MCF-7, BGC-823, A549, A375, HCT-8, HepG2, HL-60, Jurkat,
SW480, DU145, and HeLa cell lines. The data listed in Table 2
demonstrate that these compounds have good antineoplastic
activities in eleven tumor cell lines, concerning both solid
tumor and hematologic malignancy, which positively correlate
with the proteasome inhibitory activities. Six compounds and
four available cell lines were selected for IC50 determination, as
listed in Table 3. As expected, the most active compound was
found to be 10b, which exhibited significant inhibition against
all tumor cell lines tested, with the only exception being
HepG2. Respective IC50 values for cell growth inhibition were
5.34, 9.69, 5.66, and 7.03 mm for SW480, DU145, A375, and
HeLa, suggesting 10b as a promising lead compound for the
development of antitumor agents.
Docking studies and MD simulations
Thus far, many classes of inhibitors harboring an activated car-
bonyl carbon atom have been found to inhibit the 20S protea-
[10,11]
some.
In our original study, the furyl ketone group was hy-
À1
Table 2. Antiproliferative effects of compounds 10a–m tested in various tumor cell lines at 10 mgmL
.
[
a]
Compd
Inhibition [%]
HL-60
MCF-7
BGC-823
A549
HCT-8
HepG2
Jurkat
SW480
DU145
A375
HeLa
1
1
1
1
1
1
1
1
1
1
1
0a
0b
0c
0d
0 f
0g
0i
0j
0k
0l
90Æ1.2
36.4Æ0.2
51Æ0.2
86.2Æ2.8
93.1Æ1.9
23.3Æ3.9
7.2Æ3.3
12Æ0.8
7.9Æ0.3
26.7Æ0.6
86.2Æ0.4
27.6Æ0.4
16.5Æ1.3
31.5Æ0.8
33.9Æ1.7
31.6Æ1.8
À2.3Æ0.8
21.3Æ0.9
65.8Æ2.4
71.9Æ1.3
97.3Æ1.1
22.5Æ0.6
60.1Æ0.4
5Æ0.4
91.1Æ3.3
90.9Æ0.5
63.1Æ0.3
89.7Æ1.7
À3.5Æ1.5
7.6Æ2.2
83.6Æ0.8
84.4Æ0.1
À6.2Æ6.1
74.3Æ0.9
À6Æ1.3
80.1Æ0.7 81.5Æ0.1
83.1Æ0.4 82.6Æ1.4
87.2Æ0.2 94.3Æ1.3
95.1Æ0.8
48Æ0.5
17.5Æ1.7 À9.6Æ7.5
7.3Æ0.6
5.2Æ1.3
89.3Æ0.1 55.2Æ0.2
54.3Æ2.6
87.3Æ0.3 25.4Æ2.7
10.8Æ3.9 16.2Æ2.4
5.6Æ0.8 16.2Æ1.8
20.4Æ2.4 84.5Æ0.5
70.7Æ1.1 77Æ0.5
38.2Æ0.2 15.3Æ0.6 À7.9Æ7.9
1.2Æ0.7
2.5Æ0.6
0.3Æ0.7
8.1Æ1.5
5.9Æ0.6
2.0Æ0.6
19.6Æ1.8
26Æ0.1
7.5Æ0.5
7.4Æ0.1 À6.4Æ8.8
13.9Æ0.3 11.5Æ2.2
11.8Æ0.1
3.7Æ3.7
7.1Æ0.3
2.5Æ3.5
79.6Æ3.7
À3.4Æ2
6.9Æ4.5 À13.1Æ0.3 À0.5Æ0.4
1.5Æ0.1 À9.8Æ1.7 À4.5Æ4.5 63.1Æ1.6
80Æ0.8
85Æ0.3
90Æ3.7
28.9Æ0.9
52.8Æ1.3
46.4Æ1.8
31.5Æ0.9
77.5Æ0.8
1.4Æ1.7
15.9Æ0.1
28.8Æ0.4 10.6Æ0.7 À3.4Æ1.3
13.9Æ3.1 94.3Æ0.4
25.3Æ0.3 22.6Æ3.2
83.8Æ0.3 94.9Æ1.9
85.1Æ0.4 35.7Æ0.9
34.7Æ1.9 13.4Æ0.2
92.5Æ1.5 94.4Æ1.9
54.3Æ2.3
13Æ3.4
76.9Æ2.4
20.1Æ1
11.4Æ3.7
9.9Æ1.9
0m
3.8Æ3.1 À3.1Æ1.9
4.6Æ1
10.2Æ5.2
[
b]
Stauro.
92.3Æ1.5
92.1Æ3.3 95.6Æ3.8
91.8Æ2.5
95.6Æ2
74.6Æ1.8
60.7Æ0.9 50.2Æ1.9
[
a] Values represent the meanÆSD of two independent experiments, each based on four biological replicates. [b] Staurosporine (1 mm), a nonselective pro-
tein kinase inhibitor, has broad-spectrum antitumor activity and is widely used in antitumor drug screening research.
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by starting from the aforementioned pose of compound 10b
in complex with the b5 and b6 subunits of the 20S protea-
some, generated from docking. The unrestrained MD simula-
tions of the complex led to a stable trajectory of geometry and
energy terms. Calculation of RMSDs with respect to the start-
ing structure confirmed the stability of the trajectory. A plot of
RMSD values as a function of simulation time is shown in Fig-
ure 2a. We observed that the RMSD values leveled off after
1 ns and then remained stable over the final stage of the 10 ns
simulation. Throughout the MD simulation, a more stable and
reasonable binding mode for 10b with the protein was ob-
tained, and seven hydrogen bonds with Arg19, Thr21 (2),
Ala49, Ser130, Glu132, and Arg137 can be observed (Fig-
ure 2d). Among these hydrogen bonds, only one was formed
at the same site as in the docking mode, with different dis-
tance and angle values. The other two hydrogen bonds, be-
tween 10b and b5:Thr21 and b5:Gly47, were broken during
simulation, owing to movement of ligand toward the b6 subu-
nit. However, two hydrogen bonds between 10b and Thr21,
and one between 10b and Ala19 were newly generated, there-
by profoundly stabilizing the inhibitor in the ChT-L site. In ad-
dition, the ligand was predicted to form further hydrogen
bonds with b6:Ser130, Glu132, and Arg137, thus contributing
to the affinity for the b6 subunit. At the same time, the dis-
tance between the Thr1 residue and the furyl ketone moiety
remained stable during MD simulations (Figure 2b); the aver-
age value was 4.9 ꢁ, which is slightly too large to form a cova-
lent bond. All these results are consistent with the observed
biological activities and confirm a noncovalent binding mode
between inhibitor and protein.
Table 3. Inhibitory activities of selected compounds against the growth
of four tumor cell lines.
[
a]
Compd
IC50 [mm]
DU145
SW480
A375
HeLa
1
1
1
1
1
1
0a
0b
0d
0j
0k
0l
5.77Æ0.22
5.34Æ0.22
6.25Æ1.17
9.18Æ0.76
16.33Æ1.10
5.30Æ0.64
0.47Æ0.11
13.37Æ1.16
9.69Æ0.53
8.24Æ1.23
6.76Æ1.69
5.66Æ0.49
11.06Æ0.28
16.62Æ0.51
22.45Æ1.68
6.15Æ0.23
0.66Æ0.16
15.60Æ1.94
7.03Æ0.02
14.53Æ1.95
NI
[
b]
NI
NI
NI
12.36Æ1.34
0.90Æ0.34
10.83Æ1.06
0.93Æ0.09
[c]
MG132
[
a] Values represent the meanÆSD of two independent experiments,
each based on four biological replicates. [b] NI: no inhibition. [c] N-(ben-
zyloxycarbonyl)-leucinyl-leucinyl-leucinal: a potent proteasome inhibitor,
used herein as reference compound.
pothesized to form a covalent bond with the hydroxy group of
Thr1 in the active site. However, from the enzymatic and cellu-
lar assays, most of the tripeptide inhibitors were found to have
favorable potencies, whereas no inhibitory effects were found
for the dipeptide compounds. These differences and moderate
potencies caused us to speculate a noncovalent mode of inter-
action for the furan-based inhibitors in binding to the 20S pro-
teasome. To explain the SAR and to gain a better understand-
ing of the binding modes, docking studies and molecular dy-
namics simulations were carried out for the potent inhibitors.
Docking
[
12]
AutoDock 4.2 was used to dock the inhibitors into the b5
and b6 subunits of the 20S yeast proteasome, the structure of
which was obtained from the RCSB Protein Data Bank (PDB ID:
The inhibitor was well stretched into the binding pockets,
the furyl ketone moiety projected into the S1 pocket, and the
[
10]
2
4
NO8). The structure of the human 20S proteasome is quite
R group protruded into the S2 pocket with its nitrogen atom
3
similar to that of the yeast 20S proteasome, and the chymo-
hydrogen bonded to Thr21. The R 4-methoxyphenyl side
[
13]
trypsin active site is highly conserved. AutoDock uses an au-
tomated docking approach for predicting the binding modes
and changes in free energy of the ligands interacting with bio-
macromolecular targets; it was successful in application to the
chain stretched out into the subunit-specific S3 pocket, and
was found to be in close contact with the b6 subunit; the
open space of the S4 pocket was partially occupied by the N-
terminal residue (Figure 2d). In summary, the MD results of-
fered useful information for understanding the stable and
credible interaction mode between 10b and b5, b6 subunits,
and can also guide the rational design of novel inhibitors in
the future.
[
14]
area of the proteasome. Before docking, the protein and li-
[
15]
gands were prepared in Discovery Studio 2.5.
To determine the correct ligand pose among the many con-
formations generated from docking, two steps were per-
formed: First, the generated conformations were clustered to-
gether by their root-mean-square deviation (RMSD) values, and
the cluster containing the greatest number of conformations
was selected for further examination. Next, the docking confor-
mation with the lowest docked free energy was selected from
this cluster. As an example, the predicted binding mode of
Conclusions
In summary, we have identified a series of furan-based tripepti-
des as potential proteasome inhibitors with good selectivity
for the b5 subunit. Initial biological evaluations showed com-
pound 10b to be the most selective and potent inhibitor
among the acquired compounds; it effectively inhibited the b5
subunit with IC50 values of 1.64 mm in enzyme assays and
10b is shown in Figure 2c. In the simulated binding mode, b-
sheet-like hydrogen bonds between 10b and residues Thr21,
Gly47, and Ala49 of the b5 subunit are formed, which contrib-
[
16–18]
ute significantly to the potency of this compound.
3.18 mm in cell-based assays. It also inhibited proliferation in
ten tumor cell lines, suggesting 10b as a very useful com-
pound worthy of further development as an antitumor agent.
MD simulations
To obtain a more integrated and precise view of the binding
process, molecular dynamics (MD) simulations were performed
Computational studies suggested a noncovalent binding
mode, and these binding models can be valuable in guiding
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Figure 2. Results of docking and MD simulations. a) MD simulation time versus RMSD of the complex. b) Distance between the carbonyl carbon atom of the
furyl ketone group and the hydroxy oxygen atom of Thr1 during MD simulations. c) Model of predicted binding modes of compound 10b (yellow) into the
b5 (blue)/b6 (white) active site of the 20S proteasome, generated by AutoDock; key hydrogen bonds between the inhibitor and protein are shown as red
dashed lines. d) Stabilized conformation of compound 10b (magenta) derived from MD simulations.
with a reversed-phase Eclipse Plus C column (3.5 mm, 4.6ꢂ
future structural modifications and synthesis of more potent
and selective inhibitors.
18
À1
1
00 mm) at a flow rate of 1 mLmin and a gradient of solvent A
(
H O) and solvent B (CH CN). All final compounds were obtained in
2
3
a purity of no less than 95%. HPLC traces for all final products
0a–q are provided in the Supporting Information.
1
Experimental Section
(
2
S)-tert-Butyl-(1-(methoxy(methyl)amino)-4-methyl-1-oxopentan-
-yl)carbamate (1): EDCl (0.230 g, 1.2 mmol) and HOBt (0.162 g,
Chemistry
All solvents and reagents were obtained from commercial suppli-
ers. Reactions were monitored by thin-layer chromatography (TLC)
performed on silica gel GF254 purchased from Qingdao Haiyang
Chemical Co. (Qingdao, China) or Merck (Darmstadt, Germany).
Melting points (mp) were determined on an XT4A apparatus and
1.2 mmol) were added to a solution of Boc-Leu-OH (0.231 g,
1.0 mmol) and N,O-dimethylhydroxylamine hydrochloride (0.097 g,
1.0 mmol) in the presence of NMM (0.34 mL, 3.0 mmol) in 20 mL
DMF. The reaction mixture was stirred at room temperature over-
night and diluted with EtOAc (EA) and washed with 10% citric
1
13
are uncorrected. H NMR and C NMR spectra were recorded at
00/100 MHz on a Bruker Avance III 400 spectrometer. Chemical
acid, saturated aqueous NaHCO and brine in sequence, dried with
3
4
Na SO , and concentrated in vacuo to give crude product, which
2
4
shifts were referenced to the respective residual solvent signals.
High-resolution mass spectrometry (HRMS) data were obtained
using a Bruker APEX IV FTMS system. Low-resolution mass spectra
were obtained using a Waters ACQ-SQD LC–MS system. Purity anal-
ysis was performed on an Agilent 1260 series instrument equipped
was separated by silica gel chromatography to yield 1 as a colorless
1
oil (0.250 g, 91%): R =0.22 (PE/EA, 5:1); H NMR (400 MHz, CDCl ):
f
3
d=0.95 (dd, J=14.3, 6.5 Hz, 6H, CH ), 1.43 (s, 11H, Boc, CH ), 1.69–
3
2
1.76 (m, 1H, CH), 3.20 (s, 3H, NCH ), 3.79 (s, 3H, OCH ), 4.73 (d, J=
3
3
1
3
4.2 Hz, 1H, CH), 5.14 ppm (d, J=9.2 Hz, 1H, NH); C NMR
&
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(
7
100 MHz, CDCl ): d=21.5, 23.3, 24.6, 28.3, 32.0, 42.0, 48.9, 61.5,
vacuo. The resulting residue was separated by silica gel chroma-
3
+
9.3, 155.6, 173.8 ppm; HRMS (ESI) m/z [M+H] calcd for
tography to give 6a as a colorless oil (2.61 g, 85%): R =0.57 (PE/
f
+
1
C H N O : 275.19653, found: 275.19669; m/z [M+Na] calcd for
EA, 3:1); H NMR (400 MHz, [D ]DMSO): d=1.35 (s, 9H, Boc), 2.81–
1
3
27
2
4
6
C H N NaO : 297.17848, found: 297.17868.
2.99 (m, 2H, CH Ph), 3.62 (s, 3H, OCH ), 3.71 (s, 3H, OCH ), 4.16–
1
3
26
2
4
2
3
3
4
.22 (m, 1H, CH), 6.85 (d, J=8.3 Hz, 2H, Ph), 7.20 ppm (q, 3H, NH,
(S)-tert-Butyl-(1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)carba-
13
Ph); C NMR (100 MHz, [D ]DMSO): d=28.5, 36.2, 52.0, 55.3, 55.9,
6
mate (2): nBuLi (9.4 mL, 23.6 mmol) was added dropwise to a solu-
tion of furan (1.7 mL, 24.3 mmol) in THF (30 mL) at À788C. After
the addition, the pale-yellow solution was warmed to 08C and
stirred for 30 min. The resulting dark-yellow solution was re-cooled
to À788C, and a solution of 1 (1.85 g, 6.7 mmol) in THF (10 mL)
was added, and the reaction mixture was maintained at À788C for
another 2.5 h. Then, the dark-orange solution was poured into sa-
turated aqueous NH Cl (200 mL) and extracted with Et O (3ꢂ
7
3
8.6, 114.0, 129.8, 130.5, 155.8, 158.4, 173.1 ppm; MS (ESI) m/z (%):
32.40 (100) [M+Na] .
+
(S)-Methyl-2-(((benzyloxy)carbonyl)amino)-3-(4-methoxyphenyl)-
propanoate (6b): Following the same procedure as described for
the synthesis of 6a except for starting from Cbz-Tyr-OH, 6b was
obtained in 83% yield as a colorless oil: R =0.16 (PE/EA, 5:1);
f
1
H NMR (400 MHz, CDCl ): d=2.99–3.10 (m, 2H, CH Ph), 3.71 (s, 3H,
4
2
3
2
1
00 mL). The combined organic layers were washed with H O and
OCH ), 3.76 (s, 3H, OCH ), 4.59–4.64 (m, 1H, CH), 5.09 (s, 2H,
3 3
2
brine, then dried with Na SO , filtered and concentrated in vacuo.
The resulting thick oil was separated by silica gel chromatography
OCH Ph), 5.28 (d, J=8.0 Hz, 1H, NH), 6.80 (d, J=8.5 Hz, 2H, Ph),
2
7.00 (d, J=8.5 Hz, 2H, Ph), 7.32–7.33 ppm (m, 5H, Ph); C NMR
2
4
1
3
to give 2 as a yellow oil (1.71 g, 90%): R =0.59 (PE/EA, 5:1);
(100 MHz, CDCl ): d=32.6, 47.5, 50.2, 50.4, 62.2, 109.3, 122.9, 123.3,
f
3
1
H NMR (400 MHz, [D ]DMSO): d=0.90 (dd, J=19.3, 6.4 Hz, 6H,
123.4, 123.8, 125.5, 131.6, 150.9, 154.0, 167.3 ppm; MS (ESI) m/z
6
+
CH ), 1.20 (s, 1H, CH), 1.35 (s, 9H, Boc), 1.49–1.69 (m, 2H, CH ),
(%): 366.43 (95) [M+Na] .
3
2
4
7
.68–4.74 (m, 1H, CH), 6.72 (d, J=1.9 Hz, 1H, NH), 7.31 (d, J=
.6 Hz, 1H, 4’-H), 7.46 (d, J=3.4 Hz, 1H, 3’-H), 8.01 ppm (s, 1H, 5’-
(
S)-2-((tert-Butoxycarbonyl)amino)-3-(4-methoxyphenyl)propano-
1
3
ic acid (7a): 1n NaOH was added to a solution of 6a in THF until
pH 12–13, and the reaction mixture was stirred at room tempera-
ture for 2 h until acidified to pH 2–3 with 1n HCl. The aqueous
phase was extracted with EtOAc (5ꢂ10 mL), and the combined or-
ganic phases were dried with Na SO . The solvent was concentrat-
H); C NMR (100 MHz, [D ]DMSO): d=21.7, 23.5, 25.0, 28.2, 28.6,
6
5
4.8, 78.6, 113.0, 118.9, 148.3, 150.9, 156.0, 188.9 ppm; HRMS (ESI)
+
m/z [M+H] calcd for C H NO : 282.16998, found: 282.17005; m/
z [M+Na] calcd for C H NNaO : 304.15193, found: 304.15191.
15
24
4
+
15
23
4
2
4
(S)-2-Amino-1-(furan-2-yl)-4-methylpentan-1-one (3): To a solution
ed in vacuo, and the resulting residue 7a was used for the next
of 2 (337 mg, 1.2 mmol) in CH Cl (1.2 mL) TFA (0.8 mL) was added.
step without further purification.
2
2
The dark-brown solution was stirred at 08C for 2 h and then con-
centrated in vacuo. NMM (2.5 mL) was added to neutralize the re-
sidual in situ, and the resulting solution of 3 was used for the next
step without further purification.
(
S)-2-(((Benzyloxy)carbonyl)amino)-3-(4-methoxyphenyl)propa-
noic acid (7b): Following the same procedure as described for the
synthesis of 7a except for starting from 6b, 7b was obtained with-
out further purification.
(
S)-Methyl-2-amino-4-methylpentanoate (4a): SOCl2 (5 mL) was
(
S)-Methyl-2-amino-3-(4-methoxyphenyl)propanoate (4d): Fol-
carefully added to a solution of H-Leu-OH (1.31 g, 10 mmol) in dry
lowing the same procedure as described for the synthesis of 3
except for starting from 6a, 4d was obtained without further pu-
rification.
CH OH (30 mL) at À138C. The reaction solution was stirred at
3
room temperature for 5 h. Then the solution was concentrated in
vacuo, and the resulting white solid was recrystallized with CH OH
3
and Et O to give 4a as white needle-like crystals (1.30 g, 89%).
2
(
S)-Methyl-2-((S)-2-((tert-butoxycarbonyl)amino)-3-(4-methoxy-
phenyl)propanamido)-3-phenylpropanoate (8a): EDC·HCl
531 mg, 2.8 mmol) and HOBt (374 mg, 2.8 mmol) were added to
(S)-Methyl-2-amino-3-phenylpropanoate (4b): Following the
(
same procedure described for the synthesis of 4a except for start-
ing from H-Phe-OH, 4b was obtained in 84% yield as white
needle-like crystals.
a solution of 7a (the crude solution obtained previously, 2.3 mmol)
and 4b (497 mg, 2.3 mmol) in the presence of NMM (0.8 mL,
6
.9 mmol) in 20 mL DMF. The reaction mixture was stirred at room
(S)-Methyl-2-amino-3-(4-hydroxyphenyl)propanoate (4c): Follow-
temperature overnight and diluted with EtOAc and washed with
ing the same procedure described for the synthesis of 4a except
for starting from H-Tyr-OH, 4c was obtained in 81% yield as white
needle-like crystals.
10% citric acid, saturated aqueous NaHCO and brine, dried with
3
Na SO , and concentrated in vacuo to give the crude product,
2
4
which was separated by silica gel chromatography to yield 8a as
a white solid (342 mg, 75%): R =0.71 (PE/EA, 1:1); mp: 119–1208C;
f
(
S)-Methyl-2-((tert-butoxycarbonyl)amino)-3-(4-methoxyphenyl)-
propanoate (6a): To a stirred solution of Boc-Tyr-OH (2.81 g,
0 mmol) in dry DMF (40 mL) NaHCO (1.68 g, 20 mmol) and CH I
1
H NMR (400 MHz, [D ]DMSO): d=1.29 (s, 9H, Boc), 2.57–3.07 (m,
6
4
1
2
8
H, PhCH ), 3.58 (s, 3H, OCH ), 3.71 (s, 3H, OCH ), 4.08–4.14 (m,
2 3 3
H, CH), 4.48–4.53 (m, 1H, CH), 6.79 (s, 1H, NH), 6.81 (d, J=8.4 Hz,
1
3
3
(
1 mL, 16 mmol) were added sequentially. After stirring for 5 h, the
H, Ph), 7.13 (d, J=8.4 Hz, 2H, Ph), 7.22–7.30 (m, 5H, Ph),
mixture was quenched with H O (120 mL). The residue was parti-
tioned between EtOAc and H O, and the organic phase was then
washed sequentially with H O, 10% Na S0 , and brine and then
dried with Na SO , filtered, and concentrated in vacuo. Compound
13
2
.31 ppm (d, J=7.5 Hz, 1H, NH); C NMR (100 MHz, [D ]DMSO):
6
2
d=28.6, 37.0, 37.2, 52.3, 53.9, 55.4, 56.3, 78.5, 113.9, 127.0, 128.7,
29.6, 130.3, 130.6, 137.5, 155.6, 158.2, 172.3, 172.4 ppm; MS (ESI)
2
2
3
1
+
2
4
m/z (%): 479.54 (100) [M+Na] .
5
a was used in the next step without further purification. To a solu-
tion of 5a (2.95 g, 10 mmol) in dry DMF (40 mL), K CO3 (2.76 g,
(S)-Methyl-2-((S)-2-((tert-butoxycarbonyl)amino)-3-(4-methoxy-
phenyl)propanamido)-3-(4-hydroxyphenyl)propanoate (8b): Fol-
lowing the same procedure as described for the synthesis of 8a
2
2
0 mmol) and CH I (1 mL, 16 mmol) were added sequentially. After
3
stirring for 5 h, the mixture was quenched with H O (120 mL). The
2
residue was partitioned between EtOAc and H O, and the organic
except for starting from 7a and 4c, 8b was obtained in 81% yield
2
1
phase was then washed sequentially with H O, 10% Na S0 , and
as a white solid: R =0.38 (PE/EA, 1:1); mp: 144–1458C; H NMR
2
2
3
f
brine, and then dried with Na SO , filtered, and concentrated in
(400 MHz, [D ]DMSO): d=1.30 (s, 9H, Boc), 2.51–2.94 (m, 4H,
2
4
6
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1
CH Ph), 3.57 (s, 3H, OCH ), 3.71 (s, 3H, OCH ), 4.08–4.14 (m, 1H,
Rf =0.57 (PE/EA, 3:1); mp: 132–1338C; H NMR (400 MHz,
2
3
3
CH), 4.42 (dd, J=7.4, 14.0 Hz, 1H, CH), 6.66 (d, J=8.4 Hz, 2H, Ph),
.81 (d, J=8.4 Hz, 3H, NH, Ph), 7.00 (d, J=8.3 Hz, 2H, Ph), 7.13 (d,
J=8.4 Hz, 2H, Ph), 8.21 (d, J=7.5 Hz, 1H, NH), 9.23 ppm (s, 1H,
[D ]DMSO): d=2.66–3.08 (m, 4H, CH Ph), 3.58 (s, 3H, OCH ), 4.26–
6
2
3
6
4.32 (m, 1H, CH), 4.48–4.53 (m, 1H, CH), 4.93 (s, 2H, OCH Ph),
2
7.20–7.34 (m, 15H, Ph), 7.47 (d, J=8.8 Hz,1H, NH), 7.49 ppm (d, J=
1
3
13
OH); C NMR (100 MHz, [D ]DMSO): d=28.6, 36.5, 37.0, 52.2, 54.3,
7.5 Hz,1H, NH); C NMR (100 MHz, [D ]DMSO): d=37.1, 37.8, 52.3,
6
6
5
1
5.4, 56.3, 78.5, 113.9, 115.5, 127.4, 130.3, 130.5, 130.6, 155.6, 156.5,
58.2, 172.3, 172.4 ppm; MS (ESI) m/z (%): 495.54 (100) [M+Na] .
54.1, 56.3, 65.6, 126.7, 127.0, 127.9, 128.1, 128.5, 128.7, 129.5, 129.6,
+
137.5, 138.4, 156.2, 172.2, 172.3 ppm; MS (ESI) m/z (%): 483.52
+
(
100) [M+Na] .
(S)-Methyl-2-((S)-2-((tert-butoxycarbonyl)amino)-3-phenylpropa-
namido)-3-(4-methoxyphenyl)propanoate (8c): Following the
same procedure as described for the synthesis of 8a except for
(
S)-Methyl-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-(4-methoxy-
phenyl)propanamido)-3-phenylpropanoate (8h): Following the
same procedure as described for the synthesis of 8a except for
starting from 7b and 4b, 8h was obtained in 97% yield as a white
starting from Boc-Phe-OH and 4d, 8c was obtained in 77% yield
1
as a white solid: R =0.67 (PE/EA, 1:1); mp: 151–1528C; H NMR
f
(
400 MHz, [D ]DMSO): d=1.28 (s, 9H, Boc), 2.64–3.00 (m, 4H,
1
6
solid: R =0.53 (PE/EA, 1:1); mp: 161–1628C; H NMR (400 MHz,
f
CH Ph), 3.58 (s, 3H, OCH ), 3.71 (s, 3H, OCH ), 4.15–4.21 (m, 1H,
2
3
3
[D ]DMSO): d=2.58–3.07 (m, 4H, CH Ph), 3.58 (s, 3H, OCH ), 3.72
6
2
3
CH), 4.42–4.48 (m, 1H, CH), 6.83–6.87 (m, 3H, NH, Ph), 7.13–7.27
(s, 3H, OCH ), 4.19–4.25 (m, 1H, CH), 4.49 (q, J=7.8 Hz,1H, CH),
13
3
(
m, 7H, Ph), 8.28 ppm (d, J=7.7 Hz, 1H, NH); C NMR (100 MHz,
4
.93 (s, 2H, OCH Ph), 6.82 (d, J=8.3 Hz, 2H, Ph), 7.16–7.33 (m, 12H,
2
[
D ]DMSO): d=28.6, 36.4, 37.9, 52.3, 54.2, 55.4, 55.9, 78.5, 114.1,
6
Ph), 7.41 (d, J=8.8 Hz,1H, NH), 8.45 ppm (d, J=7.5 Hz, 1H, NH);
1
1
26.6, 128.4, 129.3, 129.6, 130.6, 138.5, 155.6, 158.5, 172.2,
72.3 ppm; MS (ESI) m/z (%): 479.60 (100) [M+Na] .
13
C NMR (100 MHz, [D ]DMSO): d=37.0, 37.1, 52.3, 54.1, 55.4, 56.6,
+
6
6
1
5.6, 113.9, 127.0, 127.9, 128.1, 128.7, 129.6, 130.3, 130.7, 137.4,
37.5, 156.2, 158.2, 172.3 ppm; MS (ESI) m/z (%): 513.57 (100) [M+
(S)-Methyl-2-((S)-2-((tert-butoxycarbonyl)amino)-3-(4-methoxy-
+
Na] .
phenyl)propanamido)-3-(4-methoxyphenyl)propanoate (8d): Fol-
lowing the same procedure as described for the synthesis of 8a
(S)-Methyl-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-phenylpropa-
except for starting from 7a and 4d, 8d was obtained in 93% yield
1
namido)-3-(4-hydroxyphenyl)propanoate (8i): Following the
same procedure as described for the synthesis of 8a except for
starting from Cbz-Phe-OH and 4c, 8i was obtained in 98% yield as
a white solid: R =0.35 (PE/EA, 1:1); mp: 135–1368C; H NMR
(400 MHz, [D ]DMSO): d=2.66–2.98 (m, 4H, CH Ph), 3.58 (s, 3H,
OCH ), 4.27–4.32 (m, 1H, CH), 4.39–4.45 (m, 1H, CH), 4.94 (s, 2H,
OCH Ph), 6.68 (d, J=8.3 Hz, 2H, Ph), 7.02 (d, J=8.3 Hz, 2H, Ph),
7.20–7.35 (m, 10H, Ph), 7.48 (d, J=8.8 Hz, 1H, NH), 8.41 (d, J=
7
as a white solid: R =0.55 (PE/EA, 1:1); mp: 117–1188C; H NMR
f
(
400 MHz, [D ]DMSO): d=1.29 (s, 9H, Boc), 2.58–2.99 (m, 4H,
6
CH Ph), 3.58 (s, 3H, OCH ), 3.71 (s, 6H, OCH ), 4.08–4.14 (m, 1H,
2
3
3
1
CH), 4.42–4.47 (m, 1H, CH), 6.83 (m, 5H, NH, Ph), 7.12–7.15 (m, 4H,
f
13
Ph), 8.26 ppm (d, J=7.6 Hz, 1H, NH);
C NMR (100 MHz,
6
2
[
D ]DMSO): d=28.6, 36.4, 37.0, 52.3, 54.2, 55.4, 56.2, 78.5, 113.9,
6
3
1
14.1, 129.2, 130.3, 130.6, 155.6, 158.2, 158.5, 172.3 ppm; MS (ESI)
2
+
m/z (%): 509.58 (100) [M+Na] .
13
.4 Hz, 1H, NH), 9.25 ppm (s, 1H, OH); C NMR (100 MHz,
(S)-Methyl-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-(4-methoxy-
[D ]DMSO): d=36.4, 37.8, 52.2, 54.5, 56.3, 65.7, 115.5, 126.7, 127.4,
6
phenyl)propanamido)-4-methylpentanoate (8e): Following the
same procedure as described for the synthesis of 8a except for
1
1
27.9, 128.1, 128.5, 128.7, 129.7, 130.5, 137.5, 138.5, 156.2, 156.5,
72.1, 172.4 ppm; MS (ESI) m/z (%): 499.52 (100) [M+Na] .
+
starting from 7b and 4a, 8e was obtained in 91% yield as a white
1
solid: R =0.56 (PE/EA, 1:1); mp: 98–998C; H NMR (400 MHz,
f
(S)-Methyl-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-(4-methoxy-
phenyl)propanamido)-3-(4-hydroxyphenyl)propanoate (8j): Fol-
lowing the same procedure as described for the synthesis of 8a
except for starting from 7b and 4c, 8j was obtained in 92% yield
as a white solid: R =0.24 (PE/EA, 1:1); mp: 1588C; H NMR
[
D ]DMSO): d=0.85 (d, J=6.3 Hz, 3H, CH ), 0.90 (d, J=6.4 Hz, 3H,
6 3
CH ), 1.50–1.66 (m, 3H, CH, CH ), 2.66 (dd, J=10.9, 13.5 Hz, 1H,
3
2
CH Ph), 2.92 (dd, J=3.6, 13.8 Hz, 1H, CH Ph), 3.62 (s, 3H, OCH ),
2
2
3
3
6
8
.72 (s, 3H, OCH ), 4.21–4.34 (m, 2H, CH), 4.94 (s, 2H, OCH Ph),
1
3
2
f
.83 (d, J=8.4 Hz, 2H, Ph), 7.20–7.33 (m, 7H, Ph), 7.44 (d, J=
(400 MHz, [D ]DMSO): d=2.59–2.94 (m, 4H, CH Ph), 3.57 (s, 3H,
6
2
13
.7 Hz, 1H, NH), 8.36 ppm (d, J=7.6 Hz,1H, NH);
C NMR
OCH ), 3.72 (s, 3H, OCH ), 4.19–4.25 (m, 1H, CH), 4.38–4.43 (m, 1H,
CH), 4.93 (s, 2H, OCH Ph), 6.66 (d, J=8.3 Hz, 2H, Ph), 6.81 (d, J=
2
8
7
3
3
(
100 MHz, [D ]DMSO): d=21.8, 23.2, 24.6, 37.0, 50.8, 52.3, 55.4,
6
5
1
6.6, 65.6, 113.9, 127.9, 128.1, 128.7, 130.3, 130.7, 137.5, 156.3,
58.2, 172.4, 173.3 ppm; MS (ESI) m/z (%): 479.54 (100) [M+Na] .
.4 Hz, 2H, Ph), 7.00 (d, J=8.2 Hz, 2H, Ph), 7.16–7.32 (m, 7H, Ph),
.41 (d, J=8.7 Hz, 1H, NH), 8.36 (d, J=7.4 Hz, 1H, NH), 9.24 ppm
+
13
(s, 1H, OH); C NMR (100 MHz, [D ]DMSO): d=36.4, 37.0, 52.2,
6
(S)-Methyl-2-((S)-2-(((benzyloxy)carbonyl)amino)-4-methylpenta-
5
1
4.4, 55.4, 56.6, 65.6, 113.9, 115.5, 127.4, 127.9, 128.1, 128.7, 130.3,
namido)-3-phenylpropanoate (8 f): Following the same procedure
as described for the synthesis of 8a except for starting from Cbz-
Leu-OH and 4b, 8 f was obtained in 91% yield as a white solid:
30.5, 130.7, 137.5, 156.2, 156.5, 158.2, 172.2, 172.4 ppm; MS (ESI)
+
m/z (%): 529.50 (100) [M+Na] .
1
R =0.10 (PE/EA, 8:1); mp: 81–828C; H NMR (400 MHz, [D ]DMSO):
f
6
(S)-Methyl-2-((S)-2-(((benzyloxy)carbonyl)amino)-4-methylpenta-
d=0.85 (q, J=6.6 Hz, 6H, CH ), 1.34–1.39 (m, 2H, CH ), 1.53–1.60
3
2
namido)-3-(4-methoxyphenyl)propanoate (8k): Following the
same procedure as described for the synthesis of 8a except for
starting from Cbz-Leu-OH and 4d, 8k was obtained in 75% yield
(
m, 1H, CH), 2.92–3.05 (m, 2H, CH Ph), 3.56 (s, 3H, OCH ), 4.07 (q,
2 3
J=8.9 Hz,1H, CH), 4.84 (q, J=7.7 Hz, 1H, CH), 5.02 (s, 2H, OCH Ph),
2
7
.18–7.38 (m, 11H, NH, Ph), 8.31 ppm (d, J=7.5 Hz, 1H, NH);
1
1
3
as a white solid: R
f
=0.73 (PE/EA, 1:1); mp: 91–928C; H NMR
C NMR (100 MHz, [D ]DMSO): d=22.0, 23.4, 24.6, 37.0, 41.1, 52.2,
6
(400 MHz, [D ]DMSO): d=0.90 (q, J=6.6 Hz, 6H, CH ), 1.33–1.42
6
3
5
1
3.3, 53.9, 65.8, 127.0, 128.1, 128.2, 128.7, 128.8, 129.5, 137.5, 156.2,
72.3, 172.9 ppm; MS (ESI) m/z (%): 449.49 (100) [M+Na] .
+
(m, 2H, CH ), 1.53–1.60 (m, 1H, CH), 2.85–2.97 (m, 2H, CH Ph), 3.57
2 2
(s, 3H, OCH ), 3.70 (s, 3H, OCH ), 4.03–4.09 (m, 1H, CH), 4.38–4.44
3
3
(S)-Methyl-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-phenylpropa-
(m, 1H, CH), 5.02 (s, 2H, OCH Ph), 6.81(d, J=8.4 Hz, 2H, Ph), 7.11
2
namido)-3-phenylpropanoate (8g): Following the same procedure
as described for the synthesis of 8a except for starting from Cbz-
Phe-OH and 4b, 8g was obtained in 93% yield as a white solid:
(d, J=8.4 Hz, 2H, Ph), 7.35–7.37 (m, 6H, NH, Ph), 8.24 ppm (d, J=
13
7.4 Hz, 1H, NH); C NMR (100 MHz, [D ]DMSO): d=22.0, 23.4, 24.6,
6
36.2, 41.1, 52.2, 53.3, 54.2, 55.4, 65.8, 114.1, 128.1, 128.2, 128.8,
&
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1
29.3, 130.6, 137.5, 156.2, 158.4, 172.4, 172.8 ppm; MS (ESI) m/z
tert-Butyl-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-
2-yl)amino)-3-(4-hydroxyphenyl)-1-oxopropan-2-yl)amino)-3-(4-
methoxyphenyl)-1-oxopropan-2-yl)carbamate (10b): Following
the same procedure as described for the synthesis of 10a except
+
(
%): 479.54 (100) [M+Na] .
(S)-Methyl-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-phenylpropa-
for starting from 8b, 10b was obtained in 91% yield as a white
namido)-3-(4-methoxyphenyl)propanoate (8l): Following the
same procedure as described for the synthesis of 8a except for
starting from Cbz-Phe-OH and 4d, 8l was obtained in 74% yield as
1
solid: R =0.33 (PE/EA, 1:1); mp: 143–1448C; H NMR (400 MHz,
f
[
D ]DMSO): d=0.89–0.92 (m, 6H, CH ), 1.28 (s, 9H, Boc), 1.45–1.55
6
3
1
(m, 2H, CH
2
), 1.64–1.67 (m, 1H, CH), 2.55–2.88 (m, 4H, CH Ph), 3.70
2
a white solid: R =0.16 (PE/EA, 3:1); mp: 130–1318C; H NMR
f
(s, 3H, OCH ), 4.03–4.10 (m, 1H, CH), 4.51–4.63 (m, 1H, CH), 5.00–
3
(
400 MHz, [D ]DMSO): d=2.66–3.00 (m, 4H, CH Ph), 3.58 (s, 3H,
6
2
5
6
.05 (m, 1H, CH), 6.59 (d, J=8.2 Hz, 2H, Ph), 6.73–6.84 (m, 4H, Ph),
.97 (d, J=8.2 Hz, 2H, Ph), 7.07 (d, J=8.4 Hz, 2H, 3’-H, 4’-H), 7.46
OCH ), 3.70 (s, 3H, OCH ), 4.26–4.32 (m, 1H, CH), 4.42–4.47 (m, 1H,
CH), 4.93 (s, 2H, OCH Ph), 6.83 (d, J=8.5 Hz, 2H, Ph), 7.14 (d, J=
8
8
d=36.3, 37.8, 52.3, 54.4, 55.4, 56.3, 65.6, 114.1, 126.7, 127.9, 128.1,
1
1
3
3
2
(
d, J=3.5 Hz, 1H, NH), 7.83 (d, J=8.3 Hz, 1H, NH), 8.03 (s, 1H, 5’-
.5 Hz, 2H, Ph), 7.22–7.32 (m, 10H, Ph), 7.47 (d, J=8.8 Hz, 1H, NH),
13
13
H), 8.45 (d, J=7.6 Hz, 1H, NH), 9.12 ppm (s, 1H, OH); C NMR
100 MHz, [D ]DMSO): d=21.9, 23.5, 24.8, 28.6, 37.1, 37.4, 52.9,
.43 ppm (d, J=7.4 Hz, 1H, NH); C NMR (100 MHz, [D ]DMSO):
6
(
6
5
4.0, 55.4, 56.6, 78.6, 113.1, 113.9, 115.2, 119.5, 127.8, 130.4, 130.5,
28.5, 128.7, 129.2, 129.7, 130.6, 137.5, 138.4, 156.2, 158.5, 172.2,
+
130.6, 148.6, 150.8, 155.5, 156.3, 158.2, 171.5, 171.7, 187.5 ppm;
72.3 ppm; MS (ESI) m/z (%): 513.50 (100) [M+Na] .
+
HRMS (ESI) m/z [M+H] calcd for C H N O : 622.31229, found:
6
34
44
3
8
+
22.31061, m/z [M+Na] calcd for C H N NaO : 644.29424,
34 43 3 8
(S)-Methyl-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-(4-methoxy-
found: 644.29264; HPLC: t =8.34 min (96.2% purity).
R
phenyl)propanamido)-3-(4-methoxyphenyl)propanoate (8m):
Following the same procedure as described for the synthesis of 8a
except for starting from 7b and 4d, 8m was obtained in 72%
tert-Butyl-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-
2-yl)amino)-3-(4-methoxyphenyl)-1-oxopropan-2-yl)amino)-1-
oxo-3-phenylpropan-2-yl)carbamate (10c): Following the same
procedure as described for the synthesis of 10a except for starting
yield as a white solid: R =0.52 (PE/EA, 1:1); mp: 152–1538C;
f
1
H NMR (400 MHz, [D ]DMSO): d=2.58–2.99 (m, 4H, CH Ph), 3.58 (s,
6
2
3
H, OCH ), 3.70 (s, 3H, OCH ), 3.72 (s, 3H, OCH ), 4.20–4.25 (m, 1H,
from 8c, 10c was obtained in 80% yield as a white solid: R =0.45
3
3
3
f
1
CH), 4.41–4.47 (m, 1H, CH), 4.93 (s, 2H, OCH Ph), 6.81–6.85 (m, 4H,
(PE/EA, 1:1); mp: 150–1518C; H NMR (400 MHz, [D ]DMSO): d=
2
6
Ph), 7.13–7.33 (m, 9H, Ph), 7.44 (d, J=8.8 Hz, 1H, NH), 8.43 ppm (d,
0.89–0.92 (m, 6H, CH ), 1.28 (s, 9H, Boc), 1.45–1.54 (m, 2H, CH ),
1.56–1.66 (m, 1H, CH), 2.61–2.93 (m, 4H, CH Ph), 3.69 (s, 3H,
2
3
2
1
3
J=7.5 Hz, 1H, NH); C NMR (100 MHz, [D ]DMSO): d=36.3, 37.0,
6
5
1
1
2.3, 54.3, 55.3, 55.4, 56.6, 65.6, 113.9, 114.1, 127.9, 128.1, 128.7,
29.3, 130.3, 130.6, 130.7, 137.5, 156.2, 158.2, 158.5, 172.2,
72.3 ppm; MS (ESI) m/z (%): 543.48 (100) [M+Na] .
OCH ), 4.08–4.13 (m, 1H, CH), 4.52–4.57 (m, 1H, CH), 5.01–5.06 (m,
3
1H, CH), 6.74–6.89 (m, 4H, Ph), 7.09–7.18 (m, 5H, Ph), 7.22 (d, J=
6.8 Hz, 2H, 3’-H,4’-H), 7.47 (d, J=3.6 Hz, 1H, NH), 7.90 (d, J=8.1 Hz,
+
1
H, NH), 8.05 (s, 1H, 5’-H), 8.48 ppm (d, J=7.8 Hz, 1H, NH);
1
3
C NMR (100 MHz, [D ]DMSO): d=21.3, 22.9, 24.3, 27.6, 28.0, 36.8,
6
tert-Butyl-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-
-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-3-(4-methoxy-
3
1
1
6
6
7.4, 52.3, 53.4, 54.8, 55.7, 78.0, 112.5, 113.3, 119.0, 126.0, 127.8,
2
29.0, 130.1, 138.0, 148.1, 150.2, 154.9, 157.7, 170.8, 171.1,
phenyl)-1-oxopropan-2-yl)carbamate (10a): 1n NaOH was added
to a solution of 8a (456 mg, 1 mmol) in THF until pH 12–13, and
the reaction mixture was stirred at room temperature for 2 h, fol-
lowed by acidification with 1n HCl until pH 2–3. The aqueous
phase was extracted with EtOAc (5ꢂ10 mL), and the combined or-
ganic phases were dried with Na SO . The solvent was concentrat-
+
86.9 ppm; HRMS (ESI) m/z [M+Na] calcd for C H N NaO :
34
43
3
7
+
28.29932, found: 628.29940, m/z [M+K] calcd for C H N KO :
34
43
3
7
44.27325, found: 644.27078; HPLC: t =9.76 min (99.2% purity).
R
tert-Butyl-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-
-yl)amino)-3-(4-methoxyphenyl)-1-oxopropan-2-yl)amino)-3-(4-
2
4
2
ed in vacuo, and the resulting residue 9a was used in the next
step without further purification. EDC·HCl (230 mg, 1.2 mmol) and
HOBt (162 mg, 1.2 mmol) were added to a solution of 9a (the
crude solution obtained previously, equivalent to 1 mmol 9a) and
methoxyphenyl)-1-oxopropan-2-yl)carbamate (10d): Following
the same procedure as described for the synthesis of 10a except
for starting from 8d, 10d was obtained in 66% yield as a white
1
solid: R =0.35 (PE/EA, 1:1); mp: 162–1648C; H NMR (400 MHz,
f
3
(181 mg, 1 mmol) in the presence of NMM (0.34 mL, 3 mmol) in
CDCl ) solid: d=0.87 (d, J=6.3 Hz, 3H, CH ), 0.98 (d, J=6.1 Hz, 3H,
3
3
1
0 mL DMF. The reaction mixture was stirred at room temperature
CH ), 1.33 (s, 9H, Boc), 1.40–1.45 (m, 1H, CH), 1.53–1.60 (m, 2H,
3
overnight and diluted with EtOAc and washed with 10% citric
acid, saturated aqueous NaHCO and brine, dried with Na SO , and
CH ), 2.82–3.05 (m, 4H, CH Ph), 3.75 (s, 3H, OCH ), 3.78 (s, 3H,
2
2
3
3
2
4
OCH ), 4.28–4.30 (m, 1H, CH), 4.56–4.61 (m, 1H, CH), 4.85 (s, 1H,
3
concentrated in vacuo to give the crude product, which was sepa-
NH), 5.25–5.29 (m, 1H, CH), 6.37 (s, 1H, NH), 6.47 (s, 1H, NH), 6.57
rated by silica gel chromatography to yield 10a as a white solid
(dd, J=1.7, 3.6 Hz, 1H, 4’-H), 6.72 (d, J=8.6 Hz, 2H, Ph), 6.82 (d, J=
1
(
332 mg, 55%): R =0.47 (PE/EA, 1:1); mp: 142–1438C; H NMR
f
8
.6 Hz, 2H, Ph), 6.97 (d, J=8.6 Hz, 2H, Ph), 7.10 (d, J=8.6 Hz, 2H,
(
400 MHz, [D ]DMSO): d=0.89–0.92 (m, 6H, CH ), 1.29 (s, 9H, Boc),
6
3
Ph), 7.31(d, J=3.6 Hz, 1H, 3’-H), 7.64 ppm (d, J=1.0 Hz, 1H, 5’-H);
1
.49–1.58 (m, 2H, CH ), 1.65–1.67 (m, 1H, CH), 2.57–3.00 (m, 4H,
13
2
C NMR (100 MHz, CDCl ): d=21.8, 23.2, 24.8, 28.2, 37.2, 41.8, 52.7,
3
CH Ph), 3.70 (s, 3H, OCH ), 4.02–4.10 (m, 1H, CH), 4.60–4.63 (m,
2
3
5
1
1
6
6
4.4, 55.1, 55.2, 55.9, 80.4, 112.5, 114.0, 114.2, 118.9, 128.0, 128.3,
1
H, CH), 5.01–5.07 (m, 1H, CH), 6.74–6.82 (m, 4H, Ph), 7.06–7.08
30.3, 130.3, 147.1, 150.9, 155.4, 158.7, 158.7, 170.1, 171.0,
(
m, 2H, 3’-H,4’-H), 7.20 (s, 5H, Ph), 7.47 (d, J=3.2 Hz, 1H, NH), 7.91
+
86.9 ppm; HRMS (ESI) m/z [M+Na] calcd for C H N NaO :
35
45
3
8
(
d, J=8.8 Hz, 1H, NH), 8.04 (s, 1H, 5’-H), 8.49 ppm (d, J=7.4 Hz,
+
58.30989, found: 658.31018, m/z [M+K] calcd for C H N KO :
35
45
3
8
1
3
1
3
1
1
H, NH); C NMR (100 MHz, [D ]DMSO): d=21.9, 23.5, 24.9, 28.6,
6
74.28382, found: 674.28326; HPLC: t =9.59 min (99.3% purity).
R
7.1, 38.2, 52.9, 53.7, 55.4, 56.6, 78.5, 113.1, 113.9, 119.5, 126.7,
28.4, 129.7, 130.3, 130.6, 137.8, 148.7, 150.8, 155.5, 158.2, 171.4,
Benzyl-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-
+
71.8, 187.5 ppm; HRMS (ESI) m/z [M+Na]
calcd for
yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-3-(4-methoxyphen-
yl)-1-oxopropan-2-yl)carbamate (10e): Following the same proce-
dure as described for the synthesis of 10a except for starting from
+
C H N NaO : 628.29932, found: 628.29976, m/z [M+K] calcd for
3
4
43
3
7
C H N KO : 644.27325, found: 644.27106; HPLC: t =9.68 min
3
4
43
3
7
R
(
97.7% purity).
8e, 10e was obtained in 57% yield as a white solid: R =0.38 (PE/
f
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Full Papers
1
EA, 1:1); mp: 155–1578C; H NMR (400 MHz, [D ]DMSO): d=0.83–
0
8.6 Hz, 1H, NH), 7.48 (d, J=3.4 Hz, 1H, 3’-H), 8.04 (d, J=1.9 Hz, 1H,
5’-H), 8.08 (d, J=8.2 Hz, 1H, NH), 8.46 ppm (d, J=7.5 Hz, 1H, NH);
6
.92 (m, 12H, CH ), 1.40–1.57 (m, 6H, CH, CH ), 2.64–2.89 (m, 2H,
3
2
13
CH Ph), 3.71 (s, 3H, OCH ), 4.18–4.24 (m, 1H, CH), 4.35–4.41 (m,
C NMR (100 MHz, [D ]DMSO): d=21.9, 23.5, 24.9, 37.1, 38.0, 52.9,
2
3
6
1
1
H, CH), 4.94 (s, 2H, OCH Ph), 4.99–5.04 (m, 1H, CH), 6.73 (d, J=
.6 Hz, 1H, 4’-H), 6.81 (d, J=8.6 Hz, 2H, Ph), 7.19 (d, J=8.6 Hz, 2H,
53.9, 55.4, 56.8, 65.6, 113.1, 113.9, 119.5, 126.7, 127.8, 128.1, 128.4,
128.7, 129.7, 130.3, 130.6, 137.5, 137.9, 148.7, 150.9, 156.1, 158.2,
2
+
Ph), 7.23–7.32 (m, 5H, Ph), 7.40 (d, J=8.6 Hz, 1H, NH), 7.47 (d, J=
171.4, 171.8, 187.5 ppm; HRMS (ESI) m/z [M+Na] calcd for
+
3
7
2
1
1
.5 Hz, 1H, NH), 8.01–8.03 (m, 2H, 3’-H,5’-H), 8.33 ppm (d, J=
C H N NaO : 662.28367, found: 662.28488, m/z [M+K] calcd for
37
41
3
7
1
3
.5 Hz, 1H, NH); C NMR (100 MHz, [D ]DMSO): d=21.8, 22.3, 23.4,
C H N KO : 678.25760, found: 678.25728; HPLC: t =9.76 min
37 41 3 7 R
(98% purity).
6
3.5, 24.5, 24.9, 37.0, 51.2, 53.0, 55.4, 56.7, 65.6, 113.0, 113.9, 119.3,
27.8, 128.1, 128.7, 130.4, 130.7, 137.5, 148.6, 150.9, 156.3, 158.2,
71.8, 172.4, 187.8 ppm; HRMS (ESI) m/z [M+H] calcd for
+
Benzyl-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-
yl)amino)-3-(4-hydroxyphenyl)-1-oxopropan-2-yl)amino)-1-oxo-
+
C H N O : 606.31738, found: 606.31664, m/z [M+Na] calcd for
C H N NaO : 628.29932, found: 628.29952; HPLC: t =9.63 min
3
4
44
3
7
3
-phenylpropan-2-yl)carbamate (10i): Following the same proce-
dure as described for the synthesis of 10a except for starting from
i, 10i was obtained in 77% yield as a white solid: R =0.26 (PE/
3
4
43
3
7
R
(
96% purity).
8
f
1
Benzyl-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-
yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-4-methyl-1-oxopen-
tan-2-yl)carbamate (10 f): Following the same procedure as de-
scribed for the synthesis of 10a except for starting from 8 f, 10 f
EA, 1:1); mp: 182–1848C; H NMR (400 MHz, [D ]DMSO): d=0.90–
6
0.92 (m, 6H, CH ), 1.49–1.56 (m, 2H, CH ), 1.66–1.69 (m, 1H, CH),
3
2
2.62–2.93 (m, 4H, CH Ph), 4.22–4.24 (m, 1H, CH), 4.50–4.62 (m, 1H,
2
CH), 4.92 (s, 2H, OCH Ph), 5.03–5.06 (m, 1H, CH), 6.60 (d, J=8.4 Hz,
2
was obtained in 62% yield as a white solid: R =0.52 (PE/EA, 1:1);
2H, Ph), 6.74 (dd, J=1.6, 3.5 Hz, 1H, 4’-H), 6.99 (d, J=8.4 Hz, 2H,
Ph), 7.18–7.32 (m, 10H, Ph), 7.43–7.48 (m, 2H, 3’-H, 5’-H), 7.43 (d,
J=8.8 Hz, 1H, NH), 8.02 (d, J=8.3 Hz, 1H, NH), 8.42 (d, J=7.7 Hz,
f
1
mp: 137–1388C; H NMR (400 MHz, [D ]DMSO): d=0.79–0.91 (m,
6
1
3
2H, CH ), 1.26–1.56 (m, 6H, CH, CH ), 2.73–3.00 (m, 2H, CH Ph),
.96–4.01 (m, 1H, CH), 4.54–4.60 (m, 1H, CH), 5.01 (s, 3H, CH,
3
2
2
13
1H, NH), 9.14 ppm (s, 1H, OH); C NMR (100 MHz, [D ]DMSO): d=
6
OCH Ph), 6.73 (dd, J=1.6, 3.4 Hz, 1H, 4’-H), 7.17 (s, 5H, Ph), 7.31–
21.9, 23.5, 24.8, 37.3, 37.9, 52.9, 54.3,56.6, 65.7, 113.1, 115.3, 119.5,
126.7, 127.9, 127.9, 128.1, 128.5, 128.7, 129.6, 130.6, 137.4, 138.5,
148.6, 150.9, 156.2, 156.3, 171.5, 171.6, 187.6 ppm; HRMS (ESI) m/z
2
7
8
.40 (m, 6H, 3’-H, Ph), 7.47 (d, J=3.5 Hz, 1H, NH), 7.94 (d, J=
.2 Hz, 1H, NH), 8.04 (s, 1H, 5’-H), 8.39 ppm (d, J=7.6 Hz, 1H, NH);
1
3
+
C NMR (100 MHz, [D ]DMSO): d=21.9, 22.0, 23.4, 23.5, 24.5, 24.8,
[M+H] calcd for C H N O : 626.28608, found: 626.28463, m/z
6
36 40
3
7
+
3
1
1
5
7.9, 41.2, 52.9, 53.7, 65.8, 113.1, 119.5, 126.6, 128.1, 128.2, 128.4,
[M+Na] calcd for C H N NaO : 648.26802, found: 648.26932, m/
36 39 3 7
z [M+K] calcd for C H N KO : 664.24195, found: 664.24075;
36 39 3 7
+
28.8, 129.6, 137.5, 137.9, 148.7, 150.8, 156.3, 171.4, 172.4,
+
87.5 ppm; HRMS (ESI) m/z [M+H]
calcd for C H N O :
HPLC: t =8.65 min (98.5% purity).
33
42
3
6
R
+
76.30681, found: 576.30791, m/z [M+Na]
calcd for
+
Benzyl-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-
yl)amino)-3-(4-hydroxyphenyl)-1-oxopropan-2-yl)amino)-3-(4-
methoxyphenyl)-1-oxopropan-2-yl)carbamate (10j): Following
the same procedure as described for the synthesis of 10a except
C H N NaO : 598.28876, found: 598.28673, m/z [M+K] calcd for
3
3
41
3
6
C H N KO : 614.26269, found: 614.26271; HPLC: tR =9.9 min
3
3
41
3
6
(
97.4% purity).
Benzyl-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-
yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxo-3-phenylpro-
pan-2-yl)carbamate (10g): Following the same procedure as de-
scribed for the synthesis of 10a except for starting from 8g, 10g
for starting from 8j, 10j was obtained in 65% yield as a white
1
solid: R =0.21 (PE/EA, 1:1); mp: 165–1678C; H NMR (400 MHz,
f
[D ]DMSO): d=0.89–0.91 (m, 6H, CH ), 1.48–1.56 (m, 2H, CH ),
6
3
2
1.66–1.69 (m, 1H, CH), 2.55–2.93 (m, 4H, CH Ph), 3.71 (s, 3H,
2
was obtained in 73% yield as a white solid: R =0.46 (PE/EA, 1:1);
OCH ), 4.13–4.19 (m, 1H, CH), 4.49–4.52 (m, 1H, CH), 4.92 (s, 2H,
f
3
1
mp: 168–1698C; H NMR (400 MHz, [D ]DMSO): d=0.90–0.93 (m,
OCH Ph), 5.01–5.05 (m, 1H, CH), 6.60 (d, J=8.3 Hz, 2H, Ph), 6.74
6
2
6
H, CH ), 1.49–1.57 (m, 2H, CH ), 1.66–1.69 (m, 1H, CH), 2.51–3.02
(dd, J=1.5, 3.4 Hz, 1H, 4’-H), 6.79 (d, J=8.5 Hz, 2H, Ph), 6.99 (d, J=
8.4 Hz, 2H, Ph), 7.13 (d, J=8.5 Hz, 2H, Ph), 7.22–7.32 (m, 5H, Ph),
7.42 (d, J=8.7 Hz, 1H, NH), 7.47 (d, J=3.5 Hz, 1H, NH), 8.03–8.05
(m, 2H, 3’-H, 5’-H), 8.45(d, J=7.6 Hz, 1H, NH), 9.17 ppm (s, 1H,
3
2
(
(
m, 4H, CH Ph), 4.22–4.24 (m, 1H, CH), 4.60–4.62 (m, 1H, CH), 4.92
s, 2H, OCH Ph), 5.03–5.06 (m, 1H, CH), 6.74 (dd, J=1.6, 3.5 Hz, 1H,
2
2
4
’-H), 7.21–7.32 (m, 15H, Ph), 7.43 (d, J=8.8 Hz, 1H, NH), 7.48 (d,
13
J=3.5 Hz, 1H, 3’-H), 8.05 (d, J=1.2 Hz, 1H, 5’-H), 8.10 (d, J=8.3 Hz,
1
OH); C NMR (100 MHz, [D ]DMSO): d=21.9, 23.5, 24.8, 37.1, 37.3,
6
13
H, NH), 8.46 ppm (d, J=7.7 Hz, 1H, NH); C NMR (100 MHz,
52.9, 54.2, 55.4, 56.8, 65.6, 113.1, 113.9, 115.3, 119.5, 127.8, 127.9,
128.1, 128.7, 130.4, 130.5, 130.6, 137.5, 148.6, 150.9, 156.1, 156.2,
[
D ]DMSO): d=21.9, 23.5, 24.9, 37.9, 38.0, 52.9, 53.9, 56.5, 65.6,
6
+
113.1, 119.5, 126.6, 126.7, 127.8, 128.1, 128.5, 128.7, 129.6, 129.7,
158.2, 171.5, 171.7, 187.6 ppm; HRMS (ESI) m/z [M+Na] calcd for
+
1
37.4, 137.9, 138.5, 148.7, 150.9, 156.1, 171.4, 171.7, 187.5 ppm;
C H N NaO : 678.27859, found: 678.27811, m/z [M+K] calcd for
37
41
3
8
+
HRMS (ESI) m/z [M+Na] calcd for C H N NaO : 632.27311,
found: 632.27404, m/z [M+K] calcd for C H N KO : 648.24704,
C H N KO : 694.25252, found: 694.25371; HPLC: t =8.54 min
37 41 3 8 R
(98% purity).
36
39
3
6
+
36
39
3
6
found: 648.24820; HPLC: t =9.9 min (97.2% purity).
R
Benzyl-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-
yl)amino)-3-(4-methoxyphenyl)-1-oxopropan-2-yl)amino)-4-
methyl-1-oxopentan-2-yl)carbamate (10k): Following the same
procedure as described for the synthesis of 10a except for starting
Benzyl-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-
yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-3-(4-methoxyphen-
yl)-1-oxopropan-2-yl)carbamate (10h): Following the same proce-
dure as described for the synthesis of 10a except for starting from
from 8k, 10k was obtained in 76% yield as a white solid: R =0.50
f
1
8
h, 10h was obtained in 80% yield as a white solid: R =0.36 (PE/
(PE/EA, 1:1); mp: 153–1558C; H NMR (400 MHz, [D ]DMSO): d=
f
6
1
EA, 1:1); mp: 156–1588C; H NMR (400 MHz, [D ]DMSO): d=0.89–
0.84–0.91 (m, 12H, CH ), 1.24–1.65 (m, 6H, CH, CH ), 2.66–2.93 (m,
6
3
2
0
.93 (m, 6H, CH ), 1.47–1.56 (m, 2H, CH ), 1.66–1.69 (m, 1H, CH),
2H, CH Ph), 3.68 (s, 3H, OCH ), 3.96–4.01 (m, 1H, CH), 4.49–4.55
2 3
3
2
2
.51–3.02 (m, 4H, CH Ph), 3.71 (s, 3H, OCH ), 4.14–4.19 (m, 1H,
(m, 1H, CH), 5.02 (s, 3H, CH, OCH Ph), 6.72–6.74 (m, 3H, 4’-H, Ph),
2
3
2
CH), 4.59–4.61 (m, 1H, CH), 4.92 (s, 2H, OCH Ph), 5.02–5.07 (m, 1H,
CH), 6.74 (dd, J=1.9, 3.4 Hz, 1H, 4’-H), 6.78 (d, J=8.4 Hz, 2H, Ph),
7.08 (d, J=8.3 Hz, 2H, Ph), 7.31–7.35 (m, 5H, Ph), 7.39 (d, J=
8.4 Hz, 1H, NH), 7.46 (d, J=3.4 Hz, 1H, 3’-H), 7.87 (d, J=8.1 Hz, 1H,
NH), 8.05 (s, 1H, 5’-H), 8.37 ppm (d, J=7.4 Hz, 1H, NH); C NMR
2
1
3
7
.12 (d, J=8.4 Hz, 2H, Ph), 7.15–7.32 (m, 10H, Ph), 7.37 (d, J=
&
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10
ꢀ 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Full Papers
(
100 MHz, [D ]DMSO): d=21.3, 21.4, 22.8, 22.9, 24.0, 24.2, 36.5,
2.3, 53.1, 53.3, 54.7, 65.3, 112.5, 113.2, 118.9, 127.5, 127.6, 128.2,
tert-Butyl-((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)a-
mino)-3-(4-hydroxyphenyl)-1-oxopropan-2-yl)carbamate (10o):
Following the same procedure as described for the synthesis of
10a except for starting from Boc-Tyr-OH, 10o was obtained in
6
5
1
1
6
6
29.1, 130.1, 136.9, 148.1, 150.3, 155.7, 157.6, 170.8, 171.7,
+
86.9 ppm; HRMS (ESI) m/z [M+Na] calcd for C H N NaO :
34
43
3
7
+
28.29932, found: 628.30211, m/z [M+K] calcd for C H N KO :
63% yield as a white solid: R =0.35 (PE/EA, 2:1); mp: 76–788C;
34
43
3
7
f
1
44.27325, found: 644.27183; HPLC: t =9.75 min (97.5% purity).
H NMR (400 MHz, [D ]DMSO): d=0.90–0.92 (m, 6H, CH ), 1.31 (s,
R
6
3
9
H, Boc), 1.48–1.61 (m, 2H, CH ), 1.69–1.71 (m, 1H, CH), 2.54–2.80
2
(
(
7
5
(
m, 2H, CH Ph), 4.04–4.13 (m, 1H, CH), 5.03–5.08 (m, 1H, CH), 6.61
d, J=8.1 Hz, 2H, Ph), 6.74 (s, 1H, 4’-H), 6.81 (d, J=8.5 Hz, 1H, NH),
2
Benzyl-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-
yl)amino)-3-(4-methoxyphenyl)-1-oxopropan-2-yl)amino)-1-oxo-
.01 (d, J=8.1 Hz, 2H, Ph), 7.47 (d, J=3.3 Hz, 1H, 3’-H), 8.04 (s, 1H,
3
-phenylpropan-2-yl)carbamate (10l): Following the same proce-
dure as described for the synthesis of 10a except for starting from
l, 10l was obtained in 51% yield as a white solid: R =0.46 (PE/
13
’-H), 8.26 (d, J=7.7 Hz, 1H, NH), 9.14 ppm (s, 1H, OH); C NMR
100 MHz, [D ]DMSO): d=21.8, 23.6, 24.7, 28.6, 36.9, 52.8, 56.3,
6
8
f
1
78.4, 113.1, 115.2, 119.5, 128.5, 130.5, 148.6, 150.8, 155.6, 156.2,
1
4
C H N NaO : 467.21526, found: 467.21522; HPLC: t =7.51 min
(
EA, 1:1); mp: 154–1568C; H NMR (400 MHz, [D ]DMSO): d=0.89–
6
+
72.4, 187.8 ppm; HRMS (ESI) m/z [M+H] calcd for C H N O :
24 33 2 6
0
.93 (m, 6H, CH ), 1.49–1.56 (m, 2H, CH ), 1.64–1.69 (m, 1H, CH),
3
2
+
45.23331, found: 445.23358, m/z [M+Na]
calcd for
2
.62–2.95 (m, 4H, CH Ph), 3.68 (s, 3H, OCH ), 4.22–4.25 (m, 1H,
2
3
24
32
2
6
R
CH), 4.51–4.57 (m, 1H, CH), 4.92 (s, 2H, OCH Ph), 5.02–5.07 (m, 1H,
2
97.9% purity).
CH), 6.73–6.77 (m, 3H, 4’-H, Ph), 7.12 (d, J=8.6 Hz, 2H, Ph), 7.19–
7
.32 (m, 10H, Ph), 7.46–7.49 (m, 2H, NH), 8.05–8.10 (m, 2H, 3’-H, 5’-
Benzyl ((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)ami-
no)-1-oxo-3-phenylpropan-2-yl)carbamate (10p): Following the
same procedure as described for the synthesis of 10a except for
13
H), 8.47 ppm (d, J=7.7 Hz, 1H, NH); C NMR (100 MHz, [D ]DMSO):
d=21.9, 23.5, 24.8, 37.2, 37.9, 52.9, 54.2, 55.4, 56.5, 65.6, 113.1,
6
113.9, 119.6, 126.7, 127.8, 128.1, 128.5, 128.7, 129.6, 129.7, 130.7,
starting from Cbz-Phe-OH, 10p was obtained in 87% yield as
1
1
37.4, 138.5, 148.7, 150.8, 156.2, 158.2, 171.4, 171.7, 187.5 ppm;
a white solid: R =0.67 (PE/EA, 1:1); mp: 123–1248C; H NMR
f
+
HRMS (ESI) m/z [M+Na] calcd for C H N NaO : 662.28367,
found: 662.28469, m/z [M+K] calcd for C H N KO : 678.25760,
37
41
3
7
(400 MHz, [D ]DMSO): d=0.87–0.92 (m, 6H, CH ), 1.48–1.58 (m, 2H,
6
3
+
37
41
3
7
CH ), 1.68–1.70 (m, 1H, CH), 2.65–2.96 (m, 2H, CH Ph), 4.28–4.34
2 2
found: 678.25877; HPLC: t =9.8 min (99.6% purity).
R
(m, 1H, CH), 4.92 (s, 2H, OCH Ph), 5.02–5.07 (m, 1H, CH), 6.74 (dd,
2
J=1.6, 3.6 Hz, 1H, 4’-H), 7.19–7.33 (m, 10H, Ph), 7.48–7.50 (m, 2H,
NH, 3’-H), 8.05 (d, J=0.9 Hz, 1H, 5’-H), 8.46 ppm (d, J=7.6 Hz, 1H,
Benzyl-((S)-1-(((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-
yl)amino)-3-(4-methoxyphenyl)-1-oxopropan-2-yl)amino)-3-(4-
methoxyphenyl)-1-oxopropan-2-yl)carbamate (10m): Following
the same procedure as described for the synthesis of 10a except
13
NH); C NMR (100 MHz, [D ]DMSO): d=21.9, 23.5, 24.9, 37.8, 53.1,
6
5
1
6.3, 65.6, 113.1, 119.5, 126.7, 127.9, 128.1, 128.5, 128.7, 129.6,
37.5, 138.4, 148.7, 150.8, 156.3, 172.1, 187.8 ppm; HRMS (ESI) m/z
+
[M+H] calcd for C H N O : 463.22275, found: 463.22350, m/z
for starting from 8m, 10m was obtained in 82% yield as a white
27 31
2
5
+
1
[M+Na] calcd for C H N NaO : 485.20469, found: 485.20533;
solid: R =0.26 (PE/EA, 1:1); mp: 172–1748C; H NMR (400 MHz,
27 30
2
5
f
HPLC: t =9.28 min (95.4% purity).
[
D ]DMSO): d=0.89–0.92 (m, 6H, CH ), 1.49–1.56 (m, 2H, CH ),
R
6
3
2
1
.65–1.67 (m, 1H, CH), 2.62–2.94 (m, 4H, CH Ph), 3.68 (s, 3H,
2
Benzyl ((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)ami-
no)-3-(4-hydroxyphenyl)-1-oxopropan-2-yl)carbamate (10q): Fol-
lowing the same procedure as described for the synthesis of 10a
OCH ), 3.71 (s, 3H, OCH ), 4.14–4.19 (m, 1H, CH), 4.51–4.54 (m, 1H,
CH), 4.92 (s, 2H, OCH Ph), 5.01–5.05 (m, 1H, CH), 6.73–6.80 (m, 5H,
3
3
2
4
8
5
’-H, Ph), 7.10–7.14 (m, 4H, Ph), 7.21–7.32 (m, 5H, Ph), 7.38 (d, J=
.6 Hz, 1H, NH), 7.48 (d, J=3.5 Hz, 1H, NH), 8.03–8.04 (m, 2H, 3’-H,
except for starting from Cbz-Tyr-OH, 10q was obtained in 69%
1
yield as a white solid: R =0.18 (PE/EA, 2:1); mp: 57–588C; H NMR
f
13
’-H), 8.43 ppm (d, J=7.5 Hz, 1H, NH);
C NMR (100 MHz,
(
400 MHz, [D ]DMSO): d=0.91–0.94 (m, 6H, CH ), 1.46–1.58 (m, 2H,
6
3
[
D ]DMSO): d=21.9, 23.5, 24.8, 37.1, 37.2, 52.9, 54.2, 55.4, 56.4,
6
CH ), 1.60–1.70 (m, 1H, CH), 2.54–2.85 (m, 2H, CH Ph), 4.20–4.26
2
2
5
1
1
6.8, 65.6, 113.1, 113.9, 119.6, 127.8, 127.9, 128.1, 128.7, 129.7,
30.3, 130.6, 130.7, 137.5, 137.6, 148.7, 150.9, 156.1, 158.3, 171.4,
71.7, 187.5 ppm; HRMS (ESI) m/z [M+Na]
(
m, 1H, CH), 4.95 (s, 2H, OCH Ph), 5.02–5.07 (m, 1H, CH), 6.63 (d,
2
J=8.2 Hz, 2H, Ph), 6.74 (dd, J=1.5, 3.5 Hz, 1H, 4’-H), 7.06 (d, J=
+
calcd for
8
8
1
2
1
.3 Hz, 2H, Ph), 7.23–7.35 (m, 5H, Ph), 7.40 (d, J=8.7 Hz, 1H, NH),
+
C H N NaO : 692.29424, found: 692.29410, m/z [M+K] calcd for
C H N KO : 708.26817, found: 708.26621; HPLC: t =9.65 min
3
8
43
3
8
.48 (d, J=3.6 Hz, 1H, 3’-H), 8.04 (s, 1H, 5’-H), 8.41 (d, J=7.6 Hz,
3
8
43
3
8
R
13
H, NH), 9.18 ppm (s, 1H, OH); C NMR (100 MHz, [D ]DMSO): d=
6
(
99.2% purity).
1.9, 23.5, 24.9, 37.1, 53.0, 56.7, 65.6, 113.1, 115.3, 119.4, 127.2,
27.8, 128.1, 128.4, 128.7, 130.6, 137.5, 148.6, 150.9, 156.2, 172.2,
+
tert-Butyl-((S)-1-(((S)-1-(furan-2-yl)-4-methyl-1-oxopentan-2-yl)a-
mino)-1-oxo-3-phenylpropan-2-yl)carbamate (10n): Following
the same procedure as described for the synthesis of 10a except
187.8 ppm; HRMS (ESI) m/z [M+H]
479.21766, found: 479.21861, m/z [M+Na]
calcd for C H N O :
27 31 2 6
+
calcd for
C H N NaO : 501.19961, found: 501.20030; HPLC: t =7.79 min
27
30
2
6
R
for starting from Boc-Phe-OH, 10n was obtained in 75% yield as
(97.4% purity).
1
a white solid: R =0.85 (PE/EA, 1:1); mp: 151–1528C; H NMR
f
(
400 MHz, [D ]DMSO): d=0.90–0.93 (m, 6H, CH ), 1.30 (s, 9H, Boc),
6
3
1
.45–1.61 (m, 2H, CH ), 1.69–1.72 (m, 1H, CH), 2.66–2.92 (m, 2H,
2
Computational chemistry
CH Ph), 4.17–4.23 (m, 1H, CH), 5.04–5.09 (m, 1H, CH), 6.74 (s, 1H,
2
4
3
’-H), 6.92 (d, J=8.7 Hz, 1H, NH), 7.16–7.29 (m, 5H, Ph), 7.49 (d, J=
.5 Hz, 1H, 3’-H), 8.04 (s, 1H, 5’-H), 8.32 ppm (d, J=7.8 Hz, 1H,
Protein and ligands preparation: Performed using Discovery
Studio 2.5. AutoDock 4.2 was used for all docking calculations. Mo-
lecular dynamics simulations were conducted with AMBER 11. Fig-
ures were generated with PyMOL 0.99. The proteasome crystal
structure (PDB ID: 4NO8) was used due to its relatively high resolu-
tion of 2.70 ꢁ and the structural similarity between its co-crystal-
lized ligand, a tripeptide with a ketoamide warhead, and our de-
signed furyl ketone peptides. As the active site covered two subu-
1
3
NH); C NMR (100 MHz, [D ]DMSO): d=21.8, 23.6, 24.7, 28.6, 37.7,
5
1
C H N O : 429.23840, found: 429.23866, m/z [M+Na] calcd for
C H N NaO : 451.22034, found: 451.22138; HPLC: t =9.2 min
6
2.8, 56.0, 78.5, 113.1, 119.5, 126.6, 128.4, 129.6, 138.5, 148.7, 150.8,
+
55.6, 172.2, 187.8 ppm; HRMS (ESI) m/z [M+H] calcd for
+
2
4
33
2
5
2
4
32
2
5
R
(
99.6% purity).
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Full Papers
nits (b5 and b6), we selected chains K and L for structural analysis
and docking experiments. The protein was prepared in Discovery
Studio 2.5, with amino acid residues ionized at pH 7.4, hydrogens
added, charges calculated, and the waters removed. The starting
conformation for docking was a low-energy conformer generated
in Discovery Studio’s “prepare ligands” protocol.
cence substrates were dispensed after 15 min, and luminescence
signals were recorded 20 min after reagent addition. The curve-fit-
ting software package GraphPad Prism 4.0 was used to generate
dose–response curves to determine IC50 values for individual com-
pounds tested in a dose-dependent manner.
Cell-based proteasome inhibition assays: The Proteasome-Glo
cell-based assays are homogeneous, luminescent assays that indi-
vidually measure ChT-L, T-L, or C-L protease activity associated
Docking studies: Default parameters were used as described in
the AutoDock 4.2 User’s Guide, except for those changes men-
tioned below. The preprocessed structures of the 20S proteasome
and ligands 10a–m were prepared again for docking by following
the default protocols except where noted. The energy-scoring grid
was prepared as a 40ꢂ40ꢂ40 ꢁ box with a spacing of 0.375, cen-
tered on the ligand value (11.667, À137.254, 19.568). The default
parameters for the Lamarckian genetic algorithm were used as the
protocol except for the following content: the population size was
[9]
with the proteasome complex in cultured cells. HL-60 cells were
equilibrated at 378C, 5% CO for 2 h and then the test compounds
2
were added. The cells were incubated with the drug for 2 h before
each Proteasome-Glo reagent was added. Luminescence was mea-
sured with
a Multimode Microplate Reader Varioskan Flash
(Thermo Scientific, USA) after 15 min. All data were calculated by
subtracting the blank control. The curve-fitting software package
GraphPad Prism 4.0 was used to generate dose–response curves
from which IC50 values were determined for individual compounds
tested in a dose-dependent manner.
1
50, maximum number of energy evaluations were changed to 25
million based on the number of rotatable bonds, maximum
number of generations was retained at 27000, and number of GA
runs were 100. The docked free energy was adopted in this study.
Cell viability assays: Human tumor cell lines, lung cancer (A549),
promyelocytic leukemic (HL-60), epithelial hepatoma (HepG2), mel-
anoma cancer (A375), prostate cancer (DU-145), ileocecal adenocar-
cinoma (HCT-8), breast cancer (MCF-7), colon cancer (SW480), gas-
tric carcinoma (BGC-823), cervical carcinoma (HeLa), and human
leukemia (Jurkat) were cultured in their respective media supple-
mented with 10% fetal bovine serum (FBS). The cancer cells, at
1000 cells per well, were added to a 384-well plate. After 18–24 h,
cells were treated with test compounds at corresponding concen-
trations. After an equal volume of CellTiter-Glo reagent (Prome-
MD simulations: Performed with the AMBER 11 molecular simula-
[19]
tion package.
To obtain molecular mechanical parameters for
1
0b, ab initio quantum chemical methods were employed using
[20]
the Gaussian 09 program.
The geometry was fully optimized,
and then the electrostatic potentials around them were deter-
[21]
mined at the HF/6-31G* level of theory. The RESP strategy was
used to obtain the partial atomic charges.
The starting structure of the 10b–protein complex obtained by
docking was solvated in TIP3P water using a octahedral box, which
extended 8 ꢁ away from any solute atom. To neutralize the nega-
[23]
ga) was added, plates were shaken, and luminescence was moni-
tored over time with the plates held at 378C. The relative lumines-
cence units (RLU) were measured using a Multimode Microplate
Reader Varioskan Flash (Thermo Scientific, USA), and relative cell
number was calculated by normalization to the RLU of the solvent
control-treated cells, and inhibition rate was calculated. IC50 values
were calculated from the curves generated by plotting the per-
centage of the viable cells versus the test concentration on a loga-
rithmic scale using Sigma Plot 10.0 software.
+
tive charges of simulated molecules, Na counter-ions were placed
next to each phosphate group. MD simulation was carried out by
the using SANDER module of AMBER 11. The calculations began
with 500 steps of steepest descent followed by 500 steps of conju-
gate gradient minimization with a large constraint of 500 kcal
À1 À2
mol
ꢁ
on the atoms of the complex. Then 1000 steps of steep-
est descent followed by 1500 steps of conjugate gradient minimi-
zation with no restraint on the complex atoms were performed.
Subsequently, after 20 ps of MD, during which the temperature
À1 À2
was slowly raised from 0 to 300 K with weak (10 kcalmol ꢁ ) re-
Acknowledgements
straint on the complex, the final unrestrained production simula-
tions of 10.0 ns was carried out at constant pressure (1 atm) and
temperature (300 K). In the entire simulation, SHAKE was applied
to all hydrogen atoms. Periodic boundary conditions with mini-
mum image conventions were applied to calculate the non-
bonded interactions. A cutoff of 10 ꢁ was used for the Lennard–
Jones interactions. The final conformations of the complexes were
produced from the 1000 steps of minimized averaged structure of
the last 5.0 ns of MD.
The authors acknowledge financial support from the National
Natural Science Foundation of China (21202003/21172012), the
National Basic Research Program of China (2012CB518000), and
the Specialized Research Fund for the Doctoral Program of
Higher Education of China (20120001110010).
Keywords: antitumor agents · docking · dynamic simulations ·
furans · peptides · proteasome inhibitors
Pharmacology
In vitro 20S proteasome inhibition assays: Promega Proteasome-
Glo 3 Substrate, which consists of three homogeneous biolumi-
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&
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Received: November 11, 2014
studio.
Published online on && &&, 0000
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FULL PAPERS
Q. Sun, B. Xu, Y. Niu,* F. Xu, L. Liang,
C. Wang, J. Yu, G. Yan, W. Wang, H. Jin,*
P. Xu*
Sweet 17 vs. 20S: Seventeen furan-
based peptidic molecules were de-
signed and synthesized as 20S protea-
some inhibitors, some of which showed
potency and selectivity for the b5 sub-
unit in both enzymatic and cell-based
assays. Good antineoplastic activities
were observed in multiple tumor cell
lines. Docking and molecular dynamics
simulations were also used to confirm
a noncovalent binding mode.
&& – &&
Synthesis, Bioactivity, Docking and
Molecular Dynamics Studies of Furan-
Based Peptides as 20S Proteasome
Inhibitors
&
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ꢀ 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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