Design, synthesis and biological evaluation of tripeptide boronic acid proteasome inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.08.023

A series of tripeptide boronate proteasome inhibitors were designed and synthesized on the basis of our previously built tripeptide aldehyde 3D-QSAR models. All the synthesized compounds were evaluated for their proteasome-inhibitory activities in an isolated 20S rabbit proteasome, and selected compounds were evaluated for their antitumor activities in vitro against four human cancer cell lines. Biological results showed bulky and negative substituents at P² position improved the proteasome-inhibitory potency obviously, which completely conformed to the theoretical models, while those at P³ position thoroughly deviated from the 3D-QSAR model. Most of the screened compounds showed less than 1 nM inhibitory potency and high selectivity against 20S proteasome, of which 7f is the most potent (IC₅₀ = 0.079 nM) and twofold more active than bortezomib (IC₅₀ = 0.161 nM). Cell viability indicated hydrophilic 4-hydroxyphenyl substituent at P² or P³ position was not favorable to the cellular activities. Especially for the two hematologic cancer cell lines, HL-60 and U266, 7f inhibited them at the level of less than 10 nM and was more potent than the control bortezomib. It is being considered a promising new lead to be developed for the treatment of various cancers.

Full text of DOI:10.1016/j.bmc.2009.08.023

Bioorganic & Medicinal Chemistry 17 (2009) 6851–6861
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of tripeptide boronic acid
proteasome inhibitors
*
*
Yongqiang Zhu , Shuyang Yao , Bo Xu, Zemei Ge, Jingrong Cui , Tieming Cheng, Runtao Li
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 July 2009
Revised 13 August 2009
Accepted 15 August 2009
Available online 20 August 2009
A series of tripeptide boronate proteasome inhibitors were designed and synthesized on the basis of our
previously built tripeptide aldehyde 3D-QSAR models. All the synthesized compounds were evaluated for
their proteasome-inhibitory activities in an isolated 20S rabbit proteasome, and selected compounds
were evaluated for their antitumor activities in vitro against four human cancer cell lines. Biological
results showed bulky and negative substituents at P2 position improved the proteasome-inhibitory
potency obviously, which completely conformed to the theoretical models, while those at P3 position
thoroughly deviated from the 3D-QSAR model. Most of the screened compounds showed less than
1 nM inhibitory potency and high selectivity against 20S proteasome, of which 7f is the most potent
(IC50 = 0.079 nM) and twofold more active than bortezomib (IC50 = 0.161 nM). Cell viability indicated
hydrophilic 4-hydroxyphenyl substituent at P2 or P3 position was not favorable to the cellular activities.
Especially for the two hematologic cancer cell lines, HL-60 and U266, 7f inhibited them at the level of less
than 10 nM and was more potent than the control bortezomib. It is being considered a promising new
lead to be developed for the treatment of various cancers.
Keywords:
Proteasome inhibitor
Tripeptide boronic acid
Drug design
Synthesis
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
some human diseases such as cancer, inflammation, and neurode-
generative diseases.
The ubiquitin-proteasome pathway (UPP) in eukaryotes is con-
sidered to be critical to the intracellular protein homeostasis.1
Many important biological processes2–9 such as signal transduc-
tion, cell cycle control, transcriptional regulation, inflammation,
and apoptosis are regulated by this pathway. The proteasome is a
multicatalytic core that degrades the damaged or disfolded pro-
teins. The 26S proteasome is composed of barrel-like 20S protea-
some capped by the regulatory 19S proteasome at both ends. The
19S particles are responsible for the ubiquitinated substrate recog-
nition, unfolding, and translocation of the proteins into the cata-
lytic core. In fact, the 20S complex is the real catalytic core
Several types of small molecule inhibitors of the proteasome
had been developed and biologically assayed (Fig. 1). Among these
inhibitors, a dipeptide boronic acid bortezomib (also named PS-
341) showed high selectivity, potency, stability and safety, so in
May 2003, it was approved by the FDA for the treatment of multi-
ple myeloma (MM) patients who have received one prior therapy
but failed.10 And in April 2004 and in October 2006, the drug was
permitted to be marketed in the EMEA and Japan, respectively.
On December 8, 2006, the FDA granted full approval of bortezomib
for the treatment of patients with relapsed mantle cell lymphoma
(MCL). Nowadays, the drug is being investigated on other haema-
tological maligancies and solid tumors, such as non-Hodgkin’s
lymphoma, prostate, breast and non-small-cell lung cancers in
Phase I, Phase II, and Phase III. All these facts demonstrated the
proteasome was a validated and reliable therapeutic target.
However, some clinical results showed that bortezomib led to
some serious side-effects, including fatigue, nausea, sensory neu-
ropathy, etc.11 So it is an imperative task to develop the more po-
tential proteasome inhibitor.
consisted of four stacks of subunits arranged as (a1–a7, b1–b7)2,
of which the b1, b2, and b5 subunits have the chymotripsin-like
(CT-L) activity, trypsin-like (T-L), and post-glutamyl peptide
hydrolysis activity (PGPH), respectively. Each active subunit uti-
lizes the hydrophilic c-hydroxyl group of the N-terminal threonine
(Thr1) to hydrolyze the amide bond of protein substrates. The dis-
ruption of this degradation processing with small molecule inhib-
itors against one or more catalytic b-subunit has implications in
As a continuation of our program on proteasome inhibitors,12
we had ever employed the three-dimensional quantitative struc-
ture–activity relationship (3D-QSAR) methods (including the
Comparative Molecular Field Analysis (CoMFA) and Comparative
Molecular Similarity Indices Analysis (CoMSIA) techniques) to
* Corresponding authors. Tel./fax: +86 010 82802467 (J.C.); tel.: +86 010
82801504; fax: +86 010 62346154 (R.L.).
E-mail addresses: jrcui@bju.edu.cn (J. Cui), lirt@bjmu.edu.cn (R. Li).
These authors made equal contribution to this work.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.023

6852
Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 6851–6861
O
OH
OH
O
O
H
N
H
N
B
N
N
H
N
H
N
H
N
H
O
O
O
O
O
Bortezomib
MG-132
O
H
OH
S
HO
H
Cl
NH
O
H3C
N
H
H
N
OH
O
O
H
O
OH
O
Lactacystin
NPI-0052
I
HO
O
O
O
O
H
N
H
N
O
S
O
N
O
N
H
N
N
H
N
H
O2N
H
O
O
O
O
OH
Epoxomicin
NLVS
Figure 1. Major proteasome inhibitors.
analyze the interaction mode between the 20S proteasome and tri-
peptide aldehydes. The tripeptide aldehydes were much potent
inhibitors of the 20S proteasome. But it is known that aldehyde
group is too active so that the tripeptide aldehyde had too short
half life in vivo. However, boronic acid group is comparatively sta-
ble and also a pharmacophore of bortezomib. So conjugation of the
tripeptide moiety and boronic acid may provide a good opportu-
nity to develop new inhibitors of the 20S proteasome. Based on
our study of the special characteristics of the binding domain of
the 20S catalytic core, some structurally novel tripeptide boronic
acids were designed, synthesized, and biologically evaluated.
At the same time, a newly designed synthetic route shortened
the synthetic steps and avoided the Boc group deprotection com-
pared with bortezomib standard synthesis method. The biological
results supported our previous calculation models in a certain ex-
tent and also offered some new aspects to understand the binding
mode.
by BBr3 under mild conditions to give the target tripeptide boronic
acids 7a–7m (listed in Table 1). The bortezomib was also prepared
according to the similar method.
2.2. Biological activity and SAR discussion
Proteasome inhibitory activity of the tripeptide boronates and
boronic acids was evaluated in an isolated 20S mouse proteasome,
including CT-L, T-L, and PGPH activities. The results were summa-
rized in Table 1.
In our previous 3D-QSAR models of tripeptide aldehyde12 (gen-
eral structure was shown in Table 1), both CoMFA and CoMSIA con-
tour maps had offered us the following conclusions: negative and
hydrophobic substituents at P1 and P2 positions favored the activ-
ities, and bulky, positive and hydrophobic groups at P4 position
were essential to improve the activities, however, substituents at
P3 position had no any effect on the potency. Although the warhead
of tripeptide aldehyde is different from tripeptide boronate or
boronic acid, these two types of compounds inhibit 20S protea-
some with the same mechanism by reacting with the hydroxyl
group of the N-terminal threonine of the b5 subunit to form a tet-
rahedron, which had been confirmed by X-ray diffraction.19,20 Due
to the presence of pyrazin-2-carbonyl at N-terminal and isopropyl
group at C-terminal in the marketed bortezomib, in this paper
these two moieties were still, respectively remained at P1 and P4
positions in the designed inhibitors. Substituents at P2 and P3 posi-
tions were designed according to the calculated 3D-QSAR models.
As demonstrated in Table 1, different amino acid screening of the
P2 and P3 positions, including natural phenylalanine, leucine, tyro-
sine, tryptophan, and unnatural (2-naphthyl)-alanine was carried
out in tripeptide boronates 6 and boronic acids 7. The biological re-
sults showed that all the compounds had no T-L activities. How-
ever, most of the compounds inhibited the CT-L activities with
IC50 values under 1 nM. For the PGPH activity, IC50 values lied be-
yond 2 nM level. The fact that the compounds inhibited the CT-L,
T-L, and PGPH activities so differently reflected the designed inhib-
itors were quite selective. The following discussion was based on
2. Results and discussion
2.1. Synthesis of boronic acids
The generalized synthesis of the tripeptide boronic acids is illus-
trated in Scheme 1. The key intermediate dipeptides 5a–5m were
prepared according to the conventional liquid-phase synthesis.13
The starting material L-configuration amino acid methyl esters 1a–
1m were coupled with pyrazine 2-carboxylic acid in the presence
of DCC and HOBt to give methyl esters 2a–2m, which were not sep-
arated and directly used for saponification to afford acids 3a–3m.
Another coupling of 3a–3m with
L
-P3CH2(HClꢀNH2)COOCH3 and
saponification was repeated to gain the important dipeptides 5a–
5m in high yield. The known amine boronate 8 was synthesized
according to Matteson’s procedure14–17 and coupled with dipeptides
5a–5m in the presence of 1-ethyl-3-(3-dimethylaminopropyl) car-
bodiimide hydrochloride (EDC)18 to provide pure boronates 6a–
6m (listed in Table 1) in high yield (78–87%) after chromatographic
purification. And then the protecting group pinanediol was removed

Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 6851–6861
6853
P2
P2
P2
O
O
ii
i
OCH3
OH
N
N
OCH3
N
N
.
HCl
H2N
N
H
N
H
O
O
O
iv
1
2
3
P2
P2
O
O
O
O
H
N
H
N
N
N
N
iii
N
H
OCH3
N
H
OH
O
O
P3
P3
N
4
5
P2
P2
O
O
O
O
H
N
H
N
OH
OH
O
O
N
N
N
N
vi
v
N
H
N
H
B
N
H
N
H
B
O
O
P3
P3
7
6
O
O
.
HCl
H2N
B
8
P2
P2
phenyl
P3
1a, 2a, 3a
phenyl
4a, 5a, 6a, 7a
4b, 5b, 6b, 7b
4c, 5c, 6c, 7c
4d, 5d, 6d, 7d
4e, 5e, 6e, 7e
4f, 5f, 6f, 7f
4g, 5g, 6g, 7g
4h, 5h, 6h, 7h
4i, 5i, 6i, 7i
phenyl
phenyl
1b, 2b, 3b
1c, 2c, 3c
1d, 2d, 3d
1e, 2e, 3e
1f, 2f, 3f
1g, 2g, 3g
1h, 2h, 3h
1i, 2i, 3i
2-naphthyl
phenyl
2-naphthyl
phenyl
4-hydroxylphenyl
phenyl
phenyl
4-hydroxylphenyl
(CH3)2CH
(CH3)2CH
phenyl
4-hydroxylphenyl
(CH3)2CH
(CH3)2CH
phenyl
2-naphthyl
2-naphthyl
2-naphthyl
phenyl
2-naphthyl
3-indyl
2-naphthyl
3-indyl
1j, 2j, 3j
1k, 2k, 3k
1l, 2l, 3l
phenyl
4j, 5j, 6j, 7j
4k, 5k, 6k, 7k
4l, 5l, 6l, 7l
phenyl
phenyl
3-indyl
phenyl
(CH3)2CH
(CH3)2CH
(CH3)2CH
(CH3)2CH
(CH3)2CH
2-naphthyl
1m, 2m, 3m 2-naphthyl
4m, 5m, 6m, 7m
Scheme 1. Synthesis of the tripeptide boronic acids. Reagents and conditions: (i) pyrazine 2-carboxylic acid, DCC, HOBt, NMM, THF; (ii) (1) 2 N NaOH, acetone, 0 °C; (2) 2 N
HCl, ethyl acetate; (iii) L-P3CH2(HClꢀNH2)COOCH3, DCC, HOBt, NMM, THF, 0 °C; (iv) (1) 2 N NaOH, acetone, 0 °C; (2) 2 N HCl, ethyl acetate; (v) 8, EDC, DIPEA, ꢁ20 °C; (vi) 1 N
BBr3, CH2Cl2.
the CT-L activities for such kind of compounds mainly inhibited the
b5 subunit.
Compounds 6b (IC50 = 0.220 nM) and 7b (IC50 = 0.417 nM) bearing a
much more bulky and negative 2-naphthyl groups at P2 positions
were more active than compounds 6a (IC50 = 0.402 nM) and 7a
(IC50 = 0.546 nM), both P2 positions of which were substituted by
It had been reported that the pinanediol ester of the boronic
acid nearly had the same activity and selectivity as the free boronic
acid.21 This is the case for our synthesized series of inhibitors.
Comparing pinanediol protected boronic acids 6a, 6b, and 6c with
their corresponding free acids 7a, 7b, and 7c in inhibiting the CT-L
activity, both of their IC50 were in the same level (Table 1). How-
ever, from the absolute value, the ester form was slightly more ac-
tive than the acid one, which was concord with our calculation
results and the hydrophobic characteristics of active residues in
b5 subunit.22
phenyl
groups.
Comparison
between
compounds
7k
(IC50 = 0.173 nM) and 7l (IC50 >20 nM) also accounted for this: when
the negative phenyl group at P2 position in 7k was replaced by the
positive isopropyl group (7l), the inhibition of the 20S proteasome
decreased drastically.
However, to our great surprise, the calculation models were not
consistent with experimental results on the substituents at P3 po-
sition. Probably it was due to the less varieties of substituents at P3
position of the compounds in the training set during we had ever
carried out the 3D-QSAR calculations. The biological results
showed different substituents at P3 position significantly affected
Based on our previously constructed CoMFA and CoMSIA contour
maps of tripeptide aldehyde, more steric, negative, and hydrophobic
substituentsat P2 position were introducedto increase the activities.

6854
Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 6851–6861
Table 1
General structures of tripeptide aldehyde and boronate and biological activity of bortezomib, 6a–6m, and 7a–7m (IC50, nM)
P4
CHO
P2
O
P2
O
O
O
O
O
H
N
H
N
O
O
N
N
P1
B
N
H
N
H
P
N
H
N
H
P3
P3
Tripeptide aldehyde
Tripeptide boronate
Compounds
P2
P3
P
CT-L
T-L
PGPH
6a
0.402 0.024
>20
8.330 2.493
7a
6b
7b
6c
7c
6d
7d
6e
7e
6f
H, H
H, H
H, H
H, H
H, H
H, H
H, H
0.546 0.077
0.220 0.106
0.417 0.070
0.335 0.031
0.395 0.035
0.295 0.049
0.263 0.081
0.431 0.086
0.190 0.017
0.370 0.141
0.079 0.011
0.443 0.081
0.111 0.050
0.495 0.092
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
10.907 3.206
>20
4.428 1.407
5.390 0.368
4.013 1.472
4.763 1.308
2.500 1.485
3.405 0.021
5.143 2.166
3.910 1.718
3.630 1.669
>20
HO
HO
HO
HO
7f
6g
7g
6h
3.797 0.983
>20

Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 6851–6861
6855
Table 1 (continued)
Compounds
P2
P3
P
CT-L
T-L
PGPH
>20
7h
6i
H, H
0.335 0.078
>20
>20
>20
>20
N
H
7i
6j
H, H
>20
>20
>20
>20
>20
>20
N
H
N
H
7j
H, H
H, H
H, H
H, H
>20
>20
>20
>20
>20
>20
>20
>20
>20
N
H
6k
7k
6l
0.287 0.019
0.173 0.033
>20
2.725 1.039
5.663 0.839
>20
7l
>20
>20
6m
7m
0.345 0.106
0.220 0.070
14.145
4.897
12.750 4.856
O
OH
OH
H
N
Bortezomib
0.161 0.024
>20
>20
B
N
N
N
H
O
the activities. When the phenyl substituent at P3 position of com-
pound 7e was replaced by 2-naphthyl (7f), the potency was greatly
improved (IC50 changed from 0.190 nM to 0.079 nM). So increasing
the amount and diversity of the substituents at P3 position may re-
sult in a more reliable predictive model. From the data in hand, it
seemed that steric factor instead of the electrostatic one influenced
the activities in a larger extent (comparison between compounds
7a and 7k, among 7b, 7h, and 7m). Such a deviation offered us use-
ful information to deeply understand the interaction mode be-
tween the inhibitors and b5 subunit.
combination of substituents at these two positions was beneficial
to the potency. Employment of a positive and bulky isopropyl at
P2 position and a negative and steric 2-naphthyl group at P3 posi-
tion (compound 7f, IC50 = 0.079 nM) gave an ideal result, twofold
more potent than bortezomib.
Furthermore, introduction of a hydroxyl group on the phenyl
ring whether at P2 or P3 position slightly enhanced the activity,
presumably because of the formation of hydrogen bonds between
the ligands and 20S proteasome. Both compounds 7c
(IC50 = 0.395 nM) and 7d (IC50 = 0.263 nM) harboring a hydroxyl
group at P3 and P2 position, respectively, were more active than
7a (IC50 = 0.546 nM), the P2 and P3 positions of which were substi-
tuted with phenyl groups.
However, when bulky substituents, such as 2-naphthyl group
(compound 7h, IC50 = 0.335 nM), were employed at both P2 and
P3 positions, the activity decreased in some extent. In fact, a proper

6856
Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 6851–6861
It is interesting to point out that inhibitors bearing indyl group
ninhydrin and heat as developing agent. Column chromatography
was performed on 200–300 mesh silica gel. Melting points were
determined on an X4 melting point apparatus and were uncor-
rected. 1H NMR and 13C NMR spectra were recorded at room tem-
at either P2 or P3 position, nearly had no effect on the 20S protea-
some. Compounds 7i and 7j with indolyl group at P2 and P3 posi-
tions, respectively, had the IC50 value larger than 20 nM, which
could be regarded as ineffective. The reason was being studied
by using molecular modeling techniques.
perature on
a 500 Bruker AM-500, VXR 300, or AL 300
spectrometers. Chemical shifts were expressed in d ppm and tetra-
methylsilane (TMS) was used as internal reference. Mass spectra
were obtained using ZAB-2F instruments in electrospray positive
and negative ionization modes. High-resolution mass spectra were
recorded on a ZAB-HS instrument using an electrospray source
From the screened results in enzyme, some interesting com-
pounds, such as 7b, 7c, 7d, 7f, 7g, 7h, 7m and bortezomib were se-
lected to be further evaluated in vitro against four human cell lines,
including two solid tumor cells, BGC-823 (human gastric carci-
noma cell line) cell lines and BXPC-3 (human pancreatic cancer cell
line), and two hematologic tumor cells, HL-60 (promyelocytic leu-
kemia cell line) and U266 (multi myeloma cell line) human cell
lines. Table 2 summarizes the cellular activity results. In general,
the two hematologic tumor cell lines, HL-60 and U266, were more
sensitive to such kind of inhibitors than the two solid tumors, BGC-
823 and BXPC-3. So the following discussion was mainly based on
these two cell lines unless otherwise stated. For P3 position, the
activity order is phenyl (7b) > isopropyl (7m) > 2-naphthyl (7h)
for both cell lines. At the same time, the 2-naphthyl substituent
(7g) at P3 position was more potent than 4-hydroxyphenyl one
(7c) due to its high hydrophobicity and cell permeability. However,
for P2 position, the situation varied in a certain extent. Isopropyl
substituted 7f was more active than phenyl 7g and 2-naphthyl
7h for all the cell lines. As described above, compared with hydro-
phobic 2-naphthyl substituent (7b) at P2 position, hydrophilic 4-
hydroxyphenyl group (7d) at the same position also decreased
the cellular activity. However, increasing the sample volume is
necessary for a comprehensive understanding of the SAR. Mean-
while, it is worthy to mention compound 7f. This inhibitor not only
showed the most active among all the investigated tripeptide boro-
nic acids in enzyme (Table 1), but also in cellular aspects. Espe-
cially for the two hematologic tumor cell lines, 7f inhibited both
tumor cells with IC50 less than 10 nM, which was more potent than
the standard bortezomib. So this compound was being selected as
a promising candidate to be developed.
(ESI). Elemental analyses were performed on
(Germany).
a Vario EL III
4.1.1. General procedure for the synthesis of N-pyrazin-2-yl
amino acids 5a–5m
A mixture of methyl ester 1 (1 mmol) and pyrazine 2-carboxylic
acid (1 mmol) in absolute THF (30 mL) at 0 °C was added DCC
(1 mmol) and HOBt (1.2 mmol) and the reaction was allowed to
performed until TLC showed the starting materials disappeared
(2–4 h). After the reaction finished, the resulting solid was filtered
and the solvent was removed under vacuo. The white solid was
dissolved in 50 mL ethyl acetate and washed with 5% NaHCO3,
10% citric acid, 5% NaHCO3, and brine, dried over anhydrous
Na2SO4, filtered, and evaporated to provide crude methyl ester 2
without further purification and directly used in the next reaction.
The crude product 2 (1 mmol) was dissolved in 10 mL acetone
and was added 2 N NaOH dropwise at 0 °C until TLC showed
methyl ester 2 was saponified completely (2 h). The reaction solu-
tion was acidified to pH 2. The precipitate was filtered and washed
with water until pH value reached 6–7. After dryness, the acid 3
was obtained with high purity and yield.
The process of the preparation of 2 and 3 was repeated to give
N-pyrazin-2-yl dipeptide methyl ester 4 and acid 5, respectively.
4.1.2. N-(2-Pyrazinecarbonyl)-L-phenylalanine-L-phenylalanine
(5a)
Yield 90.5%, white solid, mp: 194–196 °C; 1H NMR (DMSO-d6,
300 MHz): d 12.88 (s, 1H), 9.10 (s, 1H), 8.90–8.87 (m, 1H), 8.77–
8.73 (m, 1H), 8.75–8.65 (m, 2H), 7.24–7.14 (m, 10H), 4.84–4.80
(m, 1H), 4.53–4.50 (m, 1H), 3.14–2.89 (m, 4H).
3. Conclusions
We have studied the structure–activity relationship of a series
of tripeptide boronate and boronic acid proteasome inhibitors.
The experimental results turned out the design of substituents at
P2 position could be guided by previously constructed 3D-QSAR
model, while that of groups at P3 position was incorrectly pre-
dicted. Additional structure modifications at P3 position were
needed to clearly elucidate the SAR. Among the screened protea-
some inhibitors, the potency of 7f was twofold more active than
that of bortezomib. Cytotoxicity displayed that hydrophobic sub-
stituents instead of hydrophilic ones at P2 or P3 position were ben-
eficial for higher cellular activities. It also turned out compound 7f
was more active than the marketed bortezomib and inhibited the
two hemapotologic cancer cell lines with IC50 less than 10 nM. 7f
was being considered as a novel candidate to be developed for
the treatment of different cancers.
4.1.3. N-(2-Pyrazinecarbonyl)-L-(2-naphthyl)-alanine-L-
phenylalanine (5b)
Yield 77%, white solid, mp:196–197 °C; 1H NMR (DMSO-d6,
300 MHz):
d 9.07 (s, 1H), 8.86 (d, J = 2.7 Hz, 1H), 8.70 (d,
J = 1.2 Hz, 1H), 8.68–8.62 (m, 2H), 7.83–7.72 (m, 4H), 7.45–7.37
(m, 3H), 7.24–7.17 (m, 5H), 4.93–4.91 (m, 1H), 4.53–4.50 (m,
1H), 3.24–2.98 (m, 4H).
4.1.4. N-(2-Pyrazinecarbonyl)-L-phenylalanine-L-tyrosine (5c)
Yield 83%, foam solid. Used in the next step directly.
4.1.5. N-(2-Pyrazinecarbonyl)-L-tyrosine-L-phenylalanine (5d)
Yield 86%, foam solid. Used in the next step directly.
Table 2
Cell viability of tripeptide boronic acids and bortezomib (IC50, nM)
4. Experimental
4.1. Chemistry
Entry
BGC-823
BXPC-3
HL-60
U266
7b
7c
7d
7f
2801
5140
2460
600
31.8
445.0
256.7
21.2
10
552.5
444.7
4.6
53.0
250.0
66.2
9.9
15.8
195.0
120.0
12.2
Commercially available reagents were used directly without
any purification unless otherwise stated. Absolutely anhydrous
solvents were obtained with the proper methods introduced in
the literature. Reactions were monitored by thin-layer chromatog-
raphy carried out on silica gel plates (60F-254) using UV light as a
visualizing agent, 15% ethanolic phosphomolybdic acid and heat or
7g
7h
1040
2450
1720
2890
28.7
33.3
43.5
11.8
7.4
19.6
15.0
6.9
7m
Bortezomib

Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 6851–6861
6857
4.1.6. N-(2-Pyrazinecarbonyl)-
L
-leucine-
L
-phenylalanine (5e)
4.1.15. General procedure for the synthesis of the known salt
(8)
Yield 95.6%, sticky solid; 1H NMR (DMSO-d6, 300 MHz): d 12.61
(s, 1H), 9.20 (s, 1H), 8.98–8.91 (m, 1H), 8.77 (s, 1H), 8.66–8.62 (m,
1H), 8.46–8.44 (m, 1H), 7.22–7.15 (m, 5H), 4.61 (s, 1H), 4.48–4.46
(m, 1H), 3.10–2.87 (m, 2H), 1.98–1.92 (m, 1H), 1.62–1.59 (m, 2H),
0.92–0.88 (m, 6H).
The known salt 8 was synthesized completely according to the
methods described in the literatures.14–17
Yield 72.5%, white solid, mp: 176–178 °C; 1H NMR (DMSO-d6,
500 MHz):
d 8.12 (s, 3H), 7.45 (t, J = 50.7 Hz, 3H), 7.11 (q,
J = 8.1 Hz, 5H), 4.35 (d, J = 8.9 Hz, 1H), 3.03–2.97 (m, 2H),
2.89–2.85 (m, 1H), 2.28–2.23 (m, 4H), 2.11–2.09 (m, 1H), 1.94
(t, J = 5.4 Hz, 1H), 1.84–1.81 (m, 1H), 1.67–1.63 (m, 2H), 1.27
(s, 3H), 1.23 (s, 3H), 1.08 (d, J = 10.9 Hz, 1H), 0.78 (s, 3H); 13C
4.1.7. N-(2-Pyrazinecarbonyl)-L-leucine-L-(2-naphthyl)-alanine
(5f)
Yield 83%, white solid, mp: 99–100 °C; 1H NMR (DMSO-d6,
9.16 (s, 1H), 8.89 (d, J = 2.4 Hz, 1H), 8.70 (d,
300 MHz):
d
NMR (DMSO-d6, 125 MHz):
d
135.56, 133.80, 128.95,
J = 1.5 Hz, 1H), 8.58 (d, J = 9.0 Hz, 1H), 8.46 (d, J = 7.8 Hz, 1H),
7.79–7.73 (m, 4H), 4.60–4.57 (m, 1H), 4.55–4.53 (m, 1H), 1.54–
1.45 (m, 3H), 0.83–0.79 (m, 6H).
128.90, 128.82, 128.77, 86.49, 77.50, 50.59, 38.72, 37.67, 34.65,
34.33, 27.93, 26.70, 25.72, 23.44, 20.50; MS (ESI) m/z 266.3
(M+H)+.
4.1.8. N-(2-Pyrazinecarbonyl)-L-phenylalanine-L-(2-naphthyl)-
4.1.16. General procedure for the synthesis of the tripeptide
boronate (6)
alanine (5g)
Yield 79%, white solid, mp: 191–193 °C; 1H NMR (DMSO-d6,
300 MHz): d 9.07 (d, J = 1.2 Hz, 1H), 8.86 (d, J = 2.7 Hz, 1H), 8.68
(d, J = 1.5 Hz, 1H), 8.64–8.56 (m, 2H), 7.81–7.72 (m, 4H), 7.42–
7.40 (m, 3H), 7.17–7.11 (m, 5H), 4.83–4.79 (m, 1H), 4.59–4.55
(m, 1H), 3.26–3.02 (m, 4H).
To a solution of dipeptide 5 (1 mol) in CH2Cl2 (10 mL) at
ꢁ20 °C was added 1-ethyl-3-(3-dimethylaminopropyl) carbodiim-
ide hydrochloride (EDC) (1.5 mmol) and 1-hydroxybenzotriazole
monohydrate (HOBt) (1 mmol). Then the pinanediol leucine boro-
nate hydrochloride 8 and DIPEA (1.2 mmol) was added. The mix-
ture was allowed to warm to room temperature and stirred
overnight. The insoluble material was filtered, the solvent re-
moved under vacuum, and the residue dissolved in ethyl acetate
(25 mL). The organic layer was washed with 5% NaHCO3, 10% cit-
ric acid, 5% NaHCO3, and saturated NaCl, finally with water. The
organic layer was then dried over anhydrous Na2SO4, filtered,
and the solvent evaporated to give a white solid. The crude prod-
uct was purified by silica gel chromatography (eluted with proper
ratio of ethyl acetate and petroleum ether) to afford the desired
product 6.
4.1.9. N-(2-Pyrazinecarbonyl)-L-(2-naphthyl)-alanine-L-(2-
naphthyl)-alanine (5h)
Yield 72%, light yellow solid, mp: 208–209 °C. 1H NMR (DMSO-
d6, 300 MHz): d 9.04 (s, 1H), 8.84 (d, J = 2.4 Hz, 1H), 8.73–8.69 (m,
1H), 8.67–8.66 (m, 2H), 7.82–7.69 (m, 8H), 7.74–7.69 (m, 6H),
7.44–7.35 (m, 6H), 4.95–4.91 (m, 1H), 4.64–4.60 (m, 1H), 3.28–
3.08 (m, H).
4.1.10. N-(2-Pyrazinecarbonyl)-L-tryptophan-L-phenylalanine
(5i)
Yield 96.4%, white solid, mp: 74–76 °C; 1H NMR (DMSO-d6,
300 MHz): d 10.79 (s, 1H), 9.13 (s, 1H), 8.86 (s, 1H), 8.68 (s, 1H),
8.62–8.59 (m, 2H), 7.54–6.84 (m, 10H), 4.84 (s, 1H), 4.58–4.50
(m, 2H), 3.20–3.09 (m, 4H).
4.1.17. (+)-Pinanediol-(2-pyrazinecarbonyl)-
L-phenylalanine-L-
phenylalanine- -leucine boronate (6a)
L
Yield 82%, white solid, mp: 78–80 °C; 1H NMR (CDCl3,
500 MHz): d 9.31–9.26 (m, 1H), 8.82–8.77 (m, 1H), 8.58–8.54 (m,
1H), 8.23–8.19 (m, 1H), 7.32–6.99 (m, 10H), 6.48–6.42 (m, 1H),
5.72–5.67 (m, 1H), 4.80–4.77 (m, 1H), 4.59–4.55 (m, 1H), 4.36–
4.33 (m, 1H), 3.19–3.09 (m, 4H), 2.90–2.88 (m, 1H), 2.36–2.22
(m, 2H), 2.08–2.03 (m, 1H), 1.94–1.86 (m, 2H), 1.47–1.39 (m,
3H), 1.30–1.28 (m, 3H), 1.29–1.21 (m, 3H), 0.90–0.83 (m, 9H);
13C NMR (CDCl3, 125 MHz): d 169.86, 169.77, 163.21, 147.63,
144.33, 143.52, 142.69, 136.25, 135.94, 129.22, 129.13, 128.81,
128.45, 127.28, 126.72, 84.72, 54.73, 54.27, 51.50, 39.41, 39.37,
38.37, 38.03, 37.84, 37.64, 35.68, 28.53, 28.44, 27.05, 26.36,
26.32, 25.58, 23.96, 22.36; HRMS calcd for C38H49BN5O5 (M+1)+
666.3827, found 666.3823.
4.1.11. N-(2-Pyrazinecarbonyl)-L-phenylalanine-L-tryptophan
(5j)
Yield 93.7% light yellow solid, mp:106–108 °C; 1H NMR (DMSO-
d6, 300 MHz): d 12.59 (s, 1H), 10.87 (s, 1H), 9.16–9.12 (m, 1H),
8.89–8.87 (m, 1H), 8.75–8.72 (m, 1H), 8.66–8.59 (m, 2H), 7.56–
6.96 (m, 10H), 4.88–4.85 (m, 1H), 4.59–4.56 (m, 1H), 3.22–3.05
(m, 4H).
4.1.12. N-(2-Pyrazinecarbonyl)-L-phenylalanine-L-leucine (5k)
Yield 93.9%, pale yellow solid, mp: 142–144 °C; 1H NMR
(DMSO-d6, 300 MHz): d 12.70 (s, 1H), 9.15–9.12 (m, 1H), 8.90–
8.88 (m, 1H), 8.79–8.74 (m, 1H), 8.68–8.65 (m, 1H), 8.57–8.53
(m, 1H), 7.27–7.15 (m, 5H), 4.90–4.82 (m, 1H), 4.32–4.25 (m,
1H), 3.21–3.04 (m, 2H), 1.68–1.54 (m, 3H), 0.94–0.91 (m, 3H),
0.87–0.85 (m, 3H).
4.1.18. (+)-Pinanediol N-(2-pyrazinecarbonyl)-
alanine- -phenylalanine- -leucine boronate (6b)
Yield 87%, white solid, mp: 98–100 °C; 1H NMR (CDCl3,
300 MHz): 9.13 (s, 1H), 8.74 (d, J = 2.4 Hz, 1H), 8.48 (d,
L-(2-naphthyl)-
L
L
d
J = 2.1 Hz, 1H), 8.23–8.20 (d, J = 7.5 Hz, 1H), 7.80–7.69 (m, 4H),
7.46–7.43 (m, 2H), 7.35 (d, J = 8.4 Hz, 1H), 7.02–6.95 (m, 5H),
6.53–6.45 (m, 1H), 4.65–4.57 (m, 1H), 4.29–4.27 (m, 1H), 3.40–
3.26 (m, 2H), 3.13–3.02 (m, 1H), 2.95–2.84 (m, 1H), 2.35–2.27
(m, 1H), 2.20–2.15 (m, 1H), 2.04–1.99 (m, 1H), 1.89–1.80 (m,
1H), 1.39–1.23 (m, 12H), 0.87–0.83 (m, 9H); 13C NMR (CDCl3,
4.1.13. N-(2-Pyrazinecarbonyl)-
L-leucine-L-leucine (5l)
Yield 83%, sticky solid. Used in next step directly.
4.1.14. N-(2-Pyrazinecarbonyl)-L-(2-naphthyl)-alanine-L-
leucine (5m)
Yield 78%, white solid, mp: 98–100 °C; 1H NMR (DMSO-d6,
300 MHz): d 9.09 (d, J = 1.2 Hz, 1H), 8.86 (d, J = 2.7 Hz, 1H),
8.71–8.70 (m, 2H), 8.57 (d, J = 7.8 Hz, 1H), 7.84–7.45 (m, 4H),
7.43–7.42 (m, 3H), 5.02–4.96 (m, 1H), 4.32–4.28 (m, 1H),
1.66–1.58 (m, 3H), 0.89 (d, J = 6.6 Hz, 3H), 0.83 (d, J = 6.3 Hz,
3H).
75 MHz):
d 170.39, 169.85, 163.19, 147.57, 144.32, 143.59,
142.71, 136.13, 133.51, 133.46, 132.47, 129.22, 128.56, 128.35,
128.01, 127.66, 127.57, 127.51, 126.65, 126.24, 125.84, 54.55,
54.42, 53.64, 51.35, 39.70, 39.55, 38.19, 37.85, 37.74, 35.50,
28.61, 27.08, 26.24, 25.36, 25.21, 24.03, 22.97, 22.01; HRMS calcd
for C42H51BN5O5 (M+1)+ 716.3983, found 716.3986.

6858
Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 6851–6861
4.1.19. (+)-Pinanediol N-(2-pyrazinecarbonyl)-
L
-phenylalanine-
21.80; HRMS calcd for C39H53BN5O5 (M+1)+ 682.4140, found
682.4155.
L
-tyrosine-
L-leucine boronate (6c)
Yield 81%, light yellow solid, mp: 119–120 °C; 1H NMR
4.1.23. (+)-Pinanediol N-(2-pyrazinecarbonyl)-
-(2-naphthyl)-alanine- -leucine boronate (6g)
Yield 86%, white solid, mp: 97–98 °C; 1H NMR (CDCl3,
L-phenylalanine-
(CD3OD, 300 MHz): d 9.18 (s, 1H), 8.68 (d, J = 10.2 Hz, 1H), 8.48
(s, 1H), 8.26 (d, J = 7.5 Hz, 1H), 7.82–7.74 (m, 1H), 7.27–7.18
(m, 5H), 6.80–6.76 (m, 3H), 6.49–6.40 (m, 2H), 4.82–4.61 (m,
2H), 4.29–4.27 (d, J = 8.1 Hz, 1H), 3.13–3.06 (m, 2H), 2.95–2.84
(m, 3H), 2.35–2.27 (m, 2H), 2.04–2.02 (m, 1H), 1.85–1.80 (m,
2H), 1.43–1.26 (m, 9H), 0.88–0.83 (m, 9H); 13C NMR (CD3OD,
L
L
300 MHz): d 9.13–9.09 (m, 1H), 8.66 (d, J = 2.4 Hz, 1H), 8.34 (d,
J = 1.2 Hz, 1H), 8.10 (d, J = 7.5 Hz, 1H), 7.62–7.46 (m, 3H), 7.41–
7.21 (m, 9H), 6.53–6.45 (m, 1H), 5.99–5.78 (m, 1H), 4.76–4.72
(m, 1H), 4.25 (d, J = 8.7 Hz, 1H), 3.29–3.22 (m, 1H), 3.15 (d,
J = 6.3 Hz, 2H), 3.11–3.04 (m, 1H), 2.29–2.26 (m, 1H), 2.17–2.12
(m, 1H), 2.02–1.98 (m, 1H), 1.86–1.67 (m, 2H), 1.38–1.25 (m,
12H), 0.87–0.81 (m, 9H); 13C NMR (CDCl3, 75 MHz): d 170.53,
169.85, 163.19, 147.50, 144.04, 143.28, 143.15, 142.63, 135.96,
135.89, 133.66, 133.54, 133.20, 132.19, 129.20, 128.78, 128.14,
127.96, 127.48, 127.28, 127.21, 125.92, 125.53, 54.56, 53.36,
53.01, 51.34, 39.88, 39.70, 39.57, 38.05, 37.60, 35.57, 28.57,
27.09, 26.22, 25.32, 25.16, 24.03, 22.91, 22.01, 21.92; HRMS calcd
for C42H51BN5O5 (M+1)+ 716.3983, found 716.3996.
75 MHz):
d 170.17, 163.36, 155.62, 147.61, 143.96, 143.44,
142.83, 135.95, 130.37, 129.21, 128.78, 127.47, 127.23, 126.61,
126.44, 115.34, 85.30, 54.86, 54.74, 52.91, 51.54, 40.16, 39.82,
39.64, 38.10, 37.57, 36.97, 35.80, 35.69, 28.73, 27.10, 26.28,
25.41, 24.04, 23.06, 22.97, 22.03, 21.95; HRMS calcd for
C38H49BN5O6 (M+1)+ 682.3776, found 682.3767.
4.1.20. (+)-Pinanediol N-(2-pyrazinecarbonyl)-
phenylalanine- -leucine boronate (6d)
Yield 80%, light yellow solid, mp: 90–92 °C; 1H NMR (CD3OD,
300 MHz): 9.29 (s, 1H), 8.74 (d, J = 2.4 Hz, 1H), 8.50 (d,
L-tyrosine-L-
L
4.1.24. (+)-PinanediolN-(2-pyrazinecarbonyl)-
alanine- -(2-naphthyl)-alanine- -leucine boronate (6h)
Yield 82%, white solid, mp: 136–137 °C; 1H NMR (CD3OD,
300 MHz): 9.10 (s, 1H), 8.63 (d, J = 2.4 Hz, 1H), 8.26 (d,
L-(2-naphthyl)-
d
L
L
J = 2.1 Hz, 1H), 8.26 (d, J = 7.8 Hz, 1H), 7.11–6.99 (m, 8H), 6.74
(d, J = 8.4 Hz, 2H), 6.74–6.71 (m, 1H), 6.50–6.42 (m, 1H),
4.70–4.26 (m, 1H), 4.51–4.27 (m, 1H), 4.29–4.17 (m, 2H), 3.07–
2.93 (m, 5H), 2.37–2.31 (m, 1H), 2.21–2.16 (m, 1H), 2.06–2.03
(m, 1H), 1.90–1.80 (m, 2H), 1.40–1.26 (m, 6H), 0.90–0.79 (m,
12H); 13C NMR (CD3OD, 75 MHz): d 171.36, 170.18, 163.03,
155.47, 147.50, 144.29, 143.74, 142.73, 135.95, 130.29, 130.15,
129.31, 128.50, 127.51, 126.83, 116.47, 116.64, 85.33, 55.08,
53.19, 51.55, 39.73, 39.64, 39.40, 38.23, 38.16, 37.44, 37.14,
35.67, 28.73, 28.54, 27.14, 26.30, 25.44, 24.07, 22.98,
22.02; HRMS calcd for C38H49BN5O6 (M+1)+ 682.3776, found
682.3795.
d
J = 2.4 Hz, 1H), 8.12–8.02 (m, 1H), 7.81–7.57 (m, 6H), 7.53–7.32
(m, 8H), 7.18 (dd, J1 = 1.8 Hz, J2 = 8.4 Hz, 2H), 6.51–6.49 (m, 1H),
6.00 (d, J = 5.1 Hz, 1H), 4.87–4.72 (m, 2H), 4.26 (dd, J1 = 1.5 Hz,
J2 = 8.7 Hz, 1H), 3.32 (d, J = 6.3 Hz, 2H), 3.24–3.22 (m, 1H), 3.07–
3.00 (m, 1H), 2.33–2.25 (m, 1H), 2.18–2.13 (m, 1H), 2.04–2.00
(m, 1H), 1.87–1.67 (m, 2H), 1.36–1.25 (m, 8H), 0.87–0.74 (m,
12H); 13C NMR (CD3OD, 75 MHz): d 170.46, 169.85, 163.22,
147.46, 144.00. 143.16, 142.61, 142.49, 133.58, 133.45, 133.34,
132.50, 132.13, 128.58, 127.97, 127.71, 127.57, 127.44, 126.31.
125.90, 125.52, 85.76, 54.41, 53.30, 51.35, 39.58, 38.19, 37.98,
37.59, 35.50, 28.59, 27.10, 26.24, 25.33, 25.18, 24.04, 21.96; HRMS
calcd for C46H53BN5O5 (M+1)+ 766.4140, found 766.4148.
4.1.21. (+)-Pinanediol N-(2-pyrazinecarbonyl)-L-leucine -L-
phenylalanine- -leucine boronate (6e)
L
4.1.25. (+)-Pinanediol N-(2-pyrazinecarbonyl)-
phenylalanine- -leucine boronate (6i)
Yield 78%, light yellow solid, mp: 127–129 °C; 1H NMR (CDCl3,
300 MHz): 9.24 (s, 1H), 8.76 (d, J = 2.4 Hz, 1H), 8.51 (d,
L- tryptophan-L-
Yield 87%, white solid, mp: 63–65 °C; 1H NMR (CDCl3,
500 MHz): d 9.32 (s, 1H), 8.78–8.67 (m, 1H), 8.55–8.21 (m, 1H),
8.10–7.95 (m, 1H), 7.15–6.99 (m, 6H), 6.04–5.96 (m, 1H), 4.65–
4.34 (m, 2H), 4.30–4.15 (m, 1H), 3.22–3.01 (m, 2H), 2.37–2.22
(m, 2H), 2.12–2.04 (m, 1H), 1.93–1.90 (m, 2H), 1.76–1.63 (m,
3H), 1.48–1.39 (m, 1H), 1.28 (s, 3H), 1.29–1.22 (m, 3H), 0.94–0.90
(m, 6H), 0.86–0.83 (m, 9H); 13C NMR (CDCl3, 75 MHz): d 171.49,
170.91, 162.76, 147.12, 143.60, 141.48, 136.38, 128.13, 128.05,
127.98, 126.32, 85.18, 84.79, 53.19, 52.14, 51.22, 41.15, 41.04,
40.72, 39.78, 39.49, 35.82, 35.46, 35.28, 28.40, 26.83, 25.99,
25.40, 24.25, 23.75, 22.71, 21.78, 20.71; HRMS(ESI): calcd
C35H51BN5O5 (M+1)+ 632.3983, found 632.3984.
L
d
J = 2.4 Hz, 1H), 8.50–8.46 (m, 1H), 8.22–8.14 (m, 1H), 7.40–7.38
(m, 5H), 7.01–6.88 (m, 4H), 6.78–6.58 (m, 1H), 6.09–5.97 (m,
1H), 5.75–5.71 (m, 2H), 4.82–4.75 (m, 2H), 3.51–3.45 (m, 1H),
3.31–3.01 (m, 4H), 1.25–1.23 (m, 10H), 0.99–0.89 (m, 8H), 0.88–
0.85 (m, 6H); 13C NMR (CDCl3, 75 MHz):
d 171.77, 170.57,
163.21, 147.24, 143.48, 143.30, 142.48, 136.21, 135.38, 128.99,
128.09, 127.06, 126.38, 123.48, 121.93, 119.20, 118.45, 111.39,
109.29, 84.91, 54.22, 54.03, 52.42, 51.42, 39.95, 39.47, 37.99,
37.91, 37.19, 36.96, 35.56, 30.36, 28.61, 27.76, 27.53, 26.94,
26.19, 25.16, 22.86,21.83; HRMS(ESI): calcd C40H50BN6O5(M+1)+
705.3936, found 705.3946.
4.1.22. (+)-Pinanediol N-(2-pyrazinecarbonyl)-
L-leucine-L-(2-
naphthyl)-alanine - -leucine boronate (6f)
L
4.1.26. (+)-Pinanediol N-(2-pyrazinecarbonyl)-
-tryptophan- -leucine boronate (6j)
Yield 79%, light yellow solid, mp: 153–155 °C; 1H NMR (CDCl3,
L-phenylalanine-
Yield 85%, white solid, mp: 140–142 °C; 1H NMR (CDCl3,
300 MHz): d 9.20 (d, J = 1.5 Hz, 1H), 8.71 (d, J = 2.4 Hz, 1H),
8.40 (t, J = 1.2 Hz, 1H), 7.93 (d, J = 8.1 Hz, 1H), 7.66–7.61 (m,
4H), 7.36–7.32 (m, 3H), 6.75 (d, J = 7.8 Hz, 1H), 6.00 (d,
J = 4.8 Hz, 1H), 4.78–4.71 (m, 1H), 4.59–4.53 (m, 1H), 4.27 (d,
J = 8.7 Hz, 1H), 3.23 (d, J = 6.6 Hz, 2H), 3.14–3.10 (m, 1H),
2.28–2.20 (m, 1H), 2.17–2.08 (m, 1H), 2.00–2.05 (m, 1H), 1.87–
1.57 (m, 6H), 1.41–1.27 (m, 9H), 0.92–0.90 (m, 6H), 0.83–0.77
(m, 9H); 13C NMR (CDCl3, 75 MHz): d 171.05, 170.61, 163.13,
147.52, 144.24, 143.45, 142.59, 142.47, 133.933, 133.25,
132.22, 128.131, 127.99, 127.48, 127.26,125.95, 125.52,
85.83, 53.06, 51.98, 51.88, 51.32, 40.63, 39.68, 39.56, 38.19,
35.48, 28.56, 27.10, 26.23, 25.33, 25.22, 24.78, 24.04, 22.89,
L
L
300 MHz): d 9.08 (s, 1H), 8.73–8.69 (m, 1H), 8.48 (s, 1H), 8.34 (s,
1H), 8.11–7.94 (m, 1H), 7.32–7.21 (m, 5H), 7.08–6.88 (m, 4H),
6.62–6.42 (m, 1H), 6.01 (s, 1H), 5.65–5.58 (m, 2H), 4.38–4.29 (m,
2H), 3.20–3.01 (m, 4H), 1.40–1.24 (m, 10H), 0.99–0.89 (m, 8H),
0.83–0.80 (m, 6H); 13C NMR (CDCl3, 75 MHz): d 172.52, 170.09,
163.22, 147,23, 143.62, 143.06, 142.62, 135.90, 135.75, 129.13,
128.59, 127.13, 123.79, 121.34, 119.21, 117.82, 111.03, 108.08,
84,73, 54.69, 52.52, 51.55, 40.07, 39.74, 27.15, 38.03, 37.48,
35.79, 30.41, 28.70, 27.33, 27.00, 26.21, 25.18, 25.11, 23.92,
22.89, 21.86, 21.74; HRMS(ESI): calcd C40H50BN6O5(M+1)+
705.3936, found 705.3950.

Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 6851–6861
6859
4.1.27. (+)-Pinanediol N-(2-pyrazinecarbonyl)-
-leucine- -leucine boronate (6k)
Yield 81%, white solid, mp: 68–70 °C; 1H NMR (CDCl3,
L
-phenylalanine-
54.96, 51.70, 37.54, 37.30, 25.66, 23.17, 22.51; HRMS calcd for
C28H35BN5O5 (M+1)+ 532.2731, found 532.2717.
L
L
300 MHz): d 9.33 (s, 1H), 8.76–8.73 (d, 1H), 8.54 (s, 1H), 8.29–
8.10 (m, 1H), 7.29–7.25 (m, 5H), 6.39–6.31 (m, 2H), 4.82–4.79
(m, 1H), 4.44–4.41 (m, 1H), 4.30–4.27 (m, 1H), 3.22–3.19 (m,
3H), 2.32–2.17 (m, 3H), 2.05–2.02 (m, 1H), 1.89–1.27 (m, 14H),
0.96–0.83 (m, 15H); 13C NMR (CDCl3, 75 MHz): d 172.18, 170.33,
163.01, 147.34, 144.15, 143.84, 142.72, 135.91, 129.17,128.55,
126.99, 85.37, 84.38, 54.42, 51.38, 50,86, 40,53, 39.68, 39.48,
39.29, 38.25, 37.84, 35.60, 30.49, 28.45, 27.71, 26.95, 26.22,
25.50, 24.39, 23.86, 22.92, 22.50, 22.11, 20.98; HRMS calcd for
C35H51BN5O5 (M+1)+ 632.3983, found 632.3979.
4.1.32. N-(2-Pyrazinecarbonyl)-
L-(2-naphthyl)-alanine-L-
phenylalanine- -leucine boronic acid (7b)
L
Yield 63.5%, light yellow solid, mp: 134–135 °C; 1H NMR (CDCl3,
300 MHz): d 9.15 (s, 1H), 8.73 (d, J = 2.4 Hz, 1H), 8.46 (d, J = 2.1 Hz,
1H), 8.24–8.17 (m, 1H), 7.76–7.66 (m, 4H), 7.44–7.40 (m, 3H),
7.32–7.25 (m, 1H), 6.90–6.86 (m, 5H), 6.52–6.46 (m, 2H), 4.81–
4.67 (m, 2H), 3.31–3.26 (m, 1H), 3.11 (d, J = 6.9 Hz, 2H), 2.93–
2.89 (m, 1H), 1.92 (s, br, 2H), 1.55–1.21 (m, 3H), 0.85–0.82 (m,
6H); 13C NMR (CDCl3, 75 MHz): d 172.96, 169.99, 163.61, 147.70,
144.24, 143.30, 142.70, 137.28, 135.61, 133.47, 133.19, 132.49,
129.13, 128.64, 128.35, 128.07, 127.64, 127.55, 127.11, 126.71,
126.39, 125.92, 54.88, 51.86, 39.85, 39.34, 37.43, 37.23, 36.88,
25.68, 23.12, 22.46. HRMS calcd for C32H36BN5O5 (Mꢁ1)ꢁ
580.2727, found 580.2736.
4.1.28. (+)-Pinanediol N-(2-pyrazinecarbonyl)-
L-leucine-L-
leucine- -leucine boronate (6l)
L
Yield 86.0%, sticky solid; 1H NMR (CDCl3, 300 MHz): d 9.41 (s,
1H), 8.76–8.65 (m, 1H), 8.56–8.45 (m, 1H), 8.18–8.10 (m, 1H),
5.43–5.38 (m, 1H), 4.71–4.50 (m, 1H), 4.38–4.32 (m, 1H), 4.07–
3.97 (m, 2H), 3.86–3.84 (m, H), 2.52–2.08 (m, 3H), 2.04–1.90 (m,
6H), 1.67–1.60 (m, 2H), 1.41–1.25 (m, 10H), 0.99–0.83 (m, 21H);
13C NMR (CDCl3, 75 MHz): d 172.47, 171.55, 162.96, 147.32,
144,21, 143.83, 142.62, 85.26, 85.04, 53.76, 51.71, 51.33, 51.26,
50.75, 41.25, 41.17, 40.67, 40.52, 39.23, 35.56, 29.47, 28.47,
27.69, 26.94, 26.17, 25.63, 25,19, 24.64, 23.88, 22.04, 21.88,
20,83; HRMS(ESI): calcd C32H53BN5O5 (M+1)+ 598.4140, found
598.4144.
4.1.33. N-(2-Pyrazinecarbonyl)-L-phenylalanine-L-tyrosine-L-
leucine boronic acid (7c)
Yield 56.3%, light yellow solid, mp: 298–300 °C; 1H NMR
(CD3OD, 300 MHz): d 9.13 (s, 1H), 8.78 (t, J = 2.4 Hz, 1H), 8.66
(dd, J1 = 1.2 Hz, J2 = 2.4 Hz, 1H), 7.23–7.16 (m, 5H), 7.16–7.01 (m,
2H), 6.66–6.18 (m, 2H), 4.80–4.73 (m, 2H), 3.20–3.18 (m, 1H),
3.08–3.02 (m, 3H), 2.68–2.56 (m, 1H), 1.62–1.54 (m, 2H), 1.33–
1.26 (m, 2H), 1.14–1.04 (m, 2H), 0.89–0.83 (m, 6H); 13C NMR
(CD3OD, 75 MHz):
d 177.69, 173.05, 165.13, 164.95, 157.77,
157.68, 148.79, 145.59, 144.96, 144.79, 144.49, 137.82, 131.47,
131.35, 130.34, 129.56, 127.97, 127.40, 127.16, 116.41, 56.11,
55.90, 52.87, 52.67, 40.70, 38.82, 38.49, 37.65, 37.30, 26.83,
26.61, 23.89, 23.69, 22.31, 21.83; HRMS calcd for C28H34BN5O6
(Mꢁ1)ꢁ 546.2529, found 546.2527.
4.1.29. (+)-Pinanediol N-(2-pyrazinecarbonyl)-
L-(2-naphthyl)-
alanine- -leucine– -leucine boronate (6m)
L
L
Yield 81%, white solid, mp: 99–100 °C; 1H NMR (CDCl3,
300 MHz): d 9.31 (s, 1H), 8.73 (s, 1H), 8.49 (d, J = 1.8 Hz, 1H),
8.33 (d, J = 7.5 Hz, 1H), 7.66–7.58 (m, 4H), 7.44–7.38 (m, 3H),
6.48–6.36 (m, 2H), 4.94–4.85 (m, 1H), 4.42–4.30 (m, 1H), 4.25–
4.16(m, 1H), 3.38 (d, J = 6.3 Hz, 2H), 3.19–3.11 (m, 1H), 2.35–2.27
(m, 1H), 2.20–2.15 (m, 1H), 1.89–1.84 (m, 2H), 1.68–1.61 (m,
2H), 1.37–1.36 (m, 3H), 1.53–1.43 (m, 4H), 1.28–1.21 (m, 3H),
0.94–0.89 (m, 6H), 0.83–0.79 (m, 9H); 13C NMR (CDCl3, 75 MHz):
4.1.34. N-(2-Pyrazinecarbonyl)-L-tyrosine-L-phenylalanine-L-
leucine boronic acid (7d)
Yield 71.5%, light yellow solid, mp: 219–221 °C; 1H NMR
(CD3OD, 300 MHz): d 9.15 (s, 1H), 8.78 (d, J = 2.4 Hz, 1H), 8.66 (d,
J = 2.1 Hz, 1H), 7.24–7.19 (m, 5H), 7.13–7.00 (m, 2H), 6.97–6.62
(m, 2H), 4.80–4.71 (m, 2H), 3.11–3.06 (m, 3H), 2.93–2.87 (m,
1H), 2.64–2.54 (m, 1H), 1.62–1.54 (m, 2H), 1.40–1.29 (m, 2H),
1.16–1.11 (m, 2H), 0.92–0.81 (m, 6H); 13C NMR (CD3OD,
d
171.81, 170.10, 163.298, 147.59, 144.34, 143.72, 142.77,
133.46, 132.47, 128.56, 128.12, 127.62, 127.16, 126.18, 125.80,
85.60, 54.70, 54.53, 51.40, 51.20, 50.96, 40.89, 40.08, 39.85,
39.56, 38.18, 38.09, 35.58, 28.59, 27.08, 26.25, 25.26, 25.46,
24.51, 24.03, 23.00, 22.71, 22.15, 21.94; HRMS calcd for
C39H53BN5O5 (M+1)+ 682.4140, found 682.4152.
75 MHz):
d 177.20, 173.18, 164.92, 157.51, 148.68, 145.62,
144.93, 144.73, 137.17, 136.96, 131.36, 130.46, 130.33, 129.69,
129.62, 128.25, 128.19, 116.32, 56.34, 56.17, 52.61, 40.77, 38.46,
38.11, 37.82, 26.62, 23.86, 21.91; HRMS calcd for C30H38BN5NaO6
(M+Na+2CH2)+ 598.2813, found 598.2815.
4.1.30. General procedure for the synthesis of the tripeptide
boronic acid (7)
4.1.35. N-(2-Pyrazinecarbonyl)-L-leucine-L-phenylalanine-L-
To a stirred solution of the tripeptide boronates 6 (1 mmol) in
anhydrous CH2Cl2 was added 1 N BBr3 dissolved in CH2Cl2
(2.5 mL). The mixture was stirred at 0 °C for 2 h. The reaction
was quenched with water and extracted with CH2Cl2 (4 ꢂ 15 mL).
The combined organic layers were washed with water and brine,
dried over anhydrous Na2SO4, filtered and concentrated. The re-
sulted crude product was crystallized with ether and CH2Cl2 to give
tripetide boronic acid 7.
leucine boronic acid (7e)
Yield 68.4%, light yellow solid, mp: 113–115 °C; 1H NMR (CDCl3,
300 MHz): d 9.25–9.16 (m, 1H), 8.72 (s, 1H), 8.47 (s, 1H), 8.02–7.96
(m, 1H), 7.25–7.19 (m, 1H), 7.02–6.92 (m, 6H), 4.76–4.61 (m, 1H),
4.41–4.28 (m, 1H), 3.09–2.89 (m, 3H), 1.95–1.92 (m, 1H), 1.57–1.19
(m, 7H), 0.82–0.78 (m, 13H); 13C NMR (CDCl3, 75 MHz): d 172.04,
171.77, 163.55, 147.63, 144.25, 143.96, 142.84, 135.91, 129.95,
129.21, 128.40, 126.83, 53.29, 50.43, 40.32, 39.72, 37.06, 35.60,
25.64, 24.68, 24.19, 22.85, 22.59, 22.24, 21.73; HRMS (ESI): calcd
For C27H40BN5NaO5 (M+Na+2CH2)+ 548.3020 found:548.3027.
4.1.31. N-(2-Pyrazinecarbonyl)-
- leucine boronic acid (7a)
Yield 72.1%, light yellow solid, mp:112–114 °C; 1H NMR (CDCl3,
L-phenylalanine-L-phenylalanine-
L
4.1.36. N-(2-Pyrazinecarbonyl)-
L-leucine-L-(2-naphthyl)-
300 MHz): d 9.26–9.15 (m, 1H), 8.77 (s, 1H), 8.53 (s, 1H), 8.18 (s,
1H), 7.29–7.24 (m, 5H), 7.07–6.84 (m, 6H), 6.42–6.38 (m, 1H),
4.77–4.65 (m, 2H), 3.17–2.96 (m, 5H), 2.17–1.25 (m, 5H), 0.92–
0.90 (m, 3H), 0.88–0.86 (m, 3H); 13C NMR (CDCl3, 75 MHz): d
170.01, 169.74, 163.58, 163.32, 147.75, 144.28, 143.32, 142.72,
135.89, 135.66, 129.20, 128.88, 128.54, 128.41, 127.41, 126.75,
alanine- -leucine boronic acid (7f)
L
Yield 67.0%, light yellow solid, mp: 157–159 °C; 1H NMR (CDCl3,
300 MHz): d 9.13–9.10 (m, 1H), 8.67 (s, 1H), 8.36 (s, 1H), 7.98–7.94
(m, 2H), 7.59–7.29 (m, 7H), 7.22–7.19 (m, 2H), 4.98–4.84 (m, 1H),
4.41–4.37 (m, 1H), 3.46–3.35 (m, 2H), 3.24–3.10 (m, 1H), 1.71–1.25
(m, 8H), 0.88–0.78 (m, 12H); 13C NMR (CDCl3, 75 MHz): d 171.48,

6860
Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 6851–6861
163,92, 147,65, 144,15, 143.31, 143.07, 142.56, 133.56, 133.22,
132.25, 129.16, 128.69, 128.24, 127.51, 127.10, 125.98, 125.58,
(m, 1H), 7.32–7.22 (m, 6H), 6.99–6.91 (m, 1H), 4.93–4.57 (m, 2H),
3.21 (s, 2H), 2.85–2.58 (m, 1H), 2.18–1.25 (m, 8H), 0.96–0.82 (m,
52.95, 52.77, 51.91, 41.12, 40.23, 36.76, 25.68, 24.72, 23.12,
12H); 13C NMR (CDCl3, 75 MHz):
d 170.60, 169.97, 164.55,
ꢁ
0
22.83, 22.44, 21.69; HRMS calcd for C29H37BN5O5 (Mꢁ1)
147.49, 143.47, 135.72, 129.25, 128.96, 127.51, 124.86, 121.47,
119.49, 117.68, 111.50, 107.58, 55.78, 51.58, 50.94, 38.99, 37.06,
25.65, 25.52, 23.18, 22.61, 22.15; HRMS (ESI): calcd for
C27H40BN5NaO5 (M+Na+2CH2)+ 548.3020, found 548.3033.
546.2888; found 546.2906.
4.1.37. N-(2-Pyrazinecarbonyl)-L-phenylalanine-L-(2-naphthyl)-
alanine- -Leucine boronic acid (7g)
L
Yield 53%, light yellow solid, mp: 137–138 °C; 1H NMR (CDCl3,
300 MHz): d 9.90 (s, 1H), 8.61 (s, 1H), 8.26 (s, 1H), 8.09–7.98 (m,
1H), 7.50–7.23 (m, 12H), 7.19–7.07 (m, 1H), 6.73–6.56 (m, 2H),
4.88–4.76 (m, 1H), 4.72–4.67 (m, 1H), 3.33–3.28 (m, 1H), 3.13 (d,
J = 6.3 Hz, 2H), 3.02–2.97 (m, 1H), 2.04 (s, br, 2H), 1.51–1.42 (m,
3H), 0.87–0.80 (m, 6H); 13C NMR (CDCl3, 75 MHz): d 173.10,
170.06, 163.51, 147.59, 143.90, 142.95, 142.86, 142.64, 142.52,
135.90, 135.70, 133.45, 133.23, 133.07, 132.14, 129.29, 128.88,
128.24, 127.94, 127.46, 127.14, 125.90, 125.53, 54.94, 51.96,
42.92, 39.98, 37.26, 36.92, 25.69, 23.14, 22.56; HRMS calcd for
C32H35BN5O5 (Mꢁ1)ꢁ 580.2731, found 580.2729.
4.1.42. N-(2-Pyrazinecarbonyl)-L-leucine-L-leucine-L-leucine
boronic acid (7l)
Yield 62.8%, sticky solid; 1H NMR (CDCl3, 300 MHz): d 9.30 (s,
1H), 8.71 (s, 1H), 8.50 (s, 1H), 8.28–8.06 (m, 1H), 7.16–7.09 (m,
1H), 6.99–6.85 (m, 1H), 4.86–4.69 (m, 1H), 4.53 (s, 1H), 2.91–
2.85 (m, 1H), 2.82–1.19 (m, 11H), 0.97–0.81 (m, 18H); 13C NMR
(CDCl3, 75 MHz):
d 171.77, 171.49, 163.10, 147.51, 144.36,
144.01, 143.88, 51.89, 51.36, 44.15, 40.55, 39.76, 38.73, 38.08,
30.55, 29.62, 28.55, 25.72, 24.78, 23.02, 21.89, 20.96; HRMS calcd
for C22H37BN5O5 (Mꢁ1)ꢁ 462.2888, found 462.3825.
4.1.43. N-(2-Pyrazinecarbonyl)-
leucine- -leucine boronic acid (7m)
Yield 66%, light yellow solid, mp: 135–136 °C; 1H NMR (CDCl3,
300 MHz): 9.29 (s, 1H), 8.70 (d, J = 2.1 Hz, 1H), 8.45 (d,
L-(2-naphthyl)alanine-L-
4.1.38. N-(2-Pyrazinecarbonyl)-L-(2-naphthyl)-alanine-L-(2-
L
naphthyl)-alanine- -leucine boronic acid (7h)
L
Yield 72.1%, light yellow solid, mp: 145–146 °C; 1H NMR (CDCl3,
300 MHz): d 8.95 (s, 1H), 8.54 (s, 1H), 8.16 (s, 1H), 8.17–8.11 (m,
1H), 7.74–7.16 (m, 14H), 7.08–6.95 (m, 2H), 6.87–6.50 (m, 2H),
4.95–4.87 (m, 1H), 4.79–4.68 (m, 1H), 3.28–3.26 (m, 2H), 3.00–
2.86 (m, 1H), 2.33 (s, br, 2H), 1.38–1.35 (m, 3H), 0.81–0.79 (m,
6H); 13C NMR (CDCl3, 75 MHz): d 170.18, 169.61, 163.56, 147.51,
143.79, 141.90, 142.55, 133.42, 133.126, 133.01, 132.46, 132.07,
128.58, 128.09, 127.39, 127.14, 127.01, 126.32, 125.94, 125.83,
125.49, 54.79, 51.52, 39.78, 37.49, 37.30, 36.95, 25.64, 23.09,
22.39, 22.19; HRMS calcd for C36H37BN5O5 (Mꢁ1)ꢁ 630.2893,
found 630.2895.
d
J = 1.8 Hz, 1H), 8.40 (d, J = 7.5 Hz, 1H), 7.74–7.66 (m, 4H), 7.45–
7.42 (m, 3H), 7.37 (d, J = 8.7 Hz, 1H), 5.05–4.93 (m, 1H), 4.53–
4.45 (m, 1H), 3.38 (d, J = 8.7 Hz, 2H), 2.98–2.83 (m, 1H), 1.99 (s,
br, 2H), 1.80–1.51 (m, 1H), 1.50–1.29 (m, 6H), 0.86–0.74 (m,
12H); 13C NMR (CDCl3, 75 MHz):
d 170.59, 170.45, 163.83,
147.74, 144.27, 143.61, 142.77, 133.46, 133.27, 132.47, 128.59,
128.12, 127.63, 127.05, 126.24, 125.84, 54.76, 39.88, 37.74, 25.78,
24.61, 24.48, 23.03, 22.82, 22.63, 21.60, 21.45; HRMS calcd for
C29H37BN5O5 (Mꢁ1)ꢁ 546.2893, found 546.2903.
4.2. Biological testing
4.1.39. N-(2-Pyrazinecarbonyl)-
-leucine boronic acid (7i)
Yield 49.2%, sticky solid; 1H NMR (CDCl3, 300 MHz): d 9.72 (s,
L-tryptophan-L-phenylalanine-
L
4.2.1. Assays for proteasome activities
The enzymatic activities of the proteasome were assayed using
fluorogenic peptides: Suc-Leu-leu-Val-Tyr-AMC (Suc represents
succinyl and AMC represents 7-amido-4-methyicoumarin,
obtained from SIGMA) for chymotryptic-like (CT-L) activity, Z-
Ala-Arg-Arg-AMC (Z represents benzyloxycarbonyl, obtained from
Calbiochem) for trypsin-like (T-L) activity and Z-Leu-Leu-Glu- bNA
(bNA represents b-naphtylamide, obtained from SIGMA) for pept-
idylglutamyl peptide-hydrolyzing activity (PGPH). One microgram
of 20S proteasome purified from mouse liver was incubated with
1H), 9.28 (s, 1H), 9.17 (s, 1H), 8.64–8.56 (m, 1H), 8.06–7.97 (m,
1H), 7.19–7.16 (m, 9H), 6.89–6.78 (m, 1H), 5.05 (s, 1H), 4.98–
4.87 (m, 1H), 3.98–3.75 (m, 1H), 3.58–3.53 (m, 1H), 3.19–3.15
(m, 1H), 2.13–1.97 (m, 10H), 1.60–1.40 (m, 3H), 0.79–0.75 (m,
6H). 13C NMR (CDCl3, 75 MHz): d 171.82, 170.35, 164.19, 147.46,
143.99, 142.76, 136.29, 129.11, 128.42, 127.11, 126.68, 124.07,
122.33, 112.16, 109.91, 54.27, 53.07, 49.31, 42.29, 39.89, 39.21,
38.72, 34.16, 33.58, 29.69, 27.94, 25.61, 24.09, 30.57, 23.07; HRMS
(ESI): calcd for C32H39BN6NaO5 (M+Na+2CH2)+ 621.2973, found
621.2963.
various concentrations of compounds and 50
lM fluorogenic pep-
tides in 100 l of 20 mM Tris–HCl pH 7.8, at 37 °C for 1 h, respec-
l
tively. The fluorescence of released AMC and bNA reagents, was
measured by a spectrofluorimeter (Fluostar OPTIMA, BMG Ger-
many) at excitation/emission wavelengths of 380/440 nm and
335/410 nm, respectively. 0.1% DMSO was used as solvent control.
Compared with the fluorescence of solvent control, an inhibition
rate was calculated and thereafter the IC50 value was deduced.
4.1.40. N-(2-Pyrazinecarbonyl)-
-leucine boronic acid (7j)
Yield 42.4%, sticky solid; 1H NMR (CDCl3, 300 MHz): d 9.28–9.27
L-phenylalanine-L-tryptophan-
L
(m, 1H), 9.21–9.14 (m, 1H), 9.07–9.00 (m, 1H), 8.57–8.46 (m, 1H),
8.12–8.10 (m, 1H), 7.16–7.08 (m, 9H), 6.76–6.58 (m, 1H), 5.25–5.18
(m, 1H), 4.81–4.64 (m, 1H), 3.78–3.45 (m, 1H), 3.48–3.33 (m, 1H),
3.11–3.05 (m, 1H), 2.07–1.89 (m, 10H), 1.45–1.23 (m, 3H), 0.81–
0.79 (m, 6H). 13C NMR (CDCl3, 75 MHz): d 170.99, 169.94, 162.87,
147.61, 144.26, 142.78, 136.00, 135.92, 129.22,128.74, 127.16,
127.05, 54.59, 54.35, 51.63, 49.27, 46.08, 42.22, 41.26, 40.02,
38.26, 33.66, 26.83, 25.58, 24.62, 22.99, 22.74, 22.53, 22.16,
21.88; HRMS (ESI): calcd for C32H39BN6NaO5 (M+Na+2CH2)+
621.2973 found: 621.2971.
4.2.2. Cell culture and cytotoxicity assays
HL-60 (promyelocytic leukemia cell line), U266 (multi myeloma
cell line), and BXPC-3 (human pancreatic cancer cell line) human
cell lines were obtained from the American Type Culture Collection
(Manassas, VA). BGC-823 (human gastric carcinoma cell line) cell
line was obtained from China Pharmaceutical University. HL-60
cell was cultured in IMDM supplemented with 20% fetal bovine
serum at 37 °C in 5% CO2. BGC-823 and BXPC-3 cells were grown
in RPMI 1640 supplemented with 10% fetal bovine serum at
37 °C in 5% CO2. U266 cell was cultured in RPMI 1640 supple-
mented with 15% fetal bovine serum at 37 °C in 5% CO2.
4.1.41. N-(2-Pyrazinecarbonyl)-L-phenylalanine-L-leucine-L-
leucine boronic acid (7k)
Yield 73.2%, light yellow solid, mp: 116–118 °C; 1H NMR (CDCl3,
300 MHz): d 9.30–9.21 (m, 1H), 8.74 (s, 1H), 8.47 (s, 1H), 8.33–8.29

Y. Zhu et al. / Bioorg. Med. Chem. 17 (2009) 6851–6861
6861
4. Mitch, W. E.; Goldberg, A. L. N. Eng. J. Med. 1996, 335, 1897.
5. Hampton, R. Y.; Gardner, R. G.; Rine, J. Mol. Biol. Cell. 1996, 7, 2029.
6. Murakami, Y.; Matsufuji, S.; Kameji, T.; Hayashi, S.; Igarashi, K.; Tamura, T.;
Tanaka, K.; Ichihara, A. Nature 1992, 360, 597.
A standard MTT assay was used to measure cell growth. In brief,
a suspension of 3000 cells/150 L of medium was added to each
well of 96-well plates and allowed to grow. Twenty-four hours la-
l
7. Turner, G. C.; Du, F.; Varshavsky, A. Nature 2000, 405, 579.
8. Naidoo, N.; Song, W.; Hunter-Ensor, M.; Sehgal, A. Science 1999, 285, 1737.
9. Chain, D. G.; Schwartz, J. H.; Hegde, A. N. Mol. Neurobiol. 1999, 20, 125.
10. Richardson, P. G.; Sonneveld, P.; Schuster, M. W.; Irwin, D.; Stadtmauer, E. A.;
Facon, T.; Harousseau, J. L.; Ben-Yehu, D.; Lonial, S.; Goldschmidt, H. N. Eng. J.
Med. 2005, 352, 2487.
11. Adams, J. Drug Discovery Today 2003, 8, 307.
12. Zhu, Y. Q.; Pei, J. F.; Liu, Z. M.; Lai, L. H.; Cui, J. R.; Li, R. T. Bioorg. Med. Chem.
2006, 14, 1483.
ter, drugs prepared in medium at 10 different concentrations were
added to the corresponding plates at a volume of 50
lL per well,
and the plates were incubated for 72 h with drugs. Then 20
lL of
a solution of 5 mg/ml MTT were added to each well and incubated
for another 4 h at 37 °C. Plates were then centrifuged at 1000 rpm
at 4 °C for 5 min, and the medium was carefully discarded. The for-
mazan crystals were dissolved in 100 lL of DMSO and absorbance
13. Bodanszky, M.; Bodanszky, A. The Practice of Peptide Synthesis, 2nd ed.;
Springer: Berlin, 1994.
was read on an Infinite M200 (Tecan, Austria) microplate reader at
540 nm. The result was expressed as the mean IC50 value, which is
the average from at least three independent determinations.
14. Matteson, D. S.; Majumdar, D. Organometallics 1983, 2, 1529.
15. Matteson, D. S.; Arne, K. J. Am. Chem. Soc. 1978, 100, 1325.
16. Matteson, D. S.; Majumdar, D. J. Am. Chem. Soc. 1980, 102, 7588.
17. Matteson, D. S.; Ray, R. J. Am. Chem. Soc. 1980, 102, 7590.
18. John, C. S.; John, P. J. Am. Chem. Soc. 1965, 87, 2492.
19. Groll, M.; Ditzel, L.; Löwe, J.; Stock, D.; Bochtler, M.; Bartunik, H. D.; Huber, R.
Nature 1997, 386, 463.
20. Groll, M.; Berkers, C. R.; Ploegh, H. L.; Ovaa, H. Structure 2006, 14, 451.
21. Verdoes, M.; Florea, B. I.; enendez-Benito, V.; Maynard, C. J.; Witte, M. D.; van
der Linden, W. A.; van der Nieuwendijk, A. M. C. H.; Hofmann, T.; Berkers, C. R.;
van Leeuwen, F. W.; Groothuis, T. A.; Leeuwenburgh, M. A.; Ovaa, H.; Neefjes, J.
J.; Filipov, D. V.; van der Marel, G. A.; Dantuma, N. P.; Overkleeft, H. S. Chem.
Biol. 2006, 13, 1217.
Acknowledgement
The authors thank the Natural Science Foundation of China for
the financial support (NSFC 30772626).
References and notes
1. Ciechanover, A. EMBO J. 1998, 17, 7151.
22. Löwe, J.; Stock, D.; Jap, B.; Zwickl, P.; Baumeister, W.; Huber, R. Science 1995,
268, 533.
2. Rock, K. L.; Goldberg, A. L. Annu. Rev. Immunol. 1999, 17, 739.
3. PrÖsch, S.; Priemer, C.; HÖflich, C.; Liebenthal, C.; Babel, N.; Krüger, D. H.; Volk,
H.-D. Antiviral Ther. 2003, 8, 555.