Design, synthesis, biological evaluation, and Structure-Activity Relationship (SAR) discussion of dipeptidyl boronate proteasome inhibitors, Part I: Comprehensive understanding of the SAR of α-amino acid boronates

Article abstract of DOI:10.1021/jm9005093

New series of dipeptidyl boronate inhibitors of 20S proteasome were designed and synthesized. The comprehensive understanding of the SAR was obtained by utilizing the variation of four substituents. From the screened compounds in enzyme, novel inhibitors 49 and 50 were identified to be highly potent druglike candidates with IC₅₀ values of 1.2 and 1.6 nM, respectively, which showed better activities than the drug bortezomib on the market. Two hematologic human tumor cell lines, HL-60 and U266, were significantly sensitive to both candidates and showed nearly the same potency as the standard bortezomib with IC₅₀ values less than 10 nM. But as for most of the eight human solid tumor cell lines, both candidates were more potent than the standard with the IC₅₀ value range of 9.8-70 nM. The activity evaluation of the stereoisomers showed that changing R-isomers to S-isomers greatly reduced the potency and even induced inactivity.

Full text of DOI:10.1021/jm9005093

4192 J. Med. Chem. 2009, 52, 4192–4199
DOI: 10.1021/jm9005093
Design, Synthesis, Biological Evaluation, and Structure-Activity Relationship (SAR) Discussion of
Dipeptidyl Boronate Proteasome Inhibitors, Part I: Comprehensive Understanding of the SAR of
r-Amino Acid Boronates
Yongqiang Zhu,*,† Xin Zhao,† Xinrong Zhu,† Gang Wu,† Yuejie Li,† Yuheng Ma,† Yunxia Yuan,† Jie Yang,† Yang Hu,†
†
Li Ai, and Qingzhi Gao‡
†Jiangsu Simcere Pharmaceutical Research Institute and Jiangsu Key Laboratory of Molecular Targeted Antitumor Drug Research, No. 699-18
Xuan Wu Avenue, Xuan Wu District, Nanjing 210042, PRC, and ‡Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency School of
Pharmaceutical Science and Technology, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072, PRC
Received April 6, 2009
New series of dipeptidyl boronate inhibitors of 20S proteasome were designed and synthesized. The
comprehensive understanding of the SAR was obtained by utilizing the variation of four substituents.
From the screened compounds in enzyme, novel inhibitors 49 and 50 were identified to be highly potent
druglike candidates with IC50 values of 1.2 and 1.6 nM, respectively, which showed better activities than
the drug bortezomib on the market. Two hematologic human tumor cell lines, HL-60 and U266, were
significantly sensitive to both candidates and showed nearly the same potency as the standard
bortezomib with IC50 values less than 10 nM. But as for most of the eight human solid tumor cell
lines, both candidates were more potent than the standard with the IC50 value range of 9.8-70 nM. The
activity evaluation of the stereoisomers showed that changing R-isomers to S-isomers greatly reduced
the potency and even induced inactivity.
Introduction
proteasome was the main proteolytic component, which is
found in all eukaryotic cells and consisted of the cylinder-
shaped multicatalytic proteinase complex (MPC) 20S protea-
some and regulatory particle (RP) 19S proteasome. The 19S
proteasome locating at each end of the 20S proteasome is
made up of 18 subunits, which controls the recognition,
unfolding, and translocation of the protein substrates into
the lumen of the 20S proteasome.8 X-ray crystallography of
26S proteasome revealed that the 20S proteasome is com-
posed of 28 protein subunits arranged in four stack rings, with
each ring comprising seven R- and β-type subunits, following
an R1-7β1-7 stoichiometry.9,10 The two outer chambers are
formed by R subunits, while the central chamber, containing
the proteolytic active sites, is made up of β subunits. Three of
the 14 β subunits are responsible for the postglutamyl peptide
hydrolysis activity (PGPH, attributed to β1), trypsin-like
activity (T-L, β2), and chymotripsin-like activity (CT-L, β5),
respectively, and all these three active subunits hydrolyze
the amide bond of protein substrates with the hydrophilic
γ-hydroxyl group of the N-terminal threonine (Oγ-Thr1).
Although bortezomib is a highly potent proteasome inhi-
bitor, treatment with bortezomib resulted in some severe side
effects such as neurologic and cardiovascular adverse effects,
fatigue, nausea and vomiting, and diarrhea. Therefore, there
remains a significant demand to develop new proteasome
inhibitors with less toxicity and greater therapeutic index.
Since bortezomib was launched, various reversible or irrever-
sible proteasome inhibitors had been developed subsequently
(Figure 1).1,7 Most proteasome inhibitors are short peptides
harboring an electrophilic group at one end such as aldehyde
(MG132, Figure 1), boronic acid (bortezomib, Figure 1), and
vinyl sulfone (NLVS, Figure 1), all of which can form covalent
The development and use of proteasome inhibitors have
drawn wide attention in recent years both in fundamental and
applied sciences.1 Especially, a dipeptidyl boronic acid borte-
zomib (PS-341, Figure 1) was approved by the FDA for the
treatment of patients with relapsed and refractory multiple
myeloma (MM, approved in 2003) with at least one prior line
of therapy and for the treatment of mantle cell lymphoma
(MCL, approved in 2006).2,3 This fact indicated that the
proteasome was clinically validated as an effective target for
cancer therapy.
Three researchers won the 2004 Nobel Prize in chemistry
for discovering how the ubiquitin-proteasome pathway
(UPPa) regulated the degradation of intracellular proteins
with extreme specificity as to target, time and space.4 The
pathway plays a central role to recognize and degrade the
misfolded and abnormal proteins in most mammalian cells.5
Such a process is very important in maintaining the biological
homeostasis and regulation of different cellular processes
such as cell differentiation, cell cycle control, antigen process-
ing, and hormone metabolism.6,7 In this pathway, the 26S
*To whom correspondence should be addressed. Phone: 86-25-
85566666-1726. Fax: 86-25-85566666-1835. E-mail: zhyqscu@hotmail.com.
aAbbreviations: SAR, structure-activity relationship; IC50, inhibi-
tion constant; 3D-QSAR, three-dimensional quantitative structure-
activity relationship; DCC, N,N0-dicyclohexylcarbodiimide; HOBt,
1-hydroxybenzotriazole; EDC, 1-ethyl-3-(3- dimethylaminopropyl)car-
bodiimide; MM, multiple myeloma; UPP, ubiquitin-proteasome path-
way; MPC, multicatalytic proteinase complex; RP, regulatory particle;
CT-L, chymotripsin-like activity; T-L, trypsin-like activity; PGPH,
postglutamyl peptide hydrolysis activity; DIPEA, N,N-diisopropylethyl-
amine.
r
pubs.acs.org/jmc
Published on Web 06/18/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4193
Scheme 1. General Synthesis of N-Terminal Protected Amino
Acids 3a-ua
Figure 1. Structures of currently developed proteasome inhibitors.
bonds with the catalytic Oγ-Thr1 in the three catalytic sites.
Some nonpeptidic molecules such as NPI-0052 (in clinical
phase I, Figure 1), lactacystin (Figure 1), and omuralide
(Figure 1) inhibit the proteasome also by forming covalent
adducts.11
a Reagents and conditions: (i) P1COOH, DCC, HOBt, NMM, THF,
0 °C. (ii) (1) 2 N NaOH, acetone, 0 °C; (2) 2 N HCl, ethyl acetate, 0 °C.
We previously reported three-dimensional quantitative
structure-activity relationship (3D-QSAR) studies of tripep-
tide aldehyde proteasome inhibitors12 and subsequently car-
ried out studies on the synthesis, biological evaluation, and
SAR discussion of tripeptide boronate and boronic acid
proteasome inhibitors,13 and as a continuationof our research
work, we recently reported the 3D-QSAR studies of dipepti-
dyl boronates synthesized by Millennium Pharmaceuticals,
Inc.14 However, in the compound libraries, only the substit-
uents at P1, P2, and P4 positions were varied, which could not
offer enough information to fully understand the interaction
mode between the dipeptidyl boronates and 20S proteasome
without the important information about P3 position. What is
more, the influence of stereoisomers on the activity of 20S
proteasome had never been elucidated. To obtain compre-
hensive understanding of the effect of variations at P1, P2, P3,
and P4 positions on the activity and to summarize an accurate
and detailed SAR for the next cycle of design, a large number
of new dipeptidyl boronate inhibitors had been designed and
synthesized. After biological evaluation of these compounds
for β5 subunit of 20S proteasome, comprehensive SAR
analysis was carried out on the substituents at P1, P2, P3,
and P4 positions and the importance of variations at P3
position was elucidated for the first time. At the same time,
the influence of the stereoisomers on the binding of 20S
proteasome was also discussed. Finally, the cytotoxic activ-
ities of the two most potent inhibitors were assayed on 10
human tumor cell lines to evaluate their potentials as anti-
tumor agents. From the screened compounds, these two
candidates were selected and being evaluated in vivo.
N,N0-dicyclohexylcarbodiimide (DCC) and 1-hydroxyben-
zotriazole (HOBt) as catalytic coupling agents.15 After sa-
ponification and acidification, various aicds 3 were gained in
high yields and no isomers were detected by chiral HPLC
analysis. Whenever possible, commercially available pro-
tected amino acids 3r-u were used.
The amino boronates 11a-f hydrochloride were the key
intermediates in the total synthetic route, which were pre-
pared following procedures reported in the literature
(Scheme 2).16,17 The highly optical (þ)-pinanediol 5a and
(-)-pinanediol 5b were synthesized according to the reported
methods18,19 starting from 98% e.e. (þ)-R-pinene and 80% e.e.
(-)-R-pinene, respectively. The two isomers were obtained in
96.5% and 92.9% yields after simple distillation in vacuo,
respectively. The dimethyl dichloromethylboronate 7 was
prepared by addition of trimethoxyborane to the solution of
(dichloromethyl)lithium in THF generated in situ at -100 °C.
Transesterification of compound 7 with chiral diols 5 gave
dichlorosubstituted 8a-f with the chiral pinanediols as the
stereodirected groups in high yields after column chroma-
tography. In the presence of anhydrous zinc chloride as
catalyst, monochlorosubstituted boronates 9a-f were ob-
tained with the reaction of 8 and Grignard reagents
P3CH2MgBr. It is noteworthy to point out that the bromide
anion in the Grignard reagents should be employed instead
of chloride in order to decrease the racemation.20 Treatment
of monochlorosubstituted boronates 9a-f with lithium bis
(trimethylsilyl)amide produced protected amines 10a-f that
underwent facile deprotection in dry ethereal HCl solution to
generate the desired amino boronates 11a-f as the hydro-
chloride salts.
Results and Discussion
General synthetic pathway to obtain the dipeptidyl
boronates and boronic acids 12-53 (listed in Table 1) was
presented in Scheme 3. Coupling of amino boronates 11a-f
with various acids 3 in the presence of 1-ethyl-3-(3-dimethyl-
Chemistry. The intermediates of N-terminal protected
acids 3 were prepared as depicted in Scheme 1. From the
natural or unnatural amino acids, methyl esters 2 were
obtained using standard peptide synthesis procedures with
aminopropyl)carbodiimide hydrochloride (EDC HCl) and
3

4194 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Zhu et al.
Scheme 2. General Synthesis of Amino Boronates 11a-f Hydrochloridea
a Reagents and conditions: (i) OsO4, t-butyl alcohol, trimethylamine N-oxide dihydrate, pyridine, water, reflux, 24 h; (ii) THF, -100 °C; (iii) B-
(OCH3)3, -100 °C to room temp; (iv) 5a or 5b, THF, room temp; (v) P3CH2MgBr for 9a-e, (CH3)2CHCH2MgBr for 9f, Et2O, anhydrous ZnCl2, -78 °C
to room temp; (vi) LiN(SiMe3)2, THF, -78 °C to room temp; (vii) petroleum ether, dry HCl in Et2O, -78 °C to room temp.
HOBt afforded the dipeptidyl boronates 12-47, and the
boronate was transesterificated with isobutylboronic acid
under acid conditions to give boronic acids. It had been
reported that the boronic esters of this nature had the same
activity and specificity as their undeprotected counter-
parts,21 and our previous experimental data also supported
it.13 So in this work, we decided to leave the pinanediol group
on the boronic ester at the onset of our 20S proteasome assay
screening. Only some interesting boronic esters such as 14,
16, and 34 were selected to remove the pinanediol group to
prepare boronic acids 48, 49, and 50 for further development.
At the same time, to investigate the influence of stereoi-
somers on the biological activities, boronic acids 51, 52, and
53 were also prepared.
Biology. The capacities of dipeptidyl boronates and boro-
nic acids to inhibit the CT-L activity of 20S human protea-
some were assayed using appropriate fluorogenic substrates
(Table 1). The marketed bortezomib was used as standard.
To fully understand SAR of dipeptidyl boronates and
boronic acids, various substituents on the P1, P2, P3, and P4
positions were screened. Table 1 summarized the biological
results. First, to investigate the influence of substituents at P4
position on the potency, bortezomib and its pinanediol
protected boronate 12 were prepared. The biological data
indicated that both compounds nearly showed equal activ-
ities (IC50: 1.92 nM vs 1.90 nM). The same cases were also
accounted by compounds 16 (1.12 nM) and 49 (1.20 nM), 34
(0.76 nM) and 50 (1.60 nM), as well as 14 (3.21 nM) and 48
(4.37 nM). This result is consistent with the reported one,21
so it is reasonable to screen in vitro such kind of inhibitors
without removing the protecting pinanediol group.
Second, variations at P1 position were carried out to
elucidate the SAR of this position. Aromatic groups at P1
position such as pyrazinyl (12, 1.92 nM) and 5,6,7,8-tetra-
hedronaphthyl (34, 0.76 nM) were favored to the biological
activities. However, addition of an alkyl methyl in this
position (39, 2.34 nM) led to a minor loss of potency.
All these facts suggested that negative and aromatic substit-
uents were essential to improve the activity of inhibitors.
To obtain more potent compounds for drug development,
other tetrahedronaphthyl groups at P1 position, such as
racemized 1,2,3,4-tetrahedronaphthyl and its two isomers
(S)-1,2,3,4-tetrahedronaphthyl and (R)-1,2,3,4-tetrahedro-
naphthyl, were screened. Among these isomers, the S-one
(16, 1.12 nM) showed the most potent inhibitory activity and
the R-one (17, 5.17 nM) gave the worst result, and the
racemized one (15, 4.18 nM) fell in the middle. However,
replacement of negative groups with positive and bulky Boc

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4195
Table 1. Structures of Compounds 12-53, Bortezomib, and Their Inhibition of Human 20S Proteasome

4196 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Table 1. Continued
Zhu et al.
a IC50 value obtained for bortezomib under our experimental conditions.
match the hydrophobic residues of the β5 subunit.10,12 On
the basis of these experimental results, it can be concluded
that negative and aromatic groups with a proper volume
were favored to the potency, which was consistent with our
published theoretical results.14
Scheme 3. General Synthesis of Dipeptidyl Pinanediol Boro-
nates and Boronic Acidsa
The steric and electronic characteristics of the substituents
at P3 position also greatly affected the potency and made a
great contribution for SAR analysis, which had never been
discussed in detail previously in the literature. Data from
Table 1 offered the chance for us to summarize the SAR for
this position. On the one hand, bulky groups contributed
more to the activity than small ones. For example, steric
isopropyl substituted compound 39 (2.34 nM) was 3-fold
potent more than n-propyl substituted one (38, 8.46 nM).
The different potency between compounds 16 and 18 also
accounted for this. On the other hand, it seemed that positive
alkyl groups instead of negative aromatic ones were favored
to the potency. Both 16 and 18 were substituted by isopropyl
and n-propyl group at P3 position, respectively, and dis-
played more potent activity than phenyl substituted 19.
However, incorporation of an electrowithdrawing group
in the para-position of phenyl ring at P3 position led to
significant improvement of potency. Inhibitor 21 bearing
a fluorine atom at 4-position of phenyl ring showed
an IC50 value of 1.26 nM, removal of the fluorine atom
of 21 to give 19 caused the inhibition to decrease more
than 4-fold, and replacement of the negative fluorine atom
with a positive methyl substituent in the 4-position
of phenyl (20) even resulted in 6-fold loss in enzymatic
potency.
a Reagents and conditions: (i) EDC HCl, HOBt, DIPEA, CH2Cl2, -
15 °C to room temp; (ii) isobutylboronic acid, 2 N HCl, MeOH, hexane.
3
group dramatically decreased the potency, as evidenced by
the loss of potency shown for compounds 41-45, the IC50
values of which were all greater than 10 nM.
Substitutions at P2 position generated a distinct SAR
(Table 1). Negative phenyl substituent (34, 0.76 nM) at P2
position was much more beneficial to the potency compared
with positive methyl (33, 6.96 nM). However, with too much
bulky aromatic groups at this position, the potency of these
compounds decreased. This is indeed the case for inhibitor 35
(1.34 nM). Compared with compound 34, analogue 35
harboring a much steric naphthyl group nearly exhibited 2-
fold weaker potency, but as for alkyl substituents at P2
position, it is an exception for compound 32 (1.15 nM).
Replacement of two smaller methyl groups of 33 with a much
hindered isopropyl (32) improved the inhibition with 6-fold.
Incorporation of hydroxyl groups in the 4-position of phenyl
ring significantly resulted in diminished potency, which was
demonstrated by compounds 41 and 42, presumably because
hydrophilic characteristic of the hydroxyl group did not
The isomers of a drug always result in different or even
reverse effects such as the famous thalidomide event. So in
this work, the corresponding isomers (51, 52, and 53) of
compounds bortezomib, 49 and 50, were prepared and

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4197
Table 2. Cellular Activities of Two Drug Candidates and Bortezomib (IC50 (SD), nM)
compd
H460
SW-480
30 (2)
SKOV-3
40 (2)
A549
HELA
37.5 (2)
KB
HepG2
60 (7)
BGC
HL-60
U266
49
50
36 (3.0)
38.5 (3)
70 (2.8)
580 (9.5)
9.55 (0.62)
2.28 (0.18)
21.5 (2.6)
115 (20)
30 (1.5)
50 (10)
60 (2.3)
130 (5)
40.5 (4)
255 (7)
23.2 (2.5)
92 (10)
9.8 (1)
13.1 (1.8)
37.5 (2)
1210 (11.1)
880 (60)
9.25 (0.67)
5.50 (0.21)
2.95 (0.1)
bortezomib
59.5 (10)
2.45 (0.04)
biologically evaluated. Intriguingly, the results were surpris-
ing that the three isomers showed different influence on the
enzymatic potency. Isomers 51 and 52 greatly reduced the
IC50 values to 700 and 2290 nM, respectively, which indi-
cated the chirality of these two compounds did not strictly
match the active site of biological target 20S proteasome.
However, the activity of inhibitor 53 (9.19 nM) and its isomer
50 (1.6 nM) was not significantly different from that of other
two pairs. This is presumably due to the proper conjugation
between the aromatic phenyl ring and carboxyl bond of this
enantiomer.
For drug development, the two most potent inhibitors (49
and 50) in enzyme were further tested in 10 human tumor cell
lines to determine their effects on cancer cells. The 10 cell
lines included two hematologic tumors, such as promyelo-
cytic leukemia cell line HL-60, and multimyeloma cell line
U266, and eight solid tumors, encompassing human large
cell lung cancer cell line H460, human nonsmall cell lung
cancer cell line A549, human colon carcinoma cell line SW-
480, human ovarian carcinoma SKOV-3, human epithelia
carcinoma cell line HELA, oral cancer cell KB, human
hepatocellular liver carcinoma cell line HepG2, and human
gastric carcinoma cell line BGC-823. The cellular activities of
these two compounds and bortezomib were displayed in
Table 2. In general, two hematologic tumor cell lines were
more sensitive to such kind of inhibitors than the eight solid
tumors. All the three inhibitors (49, 50, and bortezomib)
inhibited the two hematologic tumor cell lines at the same
level with IC50 less than 10 nM, while inhibition of the eight
solid tumors was almost more than 10 nM. However, a loss
in cellular potency was observed in the solid BGC-823 tumor
cell line, which was inhibited at more than 500 nM level by
the three inhibitors.
From the highly potent compounds, two candidates 49 and 50
were further tested against 10 human main cancer cell lines.
Both inhibitors exhibited IC50 valuesless than 10 nM activities
with the same magnitudes as the marketed drug bortezomib
against two hematologic cancer cell lines. For seven solid
tumors, the two candidates possessed IC50 values ranging
from 9.8 to 70 nM and showed better activities than the drug
bortezomib, which indicated their potentials also for use as
antisolid tumor agents. At the same time, the activity differ-
ence of the stereoisomers was also investigated in this study. It
was found that changing R-isomers to S-isomers greatly
reduced the potency and even induced inactivity. Nowadays,
the in vivo biological investigation as well as the safety
evaluation of the two candidates was being conducted both
in solid and hematologic tumor models and the results will be
reported in the near future.
Experimental Section
Chemistry. Commercially available reagents were used di-
rectly without any purification unless otherwise stated. Abso-
lutely anhydrous solvents were obtained with the proper
methods introduced in the literature. Yields refer to chromato-
graphically unless otherwise stated. Reactions were monitored
by thin-layer chromatography carried out on silica gel alumi-
num sheets (60F-254) and RP-18 F254s using UV light as a
visualizing agent, 15% ethanolic phosphomolybdic acid, and
heat or ninhydrin and heat as developing agent. Column
chromatography was performed on 200-300 mesh silica gel
and ODS C-18 column. Analytical reverse phase HPLC was run
using a Kromasil 100-5C18, 4.6 mm ꢀ 250 mm column eluting
with a mixture of methanol and water containing 0.02%
triethylamine and 0.03% trifluoacetic acid. HPLC showed
purity of all the final products was greater than 95%. Melting
points were obtained on an YRT-3 melting point apparatus and
were uncorrected. 1H NMR and 13C NMR spectra were re-
corded at room temperature on a Bruker Avance 300 or Avance
500 spectrometers. Chemical shifts were reported in ppm
(δ units), and tetramethylsilane (TMS) was used as internal
reference. Coupling constants (J) are expressed in hertz. The
following abbreviations were used to designate the multiplici-
ties: s = singlet, d = doublet, t = triplet, m = multiplet, dd =
doublet of doublets. Mass spectra were obtained using Agilent
LC-MS (1956B) instruments in electrospray positive and nega-
tive ionization modes. High-resolution mass spectra were
recorded on a ZAB-HS instrument using an electrospray source
(ESI).
In comparison with bortezomib, both compounds 49 and
50 showed much higher activities against the solid tumors.
These two candidates significantly inhibited seven solid
tumor cell lines at the level of less than 100 nM. Especially
for compound 50, it acted on the oral cancer cell KB
at 9.8 nM level and inhibited HepG2 at 13.1 nM,
which displayed that this compound had the potential
for the treatment of some solid tumors, and now these
two compounds were being evaluated in vivo for some
hematologic and solid tumors. The results will be published
later.
The synthesis of the target compounds were shown below.
A typical procedure for preparation of dipeptide boronates of
13-47 was exemplified by the synthesis of compound 12.
Preparation of 48-53 was employed the similar method of
bortezomib. For description of general methods and prepara-
tion of the other key intermediates 3a-q, 5a, 5b, 8a, 8b, 9a-f,
and 11a-f, and structural characterization data for compounds
14-50, see Supporting Information.
N-[(1S)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-Hexahydro-3a,5,5-tri-
methyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]-
amino]carbonyl]-2-phenyl]-2-pyrazincarboxamide (12). Toa cooled
solution (-5 °C) of pyrazin-L-phenyl alanine (3a) (0.27 g,
1.00 mmol) dissolved in anhydrous CH2Cl2 (50 mL) was added
Conclusion
Wereport here a comprehensive understanding of SARof a
series of R-amino acids composed dipeptidyl boronates pro-
teasome inhibitors for the first time. By varying different
substituents at P1, P2, P3, and P4 positions of the backbone,
a series of structurally novel compounds were synthesized and
biologically evaluated against the 20S proteasome. Detailed
SAR discussions indicated that substituents at P3 position
were also essential for biological activities in addition to those
at P1, P2, and P4 positions, which had never been investigated.

4198 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Zhu et al.
HOBt (0.16 g, 1.20 mmol). After 20 min, the temperature of
δ 0.85-0.89 (-CH3, m, 6H), 1.58-1.59 (-CH2, m, 2H), 1.83-
1.89 (-CH, m, 1H), 2.69-2.72 (-CH2, m, 2H), 3.12-3.23
(-CH, m, 1H), 5.08 (-CH, t, J=7.4 Hz, 1H), 7.20-7.23
(-Ph, m, 1H), 7.26-7.31 (-Ph, m, 4H), 8.68 (-Pyz, dd, J1=
1.5 Hz, J2=0.96 Hz, 1H), 8.78 (-Pyz, d, J=2.5 Hz, 1H), 9.16
(-Pyz, d, J=1.4 Hz, 1H). 13C NMR (CD3OD, 125 MHz)
δ 22.45, 23.60, 26.90, 31.99, 38.46, 45.87, 52.73, 128.28, 129.73,
130.51, 137.29, 144.83, 144.88, 145.75, 148.87, 165.31, 176.99.
MS (ESI) m/z 435.1 [M þ Na þ 2CH2]þ. HRMS [M þ Naþ
2CH2]þ calcd, 435.2189; found, 435.2141.
[(1S)-1-[[(2S)-3-Phenyl-2-[((S)-(-)-1,2,3,4-tetrahedro-naphthoic-
1-carbonyl)amino]-1-oxopropyl]amino]-3-methylbutyl]boronic Acid
(52). HPLC indicates a purity of 98.0 area %. 1H NMR (CD3OD,
500 MHz) δ 0.88-0.89 (-CH3, m, 6H), 1.28-1.29 (-CH, m, 1H),
1.32-1.36 (-CH2, m, 2H), 1.60-1.64 (-CH2, m, 2H), 1.81-1.88
(-CH2, -OH, m, 3H), 2.01 (-OH, br, 1H), 2.66-2.72 (-CH2, m,
2H), 2.76-2.77 (-CH, m, 1H), 3.01 (-CH2, dd, J1=9.1 Hz, J2=
4.7 Hz, 1H), 3.16 (-CH2, dd, J1=6.5 Hz, J2=7.3 Hz, 1H), 3.68-
3.70 (-CH, m, 1H), 4.91 (-CH, dd, J1 = 6.5 Hz, J2=2.4 Hz, 1H),
6.96 (-Ph, d, J=7.6 Hz, 1H), 7.04-7.10 (-Ph, m, 3H), 7.23-7.26
(-Ph, m, 3H), 7.29-7.30 (-Ph, m, 2H). 13C NMR (CD3OD, 125
MHz) δ 22.13, 22.34, 23.71, 26.99, 28.72, 30.17, 30.74, 38.46, 40.86,
44.92, 54.78, 126.39, 126.99, 127.85, 128.19, 129.68, 130.06, 130.41,
135.22, 137.37, 138.84, 177.42, 178.15. MS (ESI) m/z 459.1
[M þ Na]þ. HRMS [M þ Na þ 2CH2]þ calcd, 487.2754; found,
487.2671.
the reaction system was cooled to -15 °C and EDC HCl (0.19 g,
3
1.00 mmol) was added. Finally the precooled (0 °C) mixture of the
known pinanediol boronate amino hydrochloride (11a) (0.30 g,
1.00 mmol) and DIPEA (0.26 mL, 1.48 mmol) in anhydrous
CH2Cl2 (10 mL) was poured. The mixture stirred at -15 °C for 1 h
and at room temperature for 2 h, finally quenched with water. The
aqueous phase was extracted with CH2Cl2 (3 ꢀ 100 mL). Com-
bined organic phase was washed with 10% citric acid, 5%
NaHCO3, and brine, dried over anhydrous Na2SO4, filtered,
and evaporated to provide crude product. ODS column chroma-
tography using acetonitrile/H2O (3:1) afforded 0.43 g (79.0%)
of 12 as a glassy solid. 1H NMR (CDCl3, 500 MHz) δ 0.82-0.85
(-CH3, m, 9H), 1.16-1.51 (-CH3, -CH2, -CH, m, 10H), 1.77-
1.92 (-CH2, -CH, m, 2H), 1.95-2.04 (-CH, m, 1H), 2.16-2.21
(-CH2, m, 1H), 2.29-2.35 (-CH2, m, 1H), 3.12-3.26 (-CH2,
-CH, m, 3H), 4.25-4.31 (-CH, m, 1H), 4.78-4.83 (-CH, m,
1H), 5.78-5.94 (-CONH, m, 1H), 7.19-7.31 (-Ph, m, 5H), 8.38
(-CONH, t, J=8.9 Hz, 1H), 8.52-8.54 (Pyz, m, 1H), 8.74 (Pyz, t,
J=1.6 Hz, 1H), 9.34-9.35 (Pyz, m, 1H). 13C NMR (CDCl3, 125
MHz) δ 22.00, 22.84, 24.00, 25.48, 26.26, 27.09, 28.59, 35.44, 35.50
(br), 38.21, 38.58, 39.52, 39.91, 51.38, 54.34, 77.94, 85.97, 126.94,
128.58, 129.45, 136.61, 142.70, 144.13, 144.30, 147.37, 162.80,
170.23. MS (ESI) m/z 519.2 [M þ H]þ. HRMS [M þ Na]þ calcd,
541.2962; found, 541.2974.
N-[(1S)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-Hexahydro-3a,5,5-tri-
methyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-2-(4-methylphe-
nyl)ethyl]amino]carbonyl]-2-phenyl]-2-pyrazincarboxamide (13).
1H NMR (CDCl3, 500 MHz) δ 0.83-0.85 (-CH3, m, 3H),
1.15-1.23 (-CH2, m, 1H), 1.27-1.29 (-CH3, m, 3H), 1.37-
1.40 (-CH3, m, 3H), 1.81-1.90 (-CH2, -CH, m, 2H), 1.99-2.03
(-CH, m, 1H), 2.15-2.16 (-CH2, m, 1H), 2.22-2.23 (-CH3, m,
3H), 2.31-2.34 (-CH2, m, 1H), 2.62-2.73 (-CH2, m, 1H),
2.87-2.90 (-CH2, m, 1H), 3.15-3.32 (-CH2, -CH, m, 3H),
4.29-4.31 (-CH, m, 1H), 4.76-4.78 (-CH, m, 1H), 5.82-5.98
(-CONH, m, 1H), 6.84-6.93 (-Ph, m, 4H), 7.18-7.28 (-Ph, m,
5H), 8.22-8.28 (-CONH, m, 1H), 8.51 (-Pyz, t, J=8.0 Hz, 1H),
8.74 (-Pyz, s, 1H), 9.30 (-Pyz, d, J=17.5 Hz, 1H). 13C NMR
(CDCl3, 125 MHz) δ 20.98, 24.06, 26.26, 27.17, 28.59, 35.44,
36.05, 38.42, 38.60, 39.34, 51.56, 54.23, 78.08, 86.11, 127.11,
128.72, 128.98, 129.19, 129.49, 129.64, 136.02, 136.45, 142.78,
144.46, 147.50, 162.82, 171.32. MS (ESI) m/z 565.3 [M -H]-.
HRMS [MþNa]þ calcd, 589.2963; found, 589.2940.
[(1S)-1-[[(2S)-3-Phenyl-2-[(5,6,7,8-tetrahedro-naphthoic-1-car-
bonyl)amino]-1-oxopropyl]amino]-3-methylbutyl]boronic Acid
(53). HPLC indicates a purity of 98.7 area %. 1H NMR
(CD3OD, 500 MHz) δ 0.90-0.93 (-CH3, m, 6H), 1.29-1.32
(-CH, m, 1H), 1.35-1.40 (-CH2, m, 2H), 1.65-1.69 (-CH2,
m, 2H), 1.72-1.75 (-CH2, m, 2H), 2.41-2.45 (-CH2, m, 1H),
2.56-2.59 (-CH2, m, 1H), 2.70-2.76 (-CH2, -CH, m, 1H),
3.04 (-CH2, dd, J1=9.6 Hz, J2=4.2 Hz, 1H), 3.22 (-CH2, dd,
J1=7.6 Hz, J2=6.2 Hz, 1H), 5.06 (-CH, dd, J1=6.3 Hz, J2= 3.3
Hz, 1H), 6.97 (-Ph, d, J=7.2 Hz, 1H), 7.05-7.11 (-Ph, m, 2H),
7.26-7.27 (-Ph, m, 1H), 7.30-7.32 (-Ph, m, 4H). 13C NMR
(CD3OD, 125 MHz) δ 22.37, 23.85, 24.08, 26.00, 27.02, 27.42,
30.65, 38.30, 40.92, 52.56, 54.77, 125.30, 126.27, 128.20, 129.67,
130.50, 131.94, 135.64, 137.38, 137.65, 139.10, 173.34, 177.50.
MS (ESI) m/z 437.1 [MþH]þ. HRMS [M þ Na þ 2CH2]þ calcd,
487.2754; found, 487.2649.
Enzyme and Inhibition Asssays. 20S proteasome activity assay
kit was purchased from Chemicon (Chemicon, USA). Other
reagents and solvents were purchased from commercial sources.
In brief, substrates and compounds were previously dissolved in
DMSO, with the final solvent concentration kept constant at
3% (v/v). The reaction buffers were (pH 7.5) 20 mM Tris, 1 mM
DTT, 10% glycerol, 0.02% (w/v) DS for CT-L activities.
Proteasome activity was determined by monitoring the hydro-
lysis of the fluorogenic substrate, Suc-Leu-Leu-Val-Tyr-AMC
(λexc = 360 and λexc = 465 nm for AMC substrates), reacting for
1 h at 37 °C in the presence of untreated (control) or proteasome
that had been incubated with different concentration of test
compounds. Fluorescence was measured using an Infinite M200
microplate reader (Tecan, Austria).
Cell Culture and Cytotoxicity Assays. HL-60 (promyelocytic
leukemia cell line), HELA (human epithelia carcinoma cell line),
and U266 (multi myeloma cell line) human cell lines were
obtained from the American Type Culture Collection (Manassas,
VA). H460 (human large cell lung cancer cell line), A549 (human
nonsmall cell lung cancer cell line), SW-480 (human colon
carcinoma cell line), SKOV-3 (human ovarian carcinoma), KB
(oral cancer cell), HepG2 (human hepatocellular liver carcinoma
cell line), and BGC-823 (human gastric carcinoma cell line) cell
lines were 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, KB, NCI-H460,
HepG2, and SW-480 cells were grown in RPMI 1640 supplemen-
ted with 10% fetal bovine serum at 37 °C in 5% CO2. U266 cell
[(1R)-1-[[(2S)-3-Phenyl-2-[(pyrazin-2-carbonyl)amino]-1-oxo-
propyl]amino]-3-methylbutyl]boronic Acid (Bortezomib). To the
solution of 12 (0.30 g, 0.58 mmol) and 2-methylpropylboronic
acid (0.30 g, 1.68 mmol) dissolved in methanol (5 mL) and
hexane (10 mL) was added 1 N HCl (1.5 mL). The reaction was
stirred at room temperature for 18 h. The methanolic phase was
washed with hexane (3 ꢀ 10 mL), and the hexane layer was
extracted with methanol (3ꢀ15 mL). The combined methanolic
layers were evaporated in vacuo, and the residue was dis-
solved in CH2Cl2 (10 mL). The solution was washed with 5%
NaHCO3 (10 mL), and the organic layer was dried over anhy-
drous Na2SO4, evaporated, and purified with chromatography
(CH3OH:CHCl3 = 1:20) to obtain 123 mg (56.4% yield) of a
1
white foam solid. HPLC indicates a purity of 99.4 area %. H
NMR (CDCl3, 500 MHz) δ 0.69-0.80 (-CH3, m, 3H), 1.11-
1.15 (-CH, m, 1H), 1.23-1.25 (-CH2, m, 2H), 1.79 (-OH, br,
2H), 3.13-3.15 (-CH, m, 1H), 3.19-3.28 (-CH2, m, 2H),
5.20 (-CH, q, J=7.9 Hz, 1H), 7.22-7.29 (-Ph, m, 5H), 7.38
(-CONH, br, 1H), 8.29 (-CONH, d, J = 8.6 Hz, 1H), 8.50
(-Pyz, m, 1H), 8.73 (-Pyz, d, J=2.4 Hz, 1H), 9.19 (-Pyz, d,
J=1.3 Hz, 1H). 13C NMR (CDCl3, 125 MHz) δ 22.16, 22.98,
25.35, 38.12, 39.66, 53.08, 127.11, 128.79, 129.45, 135.86, 142.72,
143.83, 144.36, 147.56, 162.90. MS (ESI) m/z 407.2 [M þ Na]þ.
HRMS [M þ Na þ 2CH2]þ calcd, 435.2189; found, 435.2147.
[(1S)-1-[[(2S)-3-Phenyl-2-[(pyrazin-2-carbonyl)amino]-1-ox-
opropyl]amino]-3-methylbutyl]boronic Acid (51). HPLC indi-
1
cates a purity of 97.5 area %. H NMR (CD3OD, 500 MHz)

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4199
was cultured in RPMI 1640 supplemented with 15% fetal bovine
serum at 37 °C in 5% CO2. A549 cell was cultured in F12-k
supplemented with 10% fetal bovine serum at 37 °C in 5% CO2.
SKOV-3 cell was cultured in McCoy’s 5A supplemented with
10% fetal bovine serum at 37 °C in 5% CO2. HELA cell was
cultured in EMEM supplemented with 10% fetal bovine serum at
37 °C in 5% CO2.
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Acknowledgment. We thank Prof. Luhua Lai, College of
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correct version was posted on June 24, 2009.
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Supporting Information Available: General and experimental
procedures, characterization data for 3a-q, 5a, 5b, 8a, 8b, 9a-f,
11a-f, and 14-50. This material is available free of charge via
the Internet at http://pubs.acs.org.
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