Chemical and biological evaluation of dipeptidyl boronic acid proteasome inhibitors for use in prodrugs and pro-soft drugs targeting solid tumors

Article abstract of DOI:10.1021/jm200460q

Bortezomib, a dipeptidyl boronic acid and potent inhibitor of the 26S proteasome, is remarkably effective against multiple myeloma (MM) but not against solid tumors. Dose-limiting adverse effects from "on target" inhibition of the proteasome in normal cells and tissues appear to be a key obstacle. Achieving efficacy against solid tumors therefore is likely to require making the inhibitor more selective for tumor tissue over normal tissues. The simplest strategy that might provide such tissue specificity would be to employ a tumor specific protease to release an inhibitor from a larger, noninhibitory structure. However, such release would necessarily generate an inhibitor with a free N-terminal amino group, raising a key question: Can short peptide boronic acids with N-terminal amino groups have the requisite properties to serve as warheads in prodrugs? Here we show that dipeptides of boroLeu, the smallest plausible candidates for the task, can indeed be sufficiently potent, cell-penetrating, cytotoxic, and stable to degradation by cellular peptidases to serve in this capacity.

Full text of DOI:10.1021/jm200460q

ARTICLE
pubs.acs.org/jmc
Chemical and Biological Evaluation of Dipeptidyl Boronic Acid
Proteasome Inhibitors for Use in Prodrugs and Pro-Soft Drugs
Targeting Solid Tumors
Lawrence J. Milo, Jr., Jack H. Lai, Wengen Wu, Yuxin Liu, Hlaing Maw, Youhua Li, Zhiping Jin, Ying Shu,
Sarah E. Poplawski, Yong Wu, David G. Sanford, James L. Sudmeier, and William W. Bachovchin*
Department of Biochemistry, Tufts University Sackler School of Graduate Biomedical Sciences, 136 Harrison Avenue, Boston,
Massachusetts 02111, United States
S Supporting Information
b
ABSTRACT: Bortezomib, a dipeptidyl boronic acid and potent
inhibitor of the 26S proteasome, is remarkably effective against multi-
ple myeloma (MM) but not against solid tumors. Dose-limiting
adverse effects from “on target” inhibition of the proteasome in normal
cells and tissues appear to be a key obstacle. Achieving efficacy against
solid tumors therefore is likely to require making the inhibitor more
selective for tumor tissue over normal tissues. The simplest strategy
that might provide such tissue specificity would be to employ a tumor
specific protease to release an inhibitor from a larger, noninhibitory
structure. However, such release would necessarily generate an in-
hibitor with a free N-terminal amino group, raising a key question: Can
short peptide boronic acids with N-terminal amino groups have the requisite properties to serve as warheads in prodrugs? Here we
show that dipeptides of boroLeu, the smallest plausible candidates for the task, can indeed be sufficiently potent, cell-penetrating,
cytotoxic, and stable to degradation by cellular peptidases to serve in this capacity.
’ INTRODUCTION
with the goal of lessening the PN in MM patients but also of
achieving efficacy against solid tumors. Several second generation
inhibitors have reached the clinic and are shown in Figure 1.1,4,21
Compound 3 (CEP-18770, Figure 1), like bortezomib, is a boronic
acid, and it has an inhibitory profile against the three proteasome
active sites very similar to that of bortezomib.20 The epoxy ketone
inhibitor 4 (carfilzomib, Figure 1), structurally related to the natural
product epoxomicin, is a potent, irreversible inhibitor that exhibits
specificity for β5 over β2 and β1 and a reduced tendency to inhibit
other nonproteasomal proteases.20,22ꢀ24 Proteasome inhibitor 5
(NPI-0052 or salinosporamide A, Figure 1) is a β-lactone natural
product isolated from the marine bacterium Salinispora tropica. Like
4, it is an irreversible proteasome inhibitor. It differs from 4 and
bortezomib in that it potently inhibits all three sites.20,21,25ꢀ27 Second
generation inhibitor 6 (MLN9708), the most recent entry into
clinical trials, is another boronic acid and again has IC50 values very
similar to those of bortezomib for all three sites. It differs from
bortezomib in that it has a faster off-rate from the proteasome. It is
orally available and rapidly hydrolyzes to yield 7 (MLN2238).20,22,28
The faster off-rate of 6 has been proposed to result in better
antitumor activity by permitting greater distribution of the drug into
the tissues and thereby into the tumor. Higher levels of drug and
proteasome inhibition (74.9% for 6 vs 44.8% for bortezomib) were
Bortezomib (1a, Figure 1) is the dipeptidyl boronic acid Phe-
boroLeu acylated at the N-terminus with a pyrazinoyl group and can
be written as Pyz-Phe-boroLeu. It is a potent, reversible inhibitor of
the proteolytic activity of the 26S proteasome, a multisubunit, mul-
ticatalytic complex with chymotrypsin-like (β5), trypsin-like (β2),
and caspase-like (β1) enzymatic sites, so named for the preferred
cleavages they mediate.1ꢀ4 A functional proteasome is essential for
intracellular protein homeostasis and cellular regulation.5ꢀ10 Borte-
zomib gained FDA approval for the treatment of patients with
multiple myeloma (MM) in 2003 and has since transformed their
care. In 2006, it was approved for patients with relapsed mantle cell
lymphoma (MCL).11ꢀ15 The remarkable efficacy observed for this
first-in-class agent against these hematological malignancies validated
the proteasome as an anticancer target and stimulated efforts to use
bortezomib to treat other types of cancers, especially solid tumors
such as prostate, breast, and lung. While there have been some hints
of activity against solid tumors, the hoped-for efficacy has not been
realized.16ꢀ19 Dose-limiting toxicities seem likely to be the major
obstacle to success. Even in MM and MCL, where bortezomib is
efficacious, adverse effects are a major problem. Peripheral neuro-
pathy (PN) is perhaps the most important, as it often limits the dose
and therefore the efficacy, and is the principal reason patients elect to
stop treatment.20
Multiple groups have been searching for second-generation
proteasome inhibitors with better efficacy and safety profiles,
Received: November 23, 2010
Published: June 02, 2011
r
2011 American Chemical Society
4365
dx.doi.org/10.1021/jm200460q J. Med. Chem. 2011, 54, 4365–4377
|

Journal of Medicinal Chemistry
ARTICLE
Figure 1. Structural comparison of dipeptidyl boronic acids, bortezomib (1a), and the second-generation proteasome inhibitors CEP-18770 (3),
carfilzomib (4), salinosporamide A (5), MLN9708 (6), and MLN2238 (7).
observed in mouse tumor tissue relative to blood cells, supporting
this idea.20,29
other enzymes. A tumor-activated prodrug could provide such tissue
specificity. The simplest approach would be to design a longer
boroLeu-based peptide that is itself not a protease inhibitor but is
instead a substrate for a tumor-specific protease whose action
releases a shorter peptide of boroLeu, which is a proteasome
inhibitor (Figure 2). Proteases are particularly well suited for the
“activation” role, and several “tumor specific” proteases have been
identified. These include, for example, fibroblast activation protein
(FAP), prostate specific antigen (PSA), and prostate specific
membrane antigen (PSMA).38ꢀ46
Any peptidyl boronic acid released from such constructs would
ipso facto have a free N-terminal amino group. This raises some
concerns as the free amino group might be expected to limit
potency, cell permeability, or stability, particularly as it might render
the released inhibitors susceptible to aminopeptidase degradation.
Another consideration is that it would be advantageous for the
released inhibitor to be as small as possible to allow for the
incorporation of tumor specificity into the larger peptide without
it becoming too large and cumbersome, but smallness could also
limit inhibitory potency. Dipeptidyl boronic acids are perhaps the
smallest entities that have the potential to serve in this capacity, but
they present an additional complication. On the basis of previous
findings with dipeptidyl boroPro, they are expected to undergo a
pH-dependent cyclization that could significantly attenuate their
inhibitor potency at physiological pH. But this phenomenon could
also prove useful when used as the warhead of a prodrug. The
dipeptidyl boronic acid inhibitor would be released at the tumor site
in the open-chain, enzyme inhibitor form. Cyclization subsequent to
release would then help to attenuate systemic effects as excess
inhibitor diffuses from the tumor site. Such defined and pro-
grammed loss of pharmacological activity with time is characteristic
of the action of agents termed “soft drugs”. Soft drugs need to be
applied directly to the intended site of action.47ꢀ50 Prodrugs of soft
drugs have previously been termed “pro-soft” drugs, although there
are very few working examples and little exploration of their
The above second-generation compounds are, like bortezomib,
relatively potent inhibitors of the proteasome with IC50 values in the
low nanomolar range. There are differences between them and
bortezomib, mostly in the inhibitory profiles against the three
proteasome active sites or in specificity for the proteasome over
other proteases, that could lead to improvements in safety or
efficacy. Thus far, however, the improvements appear to be incre-
mental. The one that seems to show the most promise against solid
tumors, 6, is also the only rapidly reversible inhibitor, suggesting that
potency and specificity for the proteasome may be less important in
achieving efficacy against solid tumors than other factors such as
distribution to the target tissues.20,28
Proteasome inhibition results in the buildup of misfolded and
damaged intracellular proteins, leading to the disruption of multiple
cellular signaling pathways and ultimately cell death.30ꢀ34 Multiple
myeloma cells seem to be especially sensitive to proteasome inhi-
bition, quite likely because of their overproduction of protein and
high protein turnover, resulting in greater need for proteasomal
degradation and clearance.3,11,12,14,15,19,35,36 Nevertheless, all cells,
both normal and malignant, require a functioning proteasome.
Different types of cells exhibit differences in sensitivity to protea-
some inhibition, but proteasome inhibition will, at some level and
duration, be lethal to essentially any cell. The adverse effects
observed for bortezomib and other proteasome inhibitors are quite
likely to arise from “on-target” inhibition in “off-target tissues” rather
than off-target inhibition of nonproteasomal enzymes. Neuropathic
pain, for example, which occurs to some degree with each of the
above inhibitors, has been linked to the accumulation of ubiquiti-
nated proteins in cells of the peripheral nervous system.37
Therefore, to achieve efficacy against solid tumors with a
proteasome inhibitor may require improving inhibitor selectivity for
tumor tissue over normal tissues rather than improving inhibitor
potency for the proteasome or specificity for the proteasome over
4366
dx.doi.org/10.1021/jm200460q |J. Med. Chem. 2011, 54, 4365–4377

Journal of Medicinal Chemistry
ARTICLE
Figure 2. Dipeptidyl boroLeu can be attached to the C-terminus of tumor-specific protease recognition sequences (TSPRS) and thus activated by
tumor tissue-specific proteases to release the active inhibitor within tumor and tumor-associated tissues. The active “warhead” is then available to inhibit
a target protease like the proteasome in this tissue. This dipeptidyl boronic acid is inactivated through cyclization, a reaction that is ideally favored upon
diffusion away from the intended target tissue.
potential.51ꢀ53 However, we have recently reported on the design
and characterization of a pro-soft derivative of Val-boroPro.55
Here we evaluate a series of dipeptidyl boronic acids with
respect to proteasome inhibitory potency, cell permeability,
cytotoxicity, susceptibility to degradation by cellular lysates,
which would include aminopeptidase and other cellular pepti-
dases, and intramolecular cyclization. The results show that
although dipeptidyl boronic acids vary widely with respect to
these factors, within this class there are molecules that would be
suitable for use in constructing proteasome inhibitor-based
prodrugs for the selective targeting and treatment of solid
tumors.
Figure 3. Cyclization reaction of dipeptide boronic acids.
’ RESULTS AND DISCUSSION
Proteasome Inhibitory Potency in Vitro and pH-Depen-
dent Cyclization. Previous work has shown that dipeptidyl
boronic acids containing boroPro or boroAla exist in a pH-
dependent equilibrium (Figure 3) between an open-chain, enzyme
inhibitory form (A) and a cyclic, inactive form (D). The open chain
structure is favored at low pH, but as the pH is increased and the
N-terminal ammonium ion deprotonates, the cyclic structure
becomes increasingly favored.55ꢀ61 For dipeptides of boroPro the
cyclic structures are strongly favored over the open chain structures
at physiological pH, by factors ranging from ∼100- to 2000-fold,
depending on the P2 residue.57,61 For dipeptides of boroAla,
Figure 4. In vitro proteasome inhibition by 2c is dependent on the
the cyclic structures are not as strongly favored. For example,
Ala-boroAla has a ratio of cyclic to open chain of ∼5.0 at pH 7.4
and typically varies from ∼2 to 20.56 One effect of the cyclization
reaction is to attenuate the potency of these inhibitors at physiolo-
gical pH as equilibrium is approached.57,61
Determining the proteasome inhibitor potency of a boroLeu
dipeptide therefore also requires ascertaining its cyclization
behavior, as cyclization can obscure intrinsic potency. Cycliza-
tion is a relatively slow process and can be conveniently measured
by NMR or by comparing the inhibitory potency following
preassay equilibration of the inhibitor for 24 h at pH 2.0 versus
pH 7.6, using the pH 2.0 activity as representing that of the fully
open chain species. The sensitivity and dynamic range of NMR
limit its usefulness to measurement of cyclization indexes (CIs)
pH of preassay incubation. The potency of the compound as an inhi-
bitor of the proteasome is reduced when incubated at pH 7.6 for 24 h
prior to assay.
that are <100, whereas the pH dependent enzyme assay can
determine CIs that are much larger.
Phe-boroLeu, 2c, differs from bortezomib only in that it lacks
the N-terminal pyrazinoyl group. It has a free N-terminal amino
group and therefore exhibits the expected pH-dependent inhibi-
tion (Figure 4). NMR spectroscopy was used to confirm that the
pH-dependent effect is indeed due to cyclization and that it is
fully reversible (Figures S1ꢀS5). The CI for Phe-boroLeu is
∼3.7, far less than that of boroPro dipeptides but on par with
those for boroAla dipeptides.56
4367
dx.doi.org/10.1021/jm200460q |J. Med. Chem. 2011, 54, 4365–4377

Journal of Medicinal Chemistry
ARTICLE
Table 1. Proteasome Inhibitor Potency, Cyclization Index, Cell Permeability, and Cytotoxity for Dipeptidyl boroLeu Families 1 and 2
in vitro IC50 (nM)
compd Xaa, P2 amino acid
pH 2.0
pH 7.6
CI
IC-IC50 (nM) IC-IC50/IC50
CyT50 (nM)
CyT50/IC50 CyT50/IC-IC50
P2 Amino Acids with N-Terminal Acylation
1a
1b
1c
1d
1e
Phe (bortezomib)
10 ( 1
14 ( 2
1.4 ( 0.2
2.1 ( 0.4
1.1 ( 0.2
0.8 ( 0.1
1.6 ( 0.3
27 ( 12
2.7 ( 1.2
65 ( 14
150 ( 25
15 ( 4
4 ( 3
0.4 ( 0.3
8 ( 5
0.16 ( 0.13
0.12 ( 0.07
0.08 ( 0.01
Thr
12 ( 2
16 ( 1
34 ( 2
110 ( 10
25 ( 3
18 ( 2
28 ( 2
180 ( 30
790 ( 140
2500 ( 300
520 ( 130
1800 ( 500
100 ( 50
210 ( 20
Ser
13 ( 2
β-Me-Thr
Gly
16 ( 5
Natural P2 with Free N-Termini
2a
2b
2c
2d
2e
2f
Gly
Trp
Phe
Tyr
Ala
21 ( 2
29 ( 3
37 ( 5
42 ( 4
50 ( 7
63 ( 7
66 ( 3
80 ( 9
190 ( 20
430 ( 60
100 ( 10
140 ( 20
120 ( 10
360 ( 30
770 ( 40
170 ( 20
450 ( 30
1100 ( 200
20 ( 4
>10K
>1000
>10K
>10K
>10K
>10K
>10K
>10K
>10K
>1000
>470
>30
670 ( 280
400 ( 40
90 ( 80
30 ( 10
14 ( 2
2.4 ( 2.3
4 ( 1
<0.07
<0.4
3.6 ( 0.6
3.7 ( 0.8
2.9 ( 0.4
7 ( 1
>270
>240
>200
>160
>150
>120
>50
<0.01
<0.02
<0.3
160 ( 50
3200 ( 2800
64 ( 57
Asn
Leu
Thr
Ser
12 ( 2
2g
2h
2i
2.6 ( 0.3
5.6 ( 0.7
6 ( 2
2500 ( 950
30 ( 10
<0.3
<0.2
1700 ( 1500
8.9 ( 8.0
2j
His
510 ( 130 5200 ( 1000 10 ( 3
>20
Non-Natural P2 with Free N-Termini
2k
2l
1-Nala
2-Nala
3,5-Df-Phea
Bpaa
26 ( 3
120 ( 10
220 ( 20
100 ( 10
400 ( 50
130 ( 20
300 ( 90
140 ( 10
150 ( 10
110 ( 10
150 ( 10
150 ( 10
300 ( 30
350 ( 50
540 ( 80
470 ( 60
900 ( 100
4.4 ( 0.7
7 ( 2
150 ( 30
450 ( 70
790 ( 150
1400 ( 100
>10K
6 ( 1
13 ( 4
18 ( 4
30 ( 5
>170
11 ( 1
47 ( 3
0.4 ( 0.1
1.0 ( 0.2
0.07 ( 0.01
0.06 ( 0.01
33 ( 8
2m
2n
2o
2p
2q
2r
43 ( 5
2.3 ( 0.5
7.9 ( 1.4
2.3 ( 0.5
4 ( 1
50 ( 6
4-F-Phe
Nle
60 ( 6
79 ( 9
1.3 ( 0.2
13 ( 2
<0.01
<0.1
75 ( 8
>10K
>130
950 ( 70
2-F-Phe
3,4-Df-Phea
3-F-Phe
β-Me-Thr
t-Bu-Gly
Et-Gly
Dpaa
76 ( 8
1.8 ( 0.2
1.9 ( 0.2
1.3 ( 0.1
1.7 ( 0.2
1.6 ( 0.2
2.8 ( 0.4
3.2 ( 0.5
5 ( 1
3100 ( 500
2100 ( 300
1300 ( 100
>10K
41 ( 8
26 ( 4
16 ( 2
>110
81 ( 3
2s
82 ( 6
2t
90 ( 10
100 ( 10
110 ( 10
110 ( 10
110 ( 10
170 ( 40
240 ( 30
2u
2v
2w
2x
2y
2z
2aa
>10K
>110
710 ( 50
7 ( 1
<0.07
<0.2
>10K
>90
1500 ( 300
14 ( 4
1000 ( 100
>10K
9 ( 2
>90
Nva
Pf-Phea
1100 ( 600
10 ( 6
<0.1
2.8 ( 0.7
3.7 ( 0.7
5 ( 1
3300 ( 900
>10K
20 ( 7
>40
Chg
Gla
600 ( 100 3000 ( 200
>10K
>20
Natural P2 with N-Terminal Alkylation
2ab
2ac
N,N-diMeGly
36 ( 2
39 ( 2
610 ( 40
17 ( 1
56 ( 7
>10K
>10K
>280
>260
Sar
2200 ( 200
160 ( 140
4.1 ( 3.6
<0.07
a Nal = naphthylalanyl; Bpa = biphenylalanyl; Df-Phe = difluorophenylalanyl; Dpa = diphenylalanyl; Pf-Phe = (pentafluorophenyl)alanyl; Nva =
norvalinyl; Nle = norleucyl; Chg = cyclohexylglycyl; Gla = γ-carboxyglutamyl; Sar = sarcosyl.
A series of boroLeu dipeptides were similarly examined, and
their IC50 values following pre-equilibration at pH 2.0 and pH
7.6 are listed in Table 1 together with their corresponding cycliza-
tion indices. The compounds are listed in order of decreasing
potency with respect to the pH 2.0 IC50 values because this value,
always the smaller of the two, corresponds more closely to the
intrinsic potency of the compound. Compounds 2aꢀj are dipeptides
of boroLeu with naturally occurring amino acids at P2, while
compounds 2k through 2aa have non-naturally occurring amino
acids at this position. The pH 2.0 IC50 values range from 21 to 620
4368
dx.doi.org/10.1021/jm200460q |J. Med. Chem. 2011, 54, 4365–4377

Journal of Medicinal Chemistry
ARTICLE
nM or from about 2- to 62-fold less potent than bortezomib. The pH
7.6 IC50 values are significantly higher, ranging from 100 to 2900 nM
or from 10- to 290-fold less potent than bortezomib. The pH 7.6
values would be the more relevant set for use of the compounds as
conventional proteasome inhibitors. But for use as warheads in
proteasome inhibitor prodrugs, the pH 2.0 values are more relevant,
as the open-chain form would be released from the prodrug at the
target site. The CI values range from 1.3 (2s) to20(2a). ACIof1.3is
too small to be of interest or concern. A factor of 20, however, is large
enough to provide a useful soft drug effect. Interestingly, Gly-boroLeu
(2a) has both the smallest IC50 and the largest CI of the boroLeu-
based dipeptides having a free amino group.
Several dipeptides of boroLeu substituted on the N-terminus
were also examined. Several of these, 1bꢀe, are bortezomib-like
in that they are pyrazinoylated on the N-terminus, and two, 2ab
and 2ac, are alkylated. The pyrazinoylated compounds, as
expected, do not cyclize, but the alkylated compounds do cyclize.
In fact, with a CI of 56, 2ac has the greatest CI of any of the
boroLeu-based dipeptides examined. It is also a reasonably
potent proteasome inhibitor with an IC50 of 39 nM, or only
4-fold less potent than bortezomib. The reason 1aꢀe do not
cyclize while 2ab and 2ac do is that the electron lone pair of the
N-terminal nitrogen in the acylated dipeptides is delocalized in
the amide π system and thus unavailable for nucleophilc attack
on the boron atom, whereas in the alkylated dipeptides it is in a σ
system and available for donation to the boron to form the
boronꢀnitrogen dative bond of the cyclic structure.
cell-penetrating power than those with hydrophilic side chains
appears to apply to the other compounds in Table 1.
Dipeptides of boroLeu with free N-terminal amino groups
have significantly less cell-penetrating ability than their N-term-
inally blocked counterparts, as might be expected. This is
illustrated when one compares 1a, 1b, 1d, 1e with 2c, 2h, 2t,
2a, respectively. But there are exceptions: 2i appears to be 3-fold
more cell-penetrating than 1c. While none of the dipeptides of
boroLeu are as cell penetrating as bortezomib, some are cell
penetrating enough for use as warheads in prodrugs. For
example, six (2k, 2l, 2m, 2s, 2w, 2y) have IC-IC50/IC50 values
of 20 or less, and one (2k) with a value of ∼6.0 is almost within
2-fold of bortezomib’s value of ∼2.7.
Cytotoxicity. Cytotoxicity was measured using the standard
MTT cytotoxicity assay64ꢀ66 on both HEK 293 cells and
follicular small-cell lymphocytic leukemia (FSCLL) cells. HEK
293 PS cells seem to be quite resistant to proteasome inhibition-
mediated cyctotoxicity, showing a CyT50 value of 347 nM for 1a
and values greater than 10 μM for the other compounds in
Table 1 (data not shown). However, all of the tested compounds
exhibit measurable cytotoxicity against FSCLL cells (Table 1).
Perhaps not surprisingly, bortezomib (1a) is the most cytotoxic
of the compounds tested, with a CyT50 of 4 nM. However, Phe-
boroLeu (2c) was only about 20-fold less cytotoxic with a CyT50
value of 91 nM. A CyT50 of 91 nM is most likely sufficiently
cytotoxic that Phe-boroLeu itself could be considered for use as
the warhead in a prodrug. There are, however, several dipeptidyl
boronic acids that are significantly more cytotoxic and therefore
perhaps more promising than 2c. These include 2k and 2m with
CyT50 values of 11 and 47 nM, respectively.
The relationship between cytotoxicity and proteasome inhi-
bitory potency in vitro and in cells are listed in Table 1 as the
ratios CyT50/IC50 and CyT50/IC-IC50, respectively. The
CyT50/IC-IC50 ratio is of special interest because it relates
cytotoxicity to inhibition of the proteasome achieved within
the cell. A value of 1.0 means by definition that the CyT50 and IC-
IC50 are equal. Values greater than 1.0 would suggest that more
than 50% inhibition is needed to kill 50% of the cells while values
less than 1.0 would indicate the reverse. The ratio for bortezomib
(0.16) suggests that less than 50% inhibition, perhaps around
20%, is all that is required to kill 50% of the cells. This is not too
surprising, especially as it is not entirely out of line with in vivo
PK/PD studies.20,29,67ꢀ71
However, the ratio for some of the dipeptidyl boronic acid
inhibitors is even less, sometimes dramatically so, as for example
for 2c, 2o, and 2m, and these values do seem unreasonable; i.e., it
is not likely that less than ∼5% inhibition of the proteasome
could be sufficient to trigger cell death, even in cell culture where
the inhibition is maintained over the 24 h period. What then is
the explanation for these results? One possibility is that there is a
different mechanism triggering cell death. Another is that IC-
IC50 values are significantly under-reporting the actual degree of
proteasome inhibition achieved in the cell for reasons that are not
entirely clear. One way in which this could happen is if the
inhibitors were degraded over time such that at 24 h, when the
proteasome activity is measured, there is little of the inhibitor left,
yet sufficient inhibition was attained over the first few hours to
trigger cell death. We have ruled this out by showing that the
inhibitors show no sign of decomposition after 24 h of incubation
in the cell cultures.
Cell Permeability. A number of cancers express a tumor-
specific protease on their cell surface or induce its expression on
the surface of the surrounding stromal cells (e.g., FAP).38ꢀ46 If
one of these proteases is used as the activating enzyme, the
released proteasome inhibitor would need to have cell-penetrat-
ing ability to be effective. The cell penetrating ability of the
compounds listed in Table 1 was therefore assessed. This was
done by determining their ability to inhibit the proteasome inside
cells in culture using the proteasome sensor cell line (Clontech),
which is an HEK 293 cell line stably transfected with a protea-
some-sensitive fluorescent reporter. The reporter is a fusion
protein of a green fluorescence protein with a degradation
domain (ornithine decarboxylase), which targets the protein
for degradation by the proteasome.62,63
Normally, the levels of fusion protein are low, owing to rapid
degradation by the proteasome within the cell. However, when
there is a decrease in proteasome activity, the fusion protein
concentration increases and the cell emits green fluorescence.
The amount of fluorescence is dependent upon the degree to
which the proteasome has been inhibited within the cell, allowing
a determination of an intracellular IC50 value (IC-IC50) as listed
in Table 1.62,63 The ratio IC-IC50/IC50 is a measure of cell
permeability. A value of 1.0 would correspond, in essence, to
unrestricted access or “100% permeability”. The larger the ratio,
the lower is the cell-penetrating ability of the inhibitor.
The IC-IC50/IC50 for bortezomib, 1a, is ∼2.7 indicating that
almost 3-fold higher concentrations of bortezomib are needed in
the extracellular medium to inhibit the proteasome within cells
than would be needed if it were not sequestered within the cell.
Nevertheless, this value indicates that bortezomib has signifi-
cantly more cell-penetrating ability than the other “bortezomib-
like” inhibitors (1bꢀe), which is perhaps not surprising because
it has the aromatic and hydrophobic Phe in P2, while 1bꢀe have
more hydrophilic side chains in this position. The notion that
compounds with polar or charged P2 side chains should have less
The unreasonable values of CyT50/IC-IC50 appear to occur
for inhibitors where no in-cell proteasome inhibition was
4369
dx.doi.org/10.1021/jm200460q |J. Med. Chem. 2011, 54, 4365–4377

Journal of Medicinal Chemistry
ARTICLE
Figure 5. Correlation between cytotoxicity (CyT50) and intracellular proteasome inhibitory potency as measured by the IC-IC50.
detected and only cytotoxicity values could be measured. For
those inhibitors where a clear IC-IC50 is observed, a reasonably
good correlation exists between these values and the correspond-
ing cytotoxicities (Figure 5). This strongly supports the notion
that proteasome inhibition is the mechanism triggering cytotoxi-
city for most, if not all, of the compounds examined. Note that
even for these more effective inhibitors, the IC-IC50 values are
nevertheless still about 5ꢀ10 times larger than the CyT50. We
cannot therefore be sure whether fractional inhibition of the
proteasome, about 10ꢀ30%, is sufficient to cause cell death or
whether the assay is under-reporting the inhibition actually being
achieved within the cell, at least with respect to one or more
biologically relevant substrates.
Susceptibility to Aminopeptidase Degradation. A free
N-terminal amino group might be expected to render the
dipeptide of boroLeu susceptible to degradation by aminopepti-
dases. Such susceptibility could significantly affect the suitability
of dipeptides of boroLeu as warheads in the design and con-
struction of tumor-specific proteasome inhibitor prodrug. It
could also affect some of the parameters determined here such
as cytotoxicity, intracellular inhibitory potency, and cell perme-
ability and perhaps account for some of the discrepancy between
CyT50 and IC-IC50.
To determine if degradation by aminopeptidases or other
cellular enzymes is a factor, 2a, 2c, and 2e were incubated with
intact cells (either HEK 293 or FSCLL cells) and with cell lysates
for 24 h at 10ꢀ4 M. At various time points, aliquots were removed
and analyzed by LCMS. Peaks corresponding to the parent
compound and degradation products were identified and their
areas measured. No evidence of aminopeptidase degradation, or
other degradation products, was detected for any of the dipep-
tidyl boronic acids (Figure S6a and Figure S6b). A tripeptidyl
boronic acid, Leu-Phe-boroLeu, was also examined. In contrast
with the dipeptidyl boronic acids, it was rapidly degraded by
both HEK cell lysate and FSCLL cell lysate, as determined
by the disappearance of the parent compound and correspond-
ing appearance of the products of aminopeptidase degrada-
tion (Figure S6c). Thus, dipeptidyl boronic acids appear to be
much more stable to aminopeptidases than tripeptidyl boronic
acids, perhaps owing to the non-natural amino acid moiety,
boroLeu acid, in P10.
’ CONCLUSIONS
Despite being relatively small and having a free amino group,
dipeptides of boroLeu can be sufficiently potent, cell penetrating,
cytotoxic, and resistant to degradation by cellular proteases to serve
as warheads in the construction of prodrugs designed to selectively
target the proteasome in solid tumors. The candidate that best
fulfills all of the desired properties is perhaps 2k, but 2c, 2m, 2o, and
2ac might also be suitable. The dipeptides of boroLeu can also
undergo a pH-dependent cyclization, which, although relatively
modest compared to that exhibited by dipeptides of boroPro, might
in some cases be sufficient to provide additional tissue specificity by
limiting systemic exposure to the active proteasome inhibitor
following activation at the tumor. This phenomenom could make
2a one of the better candidates, as its true cytotoxicity could be 20-
fold better than the values listed in Table 1 for a short time following
release from a prodrug at the tumor.
’ EXPERIMENTAL SECTION
Synthesis of Dipeptidyl Boronic Acids. The (þ)-pinanediol
ester of leucineboronic acid starting material was synthesized using the
methods described in the literature.72ꢀ74 Dipeptidyl boronic acids were
synthesized using standard methods, a brief description of which is given
below.72ꢀ80 Typical reaction scales were 2.0 mmol.
Synthesis of N-Pyrazinoylated Amino Acids. A solution of 1.0 equiv
of 2-pyrazinoic acid and 1.3 equiv of the desired P2 amino acid
C-terminal methyl ester containing side chain protecting groups as
necessary (see Scheme 1) in 10 mL of DMF under argon atmosphere
was cooled on an ice bath to 0 °C and allowed to stir for 10 min. To the
cooled mixture, an amount of 1.3 equiv each of HOBt, EDC, and DIPEA
was added. The mixture was stirred for 15 min, after which it was
warmed to room temperature and stirred overnight. The mixture was
diluted with 200 mL of an aqueous solution of either 10% (w/v) citric
acid or 0.1 M KHSO4 and extracted three times with 100 mL of ethyl
acetate. The organic layers were pooled, dried over MgSO4, filtered and
solvent removed under vacuum. The resulting clear oil was purified by
4370
dx.doi.org/10.1021/jm200460q |J. Med. Chem. 2011, 54, 4365–4377

Journal of Medicinal Chemistry
ARTICLE
Scheme 1. Synthesis of Dipeptidyl Boronic Acids and N-Pyrazinoylated Dipeptidyl Boronic Acids
flash chromatography using a mixture of hexanes and ethyl acetate as the
mobile phase. The purified pyrazinoylated aminoacyl ester was dissolved
in a mixture of 5 mL of THF and 5 mL of water and subsequently cooled
on an ice bath to 0 °C. To the stirring solution, an amount of 2.0 equiv of
LiOH was added. The mixture was allowed to stir at 0 °C for 3 h, after
which it was neutralized with 200 mL of KHSO4 and extracted three
times with 100 mL of ethyl acetate. The aqueous phase was collected and
lyophilized to yield the pyrazinoylated amino acid as a pale yellow
powder.
acid (þ)-pinanediol ester hydrochloride (boroLeu-pn, bLeu-pn) was added.
The mixture was cooled on an ice bath to 0 °C under argon atmosphere and
stirred for 10 min. To the cooled mixture was added 2.2 equiv of DIPEA
followed by 1.2 equiv of HATU. The mixture was stirred for 15 min, after
which it was warmed to room temperature and stirred for an additional 3 h.
The mixture was diluted with 200 mL of an aqueous solution of either 10%
(w/v) citric acid or 0.1 M KHSO4 and extracted three times with 100 mL of
ethyl acetate. The organic layers were pooled, and solvent was removed
under vacuum. The resulting pyrazinoylated, N-terminally Boc-protected or
N-terminally Cbz-protected dipeptidyl boronic acid (þ)-pinanediol esters as
clear to pale yellow oils were purified by flash chromatography using a
mixture of hexanes and ethyl acetate as the mobile phase.
Coupling to Leucineboronic Acid (þ)-Pinanediol Ester. Pyrazinoy-
lated, N-terminally Boc-protected, or N-terminally Cbz-protected amino
acid was then dissolved in 10 mL of DMF, and 1.0 equiv of leucineboronic
4371
dx.doi.org/10.1021/jm200460q |J. Med. Chem. 2011, 54, 4365–4377

Journal of Medicinal Chemistry
ARTICLE
Synthesis of Pyrazinoylated Dipeptidyl Boronic Acid (þ)-Pinanediol
Esters. This was accomplised by one of two synthetic routes. In the first
synthetic route, pyrazinoylated P2 amino acids are coupled to boroLeu-pn
as described in the above section. The second method is briefly described
here. The N-Boc-protected dipeptidyl boronic acid (þ)-pinanediol ester
was dissolved in 10 mL of a solution of 4 M HCl in THF. The mixture was
allowed to stir for 3 h at room temperature, after which the resulting white
precipitate was collected by vacuum filtration and washed 3ꢁ with cold
THF, resulting in the deprotected dipeptidyl boronic acid (þ)-pinanediol
ester. The precipitate was dried under vacuum and redissolved in 10 mL of
DMF. To the solution, an amount of 1.2 equiv of 2-pyrazinoic acid was
added. The mixture was cooled on an ice bath to 0 °C, placed under an
argon atmosphere. The mixture was allowed to stir for 10 min, after which
1.2 equiv of HATU followed by 2.2 equiv of DIPEA were added. The
mixture was allowed to stir at 0 °C for an additional 15 min before allowing it
to warm to room temperature, after which the mixture was allowed to stir for
3 h. The mixture was diluted with 200 mL of an aqueous solution of either
10% (w/v) citric acid or 0.1 M KHSO4 and extracted three times with
100 mL of ethyl acetate. The organic layers were pooled, dried over MgSO4,
filtered, and solvent was removed under vacuum. The resulting pyrazinoy-
lated dipeptidyl boronic acid (þ)-pinanediol ester as a clear to pale yellow
oil was purified by flash chromatography using a mixture of hexanes and
ethyl acetate as the mobile phase.
The final dipeptidyl boronic acids and pyrazinoylated dipeptidyl
boronic acids were purified using a Varian ProStar 210 HPLC, equipped
with a C18 reverse-phase column (Supplico Analytical Discovery C18
25 cm ꢁ 21.2 mm, 5 μm particle size, catalog no. 569226) and UV/vis
detector, using a linear gradient of increasing acetonitrile with a flow rate
of 20 mL/min. Product yields were typically between 45% and 65% after
purification. All compounds were demonstrated to have a purity of >95%
using an Agilent (formerly HP) 1100 series LC/MSD equipped with an
orthogonal electrospray mass spectometer (G2455A), autosampler/
autoinjector (G1367A WPALS), thermostated column compartment
(G1316A ColCom), sample chiller (G1330A ALSTherm), binary pump
(G1312A BinPump), degasser (G1322A), and UV/vis detector
(G1315B DAD). Samples were run over a C18 reverse-phase column
(Agilent Eclipse Plus 2.1 mm ꢁ 50 mm, 3.5 μm particle size, catalog no.
959743-902) using linear gradients of increasing acetonitrile at a flow
rate of 0.2 mL/min. The UV signals at 215 and 260 nm and the total ion
chromatogram were monitored, and purity was evaluated by examining
and integrating the resultant peaks.
NMR Characterization of Compounds. Chemical shift, cou-
pling constants, and integration data are shown below. Spectra of the
compounds with complete assignments can be found in the Supporting
Information.
((R)-3-Methyl-1-((S)-3-phenyl-2-(pyrazine-2-carboxamido)propana-
mido)butyl)boronic Acid (1a). 1H NMR (300 MHz, 80% D2O þ 20%
CD3CN) δ 9.17 (s, 1H), 8.88 (d, J = 2.4 Hz, 1H), 8.82 (s, 1H), 7.44 (dd,
J = 10.6, 5.1 Hz, 5H), 5.02 (t, J = 7.6 Hz, 1H), 3.44ꢀ3.22 (m, 2H), 2.84
(dd, J = 8.9, 6.4 Hz, 1H), 2.16ꢀ2.07 (m, 1H), 1.39 (dd, J = 12.6, 5.8 Hz,
1H), 1.34ꢀ1.19 (m, 2H), 0.89 (dd, J = 6.1, 4.3 Hz, 6H).
Removal of the N-Terminal Cbz-Protecting Group. The N-terminally
Cbz-protected dipeptidyl boronic acid (þ)-pinanediol ester was dis-
solved in 10 mL of a 3:1 mixture of ethanol and triethylamine and placed
in a hydrogenator flask. To the solution, 10% Pd/C was added and
the reaction mixture placed under a hydrogen atmosphere at 60 psi
and shaken overnight. The mixture was filtered and solvent removed
under vacuum to yield the deprotected dipeptidyl boronic acid (þ)-
pinanediol ester.
((R)-1-((2S,3R)-3-Hydroxy-2-(pyrazine-2-carboxamido)butanamido)-
3-methylbutyl)boronic Acid (1b). 1H NMR (300 MHz, D2O) δ 9.18 (s,
1H), 8.82 (d, J = 2.3 Hz, 1H), 8.77 (s, 1H), 4.46ꢀ4.32 (m, 1H), 2.80 (t,
J = 7.7 Hz, 1H), 2.04 (s, 1H), 1.53 (td, J = 13.2, 6.5 Hz, 1H), 1.46ꢀ1.31
(m, 2H), 1.27 (d, J = 6.4 Hz, 3H), 0.85 (dd, J = 6.3, 3.1 Hz, 6H).
((R)-1-((S)-3-hydroxy-2-(pyrazine-2-carboxamido)propanamido)-3-
Removal of the (þ)-Pinanediol and P2 Amino Acid Side Chain
Protecting Groups. This was accomplished using one of two methods. In
the first method, 1.0 equiv of the (þ)-pinanediol ester was dissolved in
10 mL of dichloromethane. The solution was then cooled on a dry ice/
acetone bath to ꢀ78 °C under argon atmosphere. Once cooled, an
amount of 2.0 equiv of 1 M BCl3 in DCM was added dropwise over the
course of 15 min, after which the mixture was stirred for 1 h at ꢀ78 °C.
The mixture was then allowed to warm to room temperature, and
solvent was removed by rotary evaporation. Next, an amount of 10 mL of
anhydrous methanol was added to the flask, the mixture stirred for 15
min, and the methanol removed under vacuum. The addition of 10 mL
of methanol, stirring for 15 min, and followed by removal under vacuum
were repeated an additional five times. The resulting oil was dissolved in
a minimal volume of water, frozen on dry ice, and lyophilized overnight
to yield the crude dipeptidyl boronic acid. It is worth noting that any Boc
and t-Bu protecting groups are also removed during this reaction.
In the second method Boc and t-Bu protecting groups present were
removed first. This was accomplished by dissolving 1.0 equiv of the
dipeptidyl boronic acid (þ)-pinanediol ester dissolved in 10 mL of a
solution of 4 M HCl in THF. The mixture was allowed to stir for 3 h at
room temperature, after which the resulting white precipitate was
collected by vacuum filtration and washed 3ꢁ with cold THF, resulting
in the deprotected dipeptidyl boronic acid (þ)-pinanediol ester. The
deprotected dipeptidyl boronic acid (þ)-pinanediol ester and 1.0 equiv
of phenylboronic acid were dissolved in 100 mL of water adjusted to pH 3
with 2 N HCl. Then an amount of 100 mL of either hexane or diethyl ether
was added and the reaction vigorously stirred for 30 min. The organic layer
was removed and replaced with a fresh 100 mL. The mixture was stirred
vigorously for 30 min. This procedure was repeated an additional three
times. The organic layers were pooled, dried over MgSO4, and reduced to
quantitatively yield phenylboronic acid (þ)-pinanediol ester for later
recovery of (þ)-pinanediol. The aqueous layer was reduced in volume
and lyophilized to yield the crude deprotected dipeptidyl boronic acid.
1
methylbutyl)boronic Acid (1c). H NMR (300 MHz, D2O) δ 9.21 (s,
1H), 8.85 (d, J = 2.2 Hz, 1H), 8.79 (s, 1H), 4.84 (d, J = 5.1 Hz, 1H),
4.12ꢀ3.97 (m, 2H), 2.91ꢀ2.79 (m, 1H), 1.58 (dt, J = 13.3, 6.7 Hz, 1H),
1.49ꢀ1.21 (m, 2H), 0.89 (dd, J = 6.1, 4.5 Hz, 6H).
((R)-1-((S)-3-Hydroxy-3-methyl-2-(pyrazine-2-carboxamido)buta-
namido)-3-methylbutyl)boronic Acid (1d). 1H NMR (300 MHz,
D2O) δ 9.19 (s, 1H), 8.83 (d, J = 2.3 Hz, 1H), 8.77 (s, 1H), 2.78
(dd, J = 9.0, 6.6 Hz, 1H), 1.55 (td, J = 13.2, 6.5 Hz, 1H), 1.38 (d, J = 8.1
Hz, 6H), 1.47ꢀ1.23 (m, 2H), 0.87 (dd, J = 6.4, 2.4 Hz, 6H).
(R)-(3-Methyl-1-(2-(pyrazine-2-carboxamido)acetamido)butyl)-
boronic Acid (1e). 1H NMR (300 MHz, D2O) δ 9.18 (s, 1H), 8.81 (s,
1H), 8.75 (s, 1H), 4.29 (s, 2H), 2.82 (t, J = 7.2 Hz, 1H), 1.56 (dt, J = 18.7,
6.1 Hz, 1H), 1.48ꢀ1.18 (m, 2H), 1.02ꢀ0.62 (m, 6H).
1
(R)-(1-(2-Aminoacetamido)-3-methylbutyl)boronic Acid (2a). H
NMR (300 MHz, D2O) δ 3.81 (s, 2H), 3.03 (dd, J = 8.7, 6.5 Hz, 1H),
1.70ꢀ1.51 (m, 1H), 1.52ꢀ1.43 (m, 1H), 1.43ꢀ1.26 (m, 1H), 0.88 (t, J =
6.6 Hz, 6H).
((R)-1-((S)-2-Amino-3-(1H-indol-3-yl)propanamido)-3-methylbu-
tyl)boronic Acid (2b). 1H NMR (300 MHz, D2O) δ 7.59 (d, J = 7.7 Hz,
1H), 7.47 (d, J = 8.0 Hz, 1H), 7.23 (s, 1H), 7.20 (d, J = 8.1 Hz, 1H), 7.13
(t, J = 7.3 Hz, 1H), 4.26 (dd, J = 8.8, 6.6 Hz, 1H), 3.45ꢀ3.21 (m, 2H),
2.61 (dd, J = 9.6, 6.8 Hz, 1H), 0.93 (d, J = 7.3 Hz, 2H), 0.87 (s, 1H), 0.64
(dd, J = 15.7, 6.0 Hz, 6H).
((R)-1-((S)-2-Amino-3-phenylpropanamido)-3-methylbutyl)boro-
nic Acid (2c). 1H NMR (300 MHz, 90% H2O þ 10% D2O) δ 8.63 (s,
1H), 7.50ꢀ7.32 (m, 3H), 7.32ꢀ7.19 (m, 2H), 4.21 (dd, J = 8.7, 6.7 Hz,
1H), 3.24 (dd, J = 13.6, 6.4 Hz, 1H), 3.13 (dd, J = 13.6, 9.0 Hz, 1H),
2.85ꢀ2.68 (m, 1H), 1.30ꢀ1.03 (m, 3H), 0.77 (dd, J = 8.3, 5.8 Hz, 6H).
((R)-1-((S)-2-Amino-3-(4-hydroxyphenyl)propanamido)-3-methyl-
butyl)boronic Acid (2d). 1H NMR (300 MHz, D2O) δ 7.07 (d, J = 8.3
4372
dx.doi.org/10.1021/jm200460q |J. Med. Chem. 2011, 54, 4365–4377

Journal of Medicinal Chemistry
ARTICLE
Hz, 2H), 6.80 (d, J = 8.3 Hz, 2H), 4.09 (dd, J = 9.8, 6.1 Hz, 1H), 3.12 (dt,
J = 17.2, 8.6 Hz, 1H), 2.95 (dd, J = 13.5, 10.0 Hz, 1H), 2.63 (dd, J = 10.3,
5.0 Hz, 1H), 1.17ꢀ0.83 (m, 3H), 0.68 (dd, J = 10.3, 6.1 Hz, 6H).
((R)-1-((S)-2-Aminopropanamido)-3-methylbutyl)boronic Acid (2e).
1H NMR (300 MHz, 90% H2O þ 10% D2O) δ 8.72 (s, 1H), 4.11
(q, J = 7.0 Hz, 1H), 3.04ꢀ2.84 (m, 1H), 1.69ꢀ1.42 (m, 2H), 1.53 (d, J =
7.1 Hz, 3H), 1.44ꢀ1.26 (m, 1H), 0.90 (t, J = 6.0 Hz, 6H).
Hz, 1H), 7.34ꢀ7.08 (m, 3H), 4.24 (dd, J = 9.0, 6.3 Hz, 1H), 3.25 (qd, J =
13.7, 7.9 Hz, 2H), 2.88ꢀ2.73 (m, 1H), 1.34ꢀ1.02 (m, 3H), 0.78 (dd, J =
8.5, 5.9 Hz, 6H).
((R)-1-((S)-2-Amino-3-(3,4-difluorophenyl)propanamido)-3-methyl-
butyl)boronic Acid (2r). 1H NMR (300 MHz, D2O) δ 7.17 (ddd,
J = 27.2, 18.2, 8.8 Hz, 2H), 7.00 (s, 1H), 4.13 (dd, J = 9.2, 6.3 Hz, 1H),
3.18 (dd, J = 13.7, 6.1 Hz, 1H), 3.04 (dd, J = 13.6, 9.5 Hz, 1H), 2.77 (dd,
J = 9.4, 5.4 Hz, 1H), 1.30ꢀ0.96 (m, 3H), 0.72 (t, J = 6.3 Hz, 6H).
((R)-1-((S)-2-Amino-3-(3-fluorophenyl)propanamido)-3-methylbutyl)-
boronic Acid (2s). 1H NMR (300 MHz, D2O) δ 7.33 (dd, J = 14.4, 7.6
Hz, 15H), 7.10ꢀ6.90 (m, 45H), 4.17 (dd, J = 9.0, 6.4 Hz, 7H), 3.20 (dd,
J = 13.6, 6.3 Hz, 14H), 3.07 (dd, J = 13.5, 9.4 Hz, 15H), 2.83ꢀ2.66 (m,
14H), 1.30ꢀ0.95 (m, 48H), 0.71 (t, J = 6.4 Hz, 97H).
((R)-1-((S)-2,4-Diamino-4-oxobutanamido)-3-methylbutyl)boronic
1
Acid (2f). H NMR (300 MHz, D2O) δ 4.25 (t, J = 6.4 Hz, 1H),
3.03ꢀ2.74 (m, 3H), 1.58ꢀ1.35 (m, 2H), 1.26 (dt, J = 19.7, 7.1 Hz, 1H),
0.80 (t, J = 6.1 Hz, 6H).
((R)-1-((S)-2-Amino-4-methylpentanamido)-3-methylbutyl)boro-
nic Acid (2g). 1H NMR (300 MHz, DMSO) δ 4.08 (t, J = 7.3 Hz, 1H),
2.97 (dd, J = 9.3, 6.4 Hz, 1H), 1.92ꢀ1.72 (m, 2H), 1.64 (ddd, J = 19.1,
12.8, 5.8 Hz, 2H), 1.58ꢀ1.46 (m, 1H), 1.46ꢀ1.30 (m, 1H), 1.12ꢀ0.97
(m, 6H), 0.97ꢀ0.81 (m, 6H).
((R)-1-((S)-2-Amino-3-hydroxy-3-methylbutanamido)-3-methylbutyl)-
boronic Acid (2t). 1H NMR (300 MHz, D2O) δ 8.90 (s, 1H), 3.87 (s,
1H), 2.94 (dd, J = 8.9, 6.7 Hz, 1H), 2.04 (s, 1H), 1.59 (ddd, J = 17.0, 13.0,
6.4 Hz, 1H), 1.51ꢀ1.18 (m, 2H), 1.39 (s, 3H), 1.29 (s, 3H), 0.88 (dd, J =
6.3, 3.5 Hz, 6H).
((R)-1-((S)-2-Amino-3,3-dimethylbutanamido)-3-methylbutyl)boro-
nic Acid (2u). 1H NMR (300 MHz, D2O) δ 3.75 (s, 1H), 2.86 (dd, J =
9.0, 6.7 Hz, 1H), 1.57 (dq, J = 12.9, 6.5 Hz, 1H), 1.50ꢀ1.25 (m, 2H),
1.07 (s, 9H), 0.88 (dd, J = 6.5, 2.3 Hz, 6H).
((R)-1-((S)-2-Aminobutanamido)-3-methylbutyl)boronic Acid (2v).
1H NMR 1H NMR (300 MHz, D2O) δ 3.90 (t, J = 6.6 Hz, 1H), 2.86 (dd,
J = 9.2, 6.5 Hz, 1H), 1.85 (p, J = 7.4 Hz, 2H), 1.61ꢀ1.45 (m, 1H),
1.45ꢀ1.35 (m, 1H), 1.35ꢀ1.20 (m, 1H), 0.91 (t, J = 7.5 Hz, 3H), 0.83
(dd, J = 6.2, 4.9 Hz, 6H).
((R)-1-((S)-2-Amino-3,3-diphenylpropanamido)-3-methylbutyl)-
boronic Acid (2w). 1H NMR (300 MHz, D2O) δ 7.59 (d, J = 7.3 Hz,
2H), 7.54ꢀ7.26 (m, 8H), 4.43 (d, J = 11.8 Hz, 1H), 2.69 (dd, J = 9.6, 5.6
Hz, 1H), 0.94 (ddd, J = 13.4, 9.3, 5.3 Hz, 2H), 0.86ꢀ0.75 (m, 1H), 0.68
(dd, J = 9.0, 6.2 Hz, 6H).
((R)-1-((S)-2-Aminopentanamido)-3-methylbutyl)boronic Acid (2x).
1H NMR (300 MHz, D2O) δ 3.95 (t, J = 6.8 Hz, 1H), 2.84 (dd, J = 9.3,
6.4 Hz, 1H), 1.78 (dt, J = 9.8, 5.6 Hz, 2H), 1.61ꢀ1.43 (m, 1H), 1.43ꢀ1.34
(m, 1H), 1.35ꢀ1.20 (m, 3H), 0.87 (t, J = 7.2 Hz, 3H), 0.83 (dd, J = 6.1, 5.0
Hz, 6H).
((R)-1-((S)-2-Amino-3-(perfluorophenyl)propanamido)-3-methyl-
butyl)boronic Acid (2y). 1H NMR (300 MHz, D2O) δ 4.19 (t, J = 7.5
Hz, 1H), 3.32 (d, J = 7.5 Hz, 2H), 2.99 (t, J = 7.0 Hz, 1H), 1.31 (dd, J =
12.5, 8.8 Hz, 4H), 0.83 (t, J = 6.0 Hz, 7H).
((R)-1-((2S,3R)-2-Amino-3-hydroxybutanamido)-3-methylbutyl)-
boronic Acid (2h). 1H NMR (300 MHz, D2O) δ 4.04 (p, J = 6.3 Hz,
1H), 3.75 (d, J = 6.8 Hz, 1H), 2.95ꢀ2.82 (m, 1H), 1.61ꢀ1.44 (m, 1H),
1.44ꢀ1.35 (m, 1H), 1.29 (dd, J = 13.9, 6.8 Hz, 1H), 1.21 (d, J = 6.4 Hz,
3H), 0.81 (t, J = 5.2 Hz, 6H).
((R)-1-((S)-2-Amino-3-hydroxypropanamido)-3-methylbutyl)boro-
nic Acid (2i). 1H NMR (300 MHz, D2O) δ 4.12ꢀ4.01 (m, 1H), 3.88
(qd, J = 12.3, 5.2 Hz, 2H), 2.93 (dd, J = 9.0, 6.5 Hz, 1H), 1.62ꢀ1.36 (m,
2H), 1.36ꢀ1.21 (m, 1H), 0.82 (t, J = 5.9 Hz, 6H).
((R)-1-((S)-2-Amino-3-(1H-imidazol-4-yl)propanamido)-3-methyl-
butyl)boronic Acid (2j). 1H NMR (300 MHz, D2O) δ 8.70 (s, 1H), 7.42
(s, 1H), 4.23 (dd, J = 8.2, 6.6 Hz, 1H), 3.35 (dd, J = 7.2, 2.8 Hz, 2H), 2.97
(dd, J = 9.1, 5.5 Hz, 1H), 1.40ꢀ1.15 (m, 3H), 0.81 (t, J = 6.2 Hz, 6H).
((R)-1-((S)-2-Amino-3-(naphthalen-1-yl)propanamido)-3-methyl-
butyl)boronic Acid (2k). 1H NMR (300 MHz, D2O) δ 8.16ꢀ7.88 (m,
3H), 7.75ꢀ7.38 (m, 4H), 4.37 (dd, J = 10.4, 5.9 Hz, 1H), 3.77 (dd, J = 13.6,
5.8 Hz, 1H), 3.61 (dd, J = 14.2, 10.7 Hz, 1H), 2.59 (dd, J = 9.6, 5.3 Hz, 1H),
0.99ꢀ0.84 (m, 1H), 0.79 (dd, J = 17.0, 7.1 Hz, 2H), 0.64 (t, J = 7.1 Hz, 5H).
((R)-1-((S)-2-Amino-3-(naphthalen-2-yl)propanamido)-3-methyl-
butyl)boronic Acid (2l). 1H NMR (300 MHz, D2O) δ 7.94 (t, J = 9.6 Hz,
3H), 7.79 (s, 1H), 7.69ꢀ7.48 (m, 2H), 7.42 (d, J = 8.4 Hz, 1H), 4.34 (dd,
J = 9.6, 6.0 Hz, 1H), 3.46 (dd, J = 13.4, 5.7 Hz, 1H), 3.26 (dd, J = 22.9, 9.7
Hz, 1H), 2.68 (t, J = 7.7 Hz, 1H), 0.97 (dd, J = 19.0, 11.6 Hz, 2H), 0.79
(tdd, J = 20.0, 13.0, 6.6 Hz, 2H), 0.58 (d, J = 6.3 Hz, 3H), 0.44 (d, J = 6.3
Hz, 3H), 0.34 (dd, J = 21.0, 14.4 Hz, 1H).
((R)-1-((S)-2-Amino-3-(3,5-difluorophenyl)propanamido)-3-methyl-
butyl)boronic Acid (2m). 1H NMR (300 MHz, D2O) δ 6.93 (dd, J =
15.4, 8.6 Hz, 3H), 4.21 (dd, J = 9.1, 6.4 Hz, 1H), 3.27 (dd, J = 13.6, 6.2
Hz, 1H), 3.13 (dd, J = 13.5, 9.5 Hz, 1H), 2.91ꢀ2.78 (m, 1H), 1.35ꢀ1.07
(m, 3H), 0.80 (t, J = 6.2 Hz, 6H).
((R)-1-((S)-2-Amino-2-cyclohexylacetamido)-3-methylbutyl)boro-
nic Acid (2z). 1H NMR (300 MHz, H2O þ D2O) δ 3.81 (d, J = 6.4 Hz,
1H), 2.89 (dd, J= 9.5, 6.3 Hz, 1H), 1.95ꢀ1.84(m,1H),1.86ꢀ1.61 (m, 6H),
1.62ꢀ1.52 (m, 1H), 1.52ꢀ1.42 (m, 1H), 1.42ꢀ1.31 (m, 1H), 1.31ꢀ1.16
(m, 2H), 1.15ꢀ1.00 (m, 2H), 0.90 (dd, J = 6.5, 3.9 Hz, 6H).
2-((S)-2-Amino-3-(((R)-1-borono-3-methylbutyl)amino)-3-oxopro-
pyl)malonic Acid (2aa). 1H NMR (300 MHz, D2O) δ 4.09 (t, J = 6.8 Hz,
1H), 3.00 (dd, J = 8.8, 6.4 Hz, 1H), 2.42 (d, J = 6.9 Hz, 2H), 1.52 (ddd,
J = 15.1, 11.4, 6.1 Hz, 2H), 1.37 (dt, J = 14.2, 7.1 Hz, 1H), 0.88 (t,
J = 6.0 Hz, 6H).
((R)-1-((S)-3-([1,10-Biphenyl]-4-yl)-2-aminopropanamido)-3-methyl-
butyl)boronic Acid (2n). 1H NMR (300 MHz, D2O) δ 7.73 (d, J = 5.6 Hz,
4H), 7.54 (t, J= 7.4 Hz, 2H), 7.42 (dd, J= 19.7, 7.6 Hz, 3H), 4.27 (dd, J=9.8,
5.8 Hz, 1H), 3.35 (dd, J = 13.3, 5.8 Hz, 1H), 3.16 (dd, J= 13.1, 10.4 Hz, 1H),
2.73 (dd, J = 10.0, 5.3 Hz, 1H), 1.27ꢀ0.84 (m, 3H), 0.67 (dd, J = 13.7,
6.3 Hz, 6H).
((R)-1-((S)-2-Amino-3-(4-fluorophenyl)propanamido)-3-methylbutyl)-
boronic Acid (2o). 1H NMR (300 MHz, CDCl3) δ 8.62 (s, 1H),
7.26ꢀ7.13 (m, 2H), 7.06 (t, J = 8.8 Hz, 2H), 4.14 (dd, J = 9.0, 6.4 Hz,
1H), 3.17 (dd, J = 13.7, 6.2 Hz, 1H), 3.05 (dd, J = 13.6, 9.5 Hz, 1H),
2.80ꢀ2.66 (m, 1H), 1.25ꢀ0.94 (m, 3H), 0.80ꢀ0.62 (m, 6H).
((R)-1-((S)-2-Aminohexanamido)-3-methylbutyl)boronic Acid (2p).
1H NMR (300 MHz, D2O) δ 3.92 (t, J = 6.7 Hz, 1H), 2.82 (dd, J = 9.4,
6.2 Hz, 1H), 1.78 (dd, J = 14.0, 7.3 Hz, 2H), 1.49 (ddd, J = 16.1, 11.4, 4.6 Hz,
1H), 1.38 (td, J = 9.3, 5.3 Hz, 1H), 1.32ꢀ1.15 (m, 5H), 0.79 (dd, J = 10.6,
5.4 Hz, 9H).
(R)-(1-(2-(Dimethylamino)acetamido)-3-methylbutyl)boronic Acid
(2ab). 1H NMR (300 MHz, D2O) δ 3.92 (s, 2H), 3.01 (dd, J = 9.2, 6.2
Hz, 1H), 2.86 (s, 6H), 1.65ꢀ1.38 (m, 2H), 1.32 (dd, J = 13.6, 5.9 Hz, 1H),
0.82 (t, J = 6.8 Hz, 6H).
(R)-(3-Methyl-1-(2-(methylamino)acetamido)butyl)boronic Acid
1
(2ac). H NMR (300 MHz, D2O) δ 3.80 (s, 2H), 2.95 (dd, J = 9.2,
6.1 Hz, 1H), 2.66 (s, 3H), 1.60ꢀ1.34 (m, 2H), 1.34ꢀ1.20 (m, 1H),
0.79 (t, J = 6.8 Hz, 6H).
Preparation of Inhibitors for Assay. Inhibitor was dissolved in
100% ethanol to a concentration of 0.1 M. The ethanol stock solution was
then diluted 10- to 100-fold into either pH 2.0 buffer (0.148 M HCl, 0.052
M KCl) or pH 7.6 buffer (25 mM HEPES, 0.5 mM EDTA at pH 7.6) and
allowed to stir and equilibrate overnight at room temperature. Further
((R)-1-((S)-2-Amino-3-(2-fluorophenyl)propanamido)-3-methylbutyl)-
boronic Acid (2q). 1H NMR (300 MHz, D2O) δ 7.40 (dd, J = 13.3, 6.1
4373
dx.doi.org/10.1021/jm200460q |J. Med. Chem. 2011, 54, 4365–4377

Journal of Medicinal Chemistry
ARTICLE
dilutions were carried out just prior to assay in preassay incubation buffers at
a concentration 10-fold higher than the final inhibitor concentration.
In Vitro Proteasome Inhibition Assays. Assays of the chymotryp-
sin-like proteolytic activity of the 20S proteasome using succinyl-leucyl-leucyl-
valinyl-tyrosyl-7-amino-4-methylcoumarin (Suc-LLVY-AMC, Bachem) as
the substrate have been previously described.81 As boronic acids are slow,
tight-binding inhibitors, enzyme solution was incubated with inhibitor for 10
min prior to substrate addition. The fluorescence for each well was read
(λexcitation = 360 nm and λemission = 460 nm) after an additional 30 min. Each
condition and concentration of inhibitor was prepared in quadruplicate. Data
were collected using Perkin-Elmer Wallac Vector3V 1420 multilabel counter
96- and 384-well plate reader equipped with a 355 nm excitation filter and a
460 nm emission filter and running Wallac 1420 Workstation Manager,
version 3.0.0, revsion 4, component version 3.00.0.53 software. The data were
then analyzed using Graph Prism 5.0.3, fit to a variable-slope sigmoidal
equation, and reported as % fluorescence (normalized relative fluorescence).
Wells in which reaction buffer was added in place of enzyme solution
and wells lacking SDS for enzymatic activation were used as negative
controls. As a positive control, varying concentrations of bortezomib
(1a) were used alongside each compound of interest. To ensure that the
pH 2.0 samples were not affecting the assay pH, the pH was measured
for a subset of wells for each compound and each assay in both pH 2.0
and pH 7.6 assay wells.
black, flat clear-bottom sterile tissue-culture treated polystyrene plates
(Costar catalog no. 3603) and incubated for 24 h at 37 °C and 5% CO2.
After 24 h, inhibitor dilutions were added to the plate, with gentle
mixing, yielding a total well volume of 100 μL. The cells were incubated
at 37 °C and 5% CO2 for 24 h. After 24 h, 10 μL of MTT dye solution
(5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide in 1ꢁ PBS) was added to each well. Cells were incubated with
dye for 3 h at 37 °C and 5% CO2, after which an amount of 100 μL of
MTT solubilization solution (10% (v/v) TritonX-100, 0.1 M HCl in
isopropanol) was added to each well. The plates were sealed in parafilm
and gently shaken overnight to dissolve the precipitated MTT metabo-
lite. Absorbance values for each well were obtained by reading the plates
on a 96-well plate reader at 570 nm. Each condition and concentration of
inhibitor was prepared in quadruplicate. Data were collected using either
a Perkin-Elmer Wallac Vector3V 1420 multilabel counter 96- and 384-
well plate reader equipped with 570 and 670 nm absorbance filters and
running Wallac 1420 Workstation Manager, version 3.0.0, revision 4,
component version 3.00.0.53 software or a Molecular Devices Spectra-
max 340PC monochromator-based absorbance plate reader equipped
with a peltier unit running SoftMax Pro, version 4.3 LS. The results were
then analyzed using Graph Prism 5.0.3, fit to a variable-slope sigmoidal
equation, and reported as % absorbance (normalized absorbance). Wells
containing cells and medium only and cells with medium and either pH 2
or pH 7.6 buffer only were included as negative controls. As a positive
control, a plate with dilutions of bortezomib was included with each
assay performed.
Cell Lines. The Living Colors HEK 293 ZsGreen proteasome sensor
cell line (hereafter referred to as HEK 293 PS) was obtained from
Clontech Laboratories, Inc. (catalog no. 6351535). These are HEK 293
cells that have been stably transfected with a vector that expresses a
fusion protein between the ZsGreen fluorescent protein and the
proteasome-targeting sequence of murine ornithine decarboxylase.62,63
Cells were maintained in phenol red free DMEM (Gibco catalog no.
31053) with 10% fetal bovine serum, 10 mM HEPES, 2 mM L-glutamine
(Gibco catalog no. 25030), 4.5 g/L D-glucose, 1 mM sodium pyruvate
(Gibco catalog no. 11360), 100 mgꢀ100 U/L penicillinꢀstreptomycin
(Gibco catalog no.15140), and 0.2 mg/L G418 at 37 °C and 5% CO2.
The follicular small-cell lymphocytic leukemia (FSCLL) cell line was
obtained from Dr. Jonathan Cheng, M.D., of the Fox Chase Cancer
Center, Philadelphia, PA. Cells were maintained suspended in phenol
red free RPMI-1640 (Gibco catalog no. 11835) medium with 10%
fetal bovine serum, 4.5 g/L D-glucose, 100 mgꢀ100 U/L penicillinꢀ
streptomycin, 10 mM HEPES, and 2.0 g/L sodium bicarbonate.
In-Cell Proteasome Inhibition Assays. Cells were plated at a
density of 3.2 ꢁ 104 cells per well in 96-well flat-bottom sterile tissue-
culture treated polystyrene plates (Costar catalog no. 3603) and
incubated for 24 h at 37 °C and 5% CO2. After 24 h, inhibitor dilutions
were added to the plate with gentle mixing, yielding a total well volume
of 100 μL. The cells were incubated at 37 °C and 5% CO2 for 24 h. After
24 h with inhibitor, cells were viewed via fluorescent microscope. Cell
medium was removed, and cells were lysed by adding 100 μL of cell lysis
buffer (0.025 M HEPES, 0.5 mM EDTA, and 5% (v/v) TritonX-100)
and gently shaking for 1 h. Plates were read on a 96-well fluorescence
plate reader (λex = 485 nm, λem = 535 nm). Each condition and
concentration of inhibitor was prepared in quadruplicate. Data were
collected on a Perkin-Elmer Wallac Vector3V 1420 multilabel counter
96- and 384-well plate reader equipped with a 485 nm excitation filter
and a 535 nm emission filter and running Wallac 1420 Workstation
Manager, version 3.0.0, revision 4, component version 3.00.0.53 soft-
ware. The results were then analyzed using Graph Prism 5.0.3, fit to a
variable-slope sigmoidal equation and reported as % fluorescence
(normalized relative fluorescence). Wells containing cells and medium
only and cells with medium and either pH 2 or pH 7.6 buffer only were
included as negative controls. As a positive control, a plate with dilutions
of bortezomib was included with each assay performed.
Inhibitor Degradation Assays. Intact Cells. Cells were plated at
2 ꢁ 106 cells in 900 μL of medium in a sterile 24-well tissue-culture
treated polystyrene plate (Costar no.3527) and incubated for 24 h at
37 °C þ 5% CO2. Aliqouts of 45 μL were removed at each time point,
and quenched with 5 μL of stop solution (100 μM Trp-Tyr, used as an
internal standard for LCMS, in 1 M HCl).
Cell Lysate and Conditioned Medium. Cells were grown under the
conditions described above. Medium was collected after 48 h, and cells
were collected, washed with 1ꢁx PBS, resuspended in 1ꢁ PBS þ 25%
glycerol, pH 7.2, at 107 cells/mL, and lysed using sonication. Inhibitor stocks
were prepared in 1ꢁ PBS þ 25% glycerol, pH 7.2, at 10ꢀ3 M and dilutions
made in the same buffer. Cell lysate was warmed to 37 °C, and for each
concentration of inhibitor, an amount of 20 μL was added to 180 μL
aliquots of the warmed cell lysate, and the components were mixed. At each
time point, an amount of 5 μL of the reaction was removed and added to a
mixture of 45 μL of 50 mM sodium phosphate and 5 μL of stop solution
(100 μM Trp-Tyr in 1 M HCl). Positive control samples included leucinyl-
p-nitroanilide incubated with cell lysate or conditioned medium, and
negative controls included leucinyl-p-nitroanilide or inhibitor incubated in
1ꢁ PBS þ 25% glycerol.
LCMS Analysis. Samples were then run on a ThermoQuest Finnigan
LCQ-Duo electrospray ionization LCMS equipped with a C18 reversed
phase HPLC column (Agilent Zorbax Eclipse Plus C18 narrow bore RR
2.1 cm ꢁ 50 mm, 3.5 μm particle size, part no. 959743-902), autosampler,
and UV/vis detector. We used the preprogrammed convex 10 gradient
curve of acetonitrile from 2% to 98% over 7 min at a flow of 0.2 mL/min for
the first 6.25 min. The mass spectrometer was set to run in full scan MS
positive ion mode with a mass range of 50.00ꢀ380.00 amu.
’ ASSOCIATED CONTENT
S
Supporting Information. NMR structural assignments,
b
2D NOESY NMR spectra, in-cell stability data, and cyclization
reversibility data. This material is available free of charge via the
Internet at http://pubs.acs.org.
MTT Assays. MTT assays for cytotoxicity have been described.64ꢀ66
Briefly, cells were plated at a density of 3.2 ꢁ 104 cells per well in 96-well
4374
dx.doi.org/10.1021/jm200460q |J. Med. Chem. 2011, 54, 4365–4377

Journal of Medicinal Chemistry
ARTICLE
’ AUTHOR INFORMATION
(18) Adams, J.; Palombella, V. J.; Sausville, E. A.; Johnson, J.;
Destree, A.; Lazarus, D. D.; Maas, J.; Pien, C. S.; Prakash, S.; Elliott,
P. J. Proteasome inhibitors: a novel class of potent and effective
antitumor agents. Cancer Res. 1999, 59, 2615–2622.
(19) LeBlanc, R.; Catley, L. P.; Hideshima, T.; Lentzsch, S.;
Mitsiades, C. S.; Mitsiades, N.; Neuberg, D.; Goloubeva, O.; Pien,
C. S.; Adams, J.; Gupta, D.; Richardson, P. G.; Munshi, N. C.; Anderson,
K. C. Proteasome inhibitor PS-341 inhibits human myeloma cell growth
in vivo and prolongs survival in a murine model. Cancer Res. 2002,
62, 4996–5000.
Corresponding Author
*Phone: 617-636-6881. Fax: 617-696-2409. E-mail: william.
bachovchin@tufts.edu.
’ ACKNOWLEDGMENT
This work was supported by Arisaph Pharmaceuticals.
(20) Dick, L. R.; Fleming, P. E. Building on bortezomib: second-
generation proteasome inhibitors as anti-cancer therapy. Drug Discovery
Today 2010, 15, 243–249.
(21) Moore, B. S.; Eustaquio, A. S.; McGlinchey, R. P. Advances in
and applications of proteasome inhibitors. Curr. Opin. Chem. Biol. 2008,
12, 434–440.
(22) Borissenko, L.; Groll, M. 20S proteasome and its inhibitors:
crystallographic knowledge for drug development. Chem. Rev. 2007,
107, 687–717.
’ ABBREVIATIONS USED
Chg, cyclohexyl(glycine); Gla, γ-carboxyglutamic acid; Ki, inhi-
bition constant; IC50, median inhibition concentration; CI,
cyclization index; EC50, median effective concentration
’ REFERENCES
(23) Elofsson, M.; Splittgerber, U.; Myung, J.; Mohan, R.; Crews,
C. M. Towards subunit-specific proteasome inhibitors: synthesis and
evaluation of peptide alpha0,beta0-epoxyketones. Chem. Biol. 1999,
6, 811–822.
(1) Baker, S. J.; Ding, C. Z.; Akama, T.; Zhang, Y.; Hernandez, V.;
Xia, Y. Therapeutic potential of boron-contaning compounds. Future
Med. Chem. 2009, 1, 1275–1288.
(2) Adams, J. Proteasome Inhibitors in Cancer Therapy; Humana
(24) Kuhn, D. J.; Chen, Q.; Voorhees, P. M.; Strader, J. S.; Shenk,
K. D.; Sun, C. M.; Demo, S. D.; Bennett, M. K.; van Leeuwen, F. W.;
Chanan-Khan, A. A.; Orlowski, R. Z. Potent activity of carfilzomib, a
novel, irreversible inhibitor of the ubiquitinꢀproteasome pathway,
against preclinical models of multiple myeloma. Blood 2007,
110, 3281–3290.
(25) Eustaquio, A. S.; Moore, B. S. Mutasynthesis of fluorosalinos-
poramide, a potent and reversible inhibitor of the proteasome. Angew.
Chem., Int. Ed. 2008, 47, 3936–3938.
(26) Feling, R. H.; Buchanan, G. O.; Mincer, T. J.; Kauffman, C. A.;
Jensen, P. R.; Fenical, W. Salinosporamide A: a highly cytotoxic
proteasome inhibitor from a novel microbial source, a marine bacterium
of the new genus Salinospora. Angew. Chem., Int. Ed. 2003, 42, 355–357.
(27) Craiu, A.; Gaczynska, M.; Akopian, T.; Gramm, C. F.; Fenteany,
G.; Goldberg, A. L.; Rock, K. L. Lactacystin and clasto-lactacystin beta-
lactone modify multiple proteasome beta-subunits and inhibit intracel-
lular protein degradation and major histocompatibility complex class I
antigen presentation. J. Biol. Chem. 1997, 272, 13437–13445.
(28) Kupperman, E.; Lee, E. C.; Cao, Y.; Bannerman, B.; Fitzgerald,
M.; Berger, A.; Yu, J.; Yang, Y.; Hales, P.; Bruzzese, F.; Liu, J.; Blank, J.;
Garcia, K.; Tsu, C.; Dick, L.; Fleming, P.; Yu, L.; Manfredi, M.; Rolfe, M.;
Bolen, J. Evaluation of the proteasome inhibitor MLN9708 in preclinical
models of human cancer. Cancer Res. 2010, 70, 1970–1980.
(29) Blackburn, C.; Gigstad, K. M.; Hales, P.; Garcia, K.; Jones, M.;
Bruzzese, F. J.; Barrett, C.; Liu, J. X.; Soucy, T. A.; Sappal, D. S.; Bump,
N.; Olhava, E. J.; Fleming, P.; Dick, L. R.; Tsu, C.; Sintchak, M. D.; Blank,
J. L. Characterization of a new series of non-covalent proteasome
inhibitors with exquisite potency and selectivity for the 20S beta5-
subunit. Biochem. J. 2010, 430, 461–476.
Press: Totowa, NJ, 2004; 312 pp.
(3) Voorhees, P. M.; Orlowski, R. Z. The proteasome and protea-
some inhibitors in cancer therapy. Annu. Rev. Pharmacol. Toxicol. 2006,
46, 189–213.
(4) Wehenkel, M.; Ho, Y. K.; Kim, K. B. Proteasome Inhibitors:
Recent Progress and Future Directions. In Modulation of Protein Stability
in Cancer Therapy; Rubin, E., Sakamoto, K., Eds.; Springer: New York,
2009; pp 99ꢀ119.
(5) Peters, J. M.; Franke, W. W.; Kleinschmidt, J. A. Distinct 19 and
20 S subcomplexes of the 26 S proteasome and their distribution in the
nucleus and the cytoplasm. J. Biol. Chem. 1994, 269, 7709–18.
(6) Rivett, A. J.; Palmer, A.; Knecht, E. Electron microscopic
localization of the multicatalytic proteinase complex in rat liver and in
cultured cells. J. Histochem. Cytochem. 1992, 40, 1165–1172.
(7) Scherrer, K.; Bey, F. The prosomes (multicatalytic proteinases;
proteasomes) and their relationship to the untranslated messenger
ribonucleoproteins, the cytoskeleton, and cell differentiation. Prog.
Nucleic Acid Res. Mol. Biol. 1994, 49, 1–64.
(8) Wolf, D. H.; Hilt, W. The proteasome: a proteolytic nanoma-
chine of cell regulation and waste disposal. Biochim. Biophys. Acta 2004,
1695, 19–31.
(9) Wolf, D. H.; Hilt, W. Proteasomes: The World of Regulatory
Proteolysis. In Molecular Biology Intelligence Unit; Landes Bioscience:
Austin, TX, 2000; 391 pp, http://www.landesbioscience.com/.
(10) Wolf, D. H.; Sommer, T.; Hilt, W. Death gives birth to life: the
essential role of the ubiquitinꢀproteasome system in biology. Biochim.
Biophys. Acta 2004, 1695, 1–2.
(11) Kane, R. C.; Bross, P. F.; Farrell, A. T.; Pazdur, R. Velcade: U.S.
FDA approval for the treatment of multiple myeloma progressing on
prior therapy. Oncologist 2003, 8, 508–513.
(12) Kane, R. C.; Dagher, R.; Farrell, A.; Ko, C. W.; Sridhara, R.;
Justice, R.; Pazdur, R. Bortezomib for the treatment of mantle cell
lymphoma. Clin. Cancer Res. 2007, 13, 5291–5294.
(30) Groll, M.; Clausen, T. Molecular shredders: how proteasomes
fulfill their role. Curr. Opin. Struct. Biol. 2003, 13, 665–673.
(31) Machiels, B. M.; Henfling, M. E.; Gerards, W. L.; Broers, J. L.;
Bloemendal, H.; Ramaekers, F. C.; Schutte, B. Detailed analysis of cell
cycle kinetics upon proteasome inhibition. Cytometry 1997,
28, 243–352.
(13) Goy, A. Mantle cell lymphoma: evolving novel options. Curr.
Oncol. Rep. 2007, 9, 391–398.
(32) Rechsteiner, M. Proteasomes. In Encyclopedia of Molecular Cell
Biology and Molecular Medicine, 2nd ed.; Meyers, R. A., Ed.; Wiley-VCH
Verlag: Weinheim, Germany, 2005; Vol. 11.
(14) Goy, A.; Feldman, T. Expanding therapeutic options in mantle
cell lymphoma. Clin. Lymphoma Myeloma 2007, 7 (Suppl. 5),
S184–S191.
(33) Rechsteiner, M.; Hill, C. P. Mobilizing the proteolytic machine:
cell biological roles of proteasome activators and inhibitors. Trends Cell
Biol. 2005, 15, 27–33.
(34) Rechsteiner, M.; Hoffman, L.; Dubiel, W. The multicatalytic
and 26 S proteases. J. Biol. Chem. 1993, 268, 6065–6068.
(35) Ma, M. H.; Yang, H. H.; Parker, K.; Manyak, S.; Friedman, J. M.;
Altamirano, C.; Wu, Z. Q.; Borad, M. J.; Frantzen, M.; Roussos, E.;
(15) Goy, A.; Gilles, F. Update on the proteasome inhibitor bortezomib
in hematologic malignancies. Clin. Lymphoma 2004, 4, 230–237.
(16) Giaccone, G. Clinical potential of proteasome inhibition in
solid tumours. Eur. J. Cancer 2004, 2 (Suppl.), 25–28.
(17) Lightcap, E. S.; McCormack, T. A.; Pien, C. S.; Chau, V.;
Adams, J.; Elliott, P. J. Proteasome inhibition measurements: clinical
application. Clin. Chem. 2000, 46, 673–683.
4375
dx.doi.org/10.1021/jm200460q |J. Med. Chem. 2011, 54, 4365–4377

Journal of Medicinal Chemistry
ARTICLE
Neeser, J.; Mikail, A.; Adams, J.; Sjak-Shie, N.; Vescio, R. A.; Berenson,
J. R. The proteasome inhibitor PS-341 markedly enhances sensitivity of
multiple myeloma tumor cells to chemotherapeutic agents. Clin. Cancer
Res. 2003, 9, 1136–1144.
(36) Mitsiades, C. S.; Mitsiades, N.; Hideshima, T.; Richardson,
P. G.; Anderson, K. C. Proteasome inhibition as a new therapeutic
principle in hematological malignancies. Curr Drug Targets 2006,
7, 1341–1347.
(37) Csizmadia, V.; Raczynski, A.; Csizmadia, E.; Fedyk, E. R.;
Rottman, J.; Alden, C. L. Effect of an experimental proteasome inhibitor
on the cytoskeleton, cytosolic protein turnover, and induction in the
neuronal cells in vitro. Neurotoxicology 2008, 29, 232–243.
(38) Perner, S.; Hofer, M. D.; Kim, R.; Shah, R. B.; Li, H.; Moller, P.;
Hautmann, R. E.; Gschwend, J. E.; Kuefer, R.; Rubin, M. A. Prostate-
specific membrane antigen expression as a predictor of prostate cancer
progression. Hum. Pathol. 2007, 38, 696–701.
(39) Rettig, W. J.; Garin-Chesa, P.; Beresford, H. R.; Oettgen, H. F.;
Melamed, M. R.; Old, L. J. Cell-surface glycoproteins of human
sarcomas: differential expression in normal and malignant tissues and
cultured cells. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 3110–3114.
(40) Garin-Chesa, P.; Old, L. J.; Rettig, W. J. Cell surface glycopro-
tein of reactive stromal fibroblasts as a potential antibody target in
human epithelial cancers. Proc. Natl. Acad. Sci. U.S.A. 1990, 87,
7235–7239.
(DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to
examine the role of DP-IV in T-cell function. Proc. Natl. Acad. Sci. U.S.A.
1991, 88, 1556–1559.
(56) Lai, J. H.; Zhou, Y.; Sudmeier, J. L.; Wu, W.; Sanford, D. G.;
Hliang, M.; Poplawski, S.; Bachovchin, W. W. Microscopic AcidꢀBase
Equilibria of Alanyl-boroAlanine. In Dipeptidyl Aminopeptidases in
Health and Disease; Hildebrandt, M., Klapp, B., Hoffmann, T., Demuth,
H., Eds.; Kluwer Academic/Plenum Publishers: New York, 2003; Vol.
524, pp 333ꢀ338.
(57) Snow, R. J.; Bachovchin, W. W.; Barton, R. W.; Campbell, S. J.;
Coutts, S. J.; Freeman, D. M.; Gutheil, W. G.; Kelly, T. A.; Kennedy,
C. A.; Krolikowski, D. A.; Leonard, S. F.; Pargellis, C. A.; Tong, L.;
Adams, J. Studies on proline boronic acid dipeptide inhibitors of
dipeptidyl peptidase IV: identification of a cyclic species containing a
BꢀN bond. J. Am. Chem. Soc. 1994, 116, 10860–10869.
(58) Sudmeier, J. L.; Gunther, U. L.; Gutheil, W. G.; Coutts, S. J.;
Snow, R. J.; Barton, R. W.; Bachovchin, W. W. Solution structures
of active and inactive forms of the DP IV (CD26) inhibitor Pro-
boroPro determined by NMR spectroscopy. Biochemistry 1994, 33,
12427–12438.
(59) Gunther, U. L.; Sudmeier, J. L.; Coutts, S. J.; Snow, R. J.; Barton,
R. W.; Bachovchin, W. W. Solution structures of the DP IV (CD26)
inhibitor Val-boroPro determined by NMR spectroscopy. Magn. Reson.
Chem. 1995, 33, 959–970.
(41) Levy, M. T.; McCaughan, G. W.; Abbott, C. A.; Park, J. E.;
Cunningham, A. M.; Muller, E.; Rettig, W. J.; Gorrell, M. D. Fibroblast
activation protein: a cell surface dipeptidyl peptidase and gelatinase
expressed by stellate cells at the tissue remodelling interface in human
cirrhosis. Hepatology 1999, 29, 1768–1778.
(42) Park, J. E.; Lenter, M. C.; Zimmermann, R. N.; Garin-Chesa, P.;
Old, L. J.; Rettig, W. J. Fibroblast activation protein, a dual specificity
serine protease expressed in reactive human tumor stromal fibroblasts.
J. Biol. Chem. 1999, 274, 36505–36512.
(43) Kraus, T. S.; Cohen, C.; Siddiqui, M. T. Prostate-specific
antigen and hormone receptor expression in male and female breast
carcinoma. Diagn. Pathol. 2010, 5, 63.
(44) Cheng, J. D.; Valianou, M.; Canutescu, A. A.; Jaffe, E. K.; Lee,
H. O.; Wang, H.; Lai, J. H.; Bachovchin, W. W.; Weiner, L. M.
Abrogation of fibroblast activation protein enzymatic activity attenuates
tumor growth. Mol. Cancer Ther. 2005, 4, 351–360.
(60) Sudmeier, J. L.; Zhou, Y.; Lai, J. H.; Maw, H. H.; Wu, W.;
Bachovchin, W. W. Autochelation in dipeptide boronic acids: pH-
dependent structures and equilibria of Asp-boroPro and His-boroPro
by NMR spectroscopy. J. Am. Chem. Soc. 2005, 127, 8112–8119.
(61) Kelly, T. A.; Adams, J.; Bachovchin, W. W.; Barton, R. W.;
Campbell, S. J.; Coutts, S. J.; Kennedy, C. A.; Snow, R. J. Immunosup-
pressive boronic acid dipeptides: correlation between conformation and
activity. J. Am. Chem. Soc. 1993, 115, 12637–12638.
(62) BD Living Colors cell lines. Clonetechniques 2004, XIX, 2ꢀ4.
(63) Proteasome sensor vector. Clonetechniques 2003, XVIII, 14.
(64) Mosmann, T. Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. J. Immunol.
Methods 1983, 65, 55–63.
(65) Cory, A. H.; Owen, T. C.; Barltrop, J. A.; Cory, J. G. Use of an
aqueous soluble tetrazolium/formazan assay for cell growth assays in
culture. Cancer Commun. 1991, 3, 207–212.
(45) Silver, D. A.; Pellicer, I.; Fair, W. R.; Heston, W. D.; Cordon-
Cardo, C. Prostate-specific membrane antigen expression in normal and
malignant human tissues. Clin. Cancer Res. 1997, 3, 81–85.
(46) Kidwai, N.; Gong, Y.; Sun, X.; Deshpande, C. G.; Yeldandi,
A. V.; Rao, M. S.; Badve, S. Expression of androgen receptor and
prostate-specific antigen in male breast carcinoma. Breast Cancer Res.
2004, 6, R18–R23.
(66) Wilson, A. P. Cytotoxicity and Viability Assays in Animal Cell
Culture: A Practical Approach, 3rd ed.; Oxford University Press: Oxford,
U.K., 2000; Vol. 1.
(67) Blackburn, C.; Barrett, C.; Blank, J. L.; Bruzzese, F. J.; Bump,
N.; Dick, L. R.; Fleming, P.; Garcia, K.; Hales, P.; Hu, Z.; Jones, M.; Liu,
J. X.; Sappal, D. S.; Sintchak, M. D.; Tsu, C.; Gigstad, K. M. Optimization
of a series of dipeptides with a P3 threonine residue as non-covalent
inhibitors of the chymotrypsin-like activity of the human 20S protea-
some. Bioorg. Med. Chem. Lett. 2010, 20, 6581–6586.
(47) Bodor, N. Soft drugs: principles and methods for the design of
safe drugs. Med. Res. Rev. 1984, 4, 449–469.
(48) Bodor, N. The soft drugapproach. CHEMTECH 1984, 14, 28–38.
(49) Bodor, N.; Buchwald, P. Soft drug design: general principles
and recent applications. Med. Res. Rev. 2000, 20, 58–101.
(50) Bodor, N.; Buchwald, P. Designing safer (soft) drugs by
avoiding the formation of toxic and oxidative metabolites. Mol. Biotech-
nol. 2004, 26, 123–132.
(51) Bodor, N.; Sloan, K. B. Soft drugs V: thiazolidine-type deriva-
tives of progesterone and testosterone. J. Pharm. Sci. 1982, 71, 514–520.
(52) Robinson, J. R.; Lee, V. H. L. Controlled Drug Delivery: Funda-
mentals and Applications, 2nd ed.; Dekker: New York, 1987; 716 pp.
(53) Bodor, N.; Sloan, K. B.; Little, R. J.; Selk, S. H.; Caldwell, L. Soft
drugs 4. 3-Spirothiazolidines of hydrocortisone and its derivatives. Int. J.
Pharm. 1982, 10, 307–321.
(54) Poplawski, S. E.; Lai, J. H.; Sanford, D. G.; Sudmeier, J. L.; Wu,
W.; Bachovchin, W. W. Pro-soft Val-boroPro: a strategy for enhancing in
vivo performance of boronic acid inhibitors of serine proteases. J. Med.
Chem. 2011, 54, 2022–2028.
(55) Flentke, G. R.; Munoz, E.; Huber, B. T.; Plaut, A. G.; Kettner,
C. A.; Bachovchin, W. W. Inhibition of dipeptidyl aminopeptidase IV
(68) Potts, B. C.; Lam, K. S. Generating a generation of proteasome
inhibitors: from microbial fermentation to total synthesis of salinospor-
amide a (marizomib) and other salinosporamides. Mar. Drugs 2010,
8, 835–880.
(69) Ling, T.; Potts, B. C.; Macherla, V. R. Concise formal synthesis
of (ꢀ)-salinosporamide A (marizomib) using a regio- and stereoselec-
tive epoxidation and reductive oxirane ring-opening strategy. J. Org.
Chem. 2010, 75, 3882–3885.
(70) Mayer, A. M.; Glaser, K. B.; Cuevas, C.; Jacobs, R. S.; Kem, W.;
Little, R. D.; McIntosh, J. M.; Newman, D. J.; Potts, B. C.; Shuster, D. E.
The odyssey of marine pharmaceuticals: a current pipeline perspective.
Trends Pharmacol. Sci. 2010, 31, 255–265.
(71) Singh, A. V.; Palladino, M. A.; Lloyd, G. K.; Potts, B. C.;
Chauhan, D.; Anderson, K. C. Pharmacodynamic and efficacy studies of
the novel proteasome inhibitor NPI-0052 (marizomib) in a human plasma-
cytoma xenograft murine model. Br. J. Haematol. 2010, 149, 550–559.
(72) Matteson, D. S.; Jesthi, P. K.; Sadhu, K. M. Synthesis and
properties of pinanediol R-amido boronic esters. Organometallics 1984,
3, 1284–1288.
4376
dx.doi.org/10.1021/jm200460q |J. Med. Chem. 2011, 54, 4365–4377

Journal of Medicinal Chemistry
ARTICLE
(73) Matteson, D. S.; Sadhu, K. M.; Ray, R.; Peterson, M. L.;
Majumdar, D.; Hurst, G. D.; Jesthi, P. K.; Tsai, D. J. S.; Erdik, E.
Asymmetric synthesis with boronic esters. Pure Appl. Chem. 1985,
57, 1741–1748.
(74) Matteson, D. S.; Hurst, G. D. A practical synthesis of
(dichloromethyl)- and (1,1-dichloroethyl)boronic esters. Organometal-
lics 1986, 5, 1465–1467.
(75) Matteson, D. S. R-Amido boronic acids: a synthetic challenge
and their properties as serine protease inhibitors. Med. Res. Rev. 2007,
28, 233–246.
(76) Adams, J.; Behnke, M.; Chen, S.; Cruickshank, A. A.; Dick,
L. R.; Grenier, L.; Klunder, J. M.; Ma, Y. T.; Plamondon, L.; Stein, R. L.
Potent and selective inhibitors of the proteasome: dipeptidyl boronic
acids. Bioorg. Med. Chem. Lett. 1998, 8, 333–338.
(77) Hall, D. G. Boronic Acids: Preparation and Applications in
Organic Synthesis and Medicine; Wiley-VCH Verlag GmbH: Weinheim,
Germany, 2005; 549 pp.
(78) Mantri, P.; Duffy, D. E.; Kettner, C. A. New asymmetric
synthesis of R-aminoboronic acids containing functionalized side chains.
J. Org. Chem. 1996, 61, 5690–5692.
(79) Coutts, S. J.; Kelly, T. A.; Snow, R. J.; Kennedy, C. A.; Barton,
R. W.; Adams, J.; Krolikowski, D. A.; Freeman, D. M.; Campbell, S. J.;
Ksiazek, J. F.; Bachovchin, W. W. Structureꢀactivity relationships of
boronic acid inhibitors of dipeptidyl peptidase IV. 1. Variation of the P2
position of Xaa-boroPro dipeptides. J. Med. Chem. 1996, 39, 2087–2094.
(80) Coutts, S. J.; Adams, J.; Krolikowski, D.; Snow, R. J. Two
efficient methods for the cleavage of pinanediol boronate esters yielding
the free boronic acids. Tetrahedron Lett. 1994, 35, 5109–5112.
(81) Stein, R. L.; Melandri, F.; Dick, L. Kinetic characterization of
the chymotryptic activity of the 20S proteasome. Biochemistry 1996,
35, 3899–3908.
4377
dx.doi.org/10.1021/jm200460q |J. Med. Chem. 2011, 54, 4365–4377