Synthesis and biological characterization of protease-activated prodrugs of doxazolidine

Article abstract of DOI:10.1021/jm300714p

Doxazolidine (doxaz) is a new anthracycline anticancer agent. While structurally similar to doxorubicin (dox), doxaz acts via a distinct mechanism to selectively enhance anticancer activity over cardiotoxicity, the most significant clinical impediment to successful anthracycline treatment. Here, we describe the synthesis and characterization of a prodrug platform designed for doxaz release mediated by secreted proteolytic activity, a common association with invasiveness and poor prognosis in cancer patients. GaFK-Doxaz is hydrolyzable by the proteases plasmin and cathepsin B, both strongly linked with cancer progression, as well as trypsin. We demonstrate that activation of GaFK-Doxaz releases highly potent doxaz that powerfully inhibits the growth of a wide variety of cancer cells (average IC₅₀ of 8 nM). GaFK-Doxaz is stable in human plasma and is poorly membrane permeable, thereby limiting activation to locally secreted proteolytic activity and reducing the likelihood of severe side effects.

Full text of DOI:10.1021/jm300714p

Article
pubs.acs.org/jmc
Synthesis and Biological Characterization of Protease-Activated
Prodrugs of Doxazolidine
Benjamin L. Barthel, Daniel L. Rudnicki, Thomas Price Kirby, Sean M. Colvin, David J. Burkhart,†
and Tad H. Koch*
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, United States
S
* Supporting Information
ABSTRACT: Doxazolidine (doxaz) is a new anthracycline
anticancer agent. While structurally similar to doxorubicin
(dox), doxaz acts via a distinct mechanism to selectively
enhance anticancer activity over cardiotoxicity, the most
significant clinical impediment to successful anthracycline
treatment. Here, we describe the synthesis and characterization
of a prodrug platform designed for doxaz release mediated by
secreted proteolytic activity, a common association with
invasiveness and poor prognosis in cancer patients. GaFK-
Doxaz is hydrolyzable by the proteases plasmin and cathepsin
B, both strongly linked with cancer progression, as well as
trypsin. We demonstrate that activation of GaFK-Doxaz
releases highly potent doxaz that powerfully inhibits the growth
of a wide variety of cancer cells (average IC50 of 8 nM). GaFK-
Doxaz is stable in human plasma and is poorly membrane permeable, thereby limiting activation to locally secreted proteolytic
activity and reducing the likelihood of severe side effects.
1 equiv of formaldehyde, which links the 3′-amino and 4′-
hydroxyl groups to form an oxazolidine ring (Scheme 1). Doxaz
is much more toxic to cancer cells compared to dox, with IC50
values between 1 and 4 orders of magnitude lower, and its
potency is not affected by P170GP expression. The increased
cytotoxic activity of doxaz is tumor selective because doxaz and
dox are equally toxic against a rat cardiomyocyte cell line. These
observations and additional investigations indicated that doxaz is
mechanistically distinct from dox and is not dependent upon
topoisomerase II.11 Instead, doxaz is a DNA alkylating agent at
sites of 5′-GC-3′, where it forms a covalent bond via the
formaldehyde carbon to one guanine and hydrogen-bonds to the
guanine on the opposite strand.10,12
The distinct advantages that doxaz has over dox make it an
attractive potential clinical agent. However, doxaz is very short-
lived; under physiological conditions, hydrolysis and loss of the
formaldehyde result in its conversion to dox with a half-life of
approximately 3 min.10 Therefore, we undertook the develop-
ment of various prodrug strategies, in which doxaz is inactivated
and stabilized until being selectively released by environmental
conditions present in the microenvironment of the tumor. A first-
generation doxaz prodrug, pentyl PABC-Doxaz (PPD), was
designed for activation by human carboxylesterase 2 (CES2,
hiCE) and demonstrated an in vivo antitumor efficacy against
CES2-positive xenografts that was better than or equal to that of
INTRODUCTION
■
Doxorubicin (dox) is a successful and widely used anthracycline
anticancer agent and is a common component in many protocols
for treatment of both solid and nonsolid tumors. Its efficacy is
tempered by a considerable dose-dependent cardiotoxic side
effect, potentially leading to drug-refractory congestive heart
failure years after the cessation of treatment.1,2 Additionally,
tumors can gain resistance to dox therapy, primarily via the
multidrug-resistance phenotype (MDR) and elevated expression
of P170 glycoprotein (P170GP).3 Consequently, the clinical
attitude toward dox is one of ambivalence. Although a large body
of evidence suggests that the cardiotoxicity and antitumor
activity are derived from distinct mechanisms (oxidative damage
and topoisomerase II inhibition, respectively),2,4,5 extensive
research into compounds and formulations that selectively
enhance the anticancer activity has produced few clinically
relevant variants. Commonly, patients are treated with an
additional, iron-sequestering compound, dexrazoxane (ICRF-
187), to dampen the iron-dependent redox chemistry that leads
to the liberation of reactive oxygen species and damage to cardiac
tissue.6 Formulations in which the drug is encapsulated in a
naked liposome or a polyethylene glycol (PEG) coated liposome
have successfully improved the therapeutic index but, particularly
for PEG-coated formulations, introduce a dose-limiting hand−
foot syndrome side effect.7−9
In 2005, we described a variant of dox that demonstrated a
selective enhancement of antitumor activity.10 Termed dox-
azolidine (doxaz), the new compound is a conjugate of dox with
Received: May 21, 2012
Published: June 28, 2012
© 2012 American Chemical Society
6595
dx.doi.org/10.1021/jm300714p | J. Med. Chem. 2012, 55, 6595−6607

Journal of Medicinal Chemistry
Article
a
Scheme 1. Synthesis of doxaz and Structure of Protease-Activated doxaz Prodrugs
a
Doxorubicin was converted to doxazolidine by reaction with a molecule of formaldehyde at the exocyclic amine and alcohol groups of the
daunosamine sugar and loss of water. This results in an oxazolidine ring, which can be readily hydrolyzed, losing the formaldehyde, back to dox with
a physiological half-life of approximately 3 min at 37 °C and pH 7.10 Enzyme-activated prodrugs of doxaz, which are stabilized by acylation of the
oxazolidine nitrogen, incorporate an enzyme-cleavable trigger (R) separated from the drug by a spacer derived from p-aminobenzyl alcohol.
Protease-activated doxaz prodrug containing the triggers D-ala-L-Phe-L-Lys (aFK-Doxaz, 1) and N-acetyl-Gly-D-ala-L-Phe-L-Lys (GaFK-Doxaz, 2) are
described here, while the carboxylesterase-activated doxaz prodrug PPD, where the trigger is pentyloxycarbonyl, has been described previously.13
a
Scheme 2. Protease Activation of doxaz Prodrugs
a
Activation occurs through protease-mediated hydrolysis of the Lys-PABC amide bond. The resulting unstable intermediate undergoes a
spontaneous 1,6 elimination, releasing an iminoquinone methide and the unstable carbamic acid of doxaz. Spontaneous decarboxylation liberates free
doxaz, which readily cross-links DNA or hydrolyzes to dox under physiological conditions.
dox.13−15 Moreover, PPD was effective without producing the
severe cardiac damage characteristic of dox. However, despite
being important for activation of the clinical anticancer agents
irinotecan and capecitabine, CES2 is not a reliable marker for
tumor tissue.16 PPD, therefore, is limited in its scope and not a
candidate for wide-spectrum therapy but is a proof of concept for
anticancer agents based upon prodrug delivery of doxaz.
Proteolytic activity, on the other hand, is a very common and
widespread indicator of neoplastic tissues. In addition to creating
the physical space required for invasion and metastasis, tumor-
6596
dx.doi.org/10.1021/jm300714p | J. Med. Chem. 2012, 55, 6595−6607

Journal of Medicinal Chemistry
Article
a
Scheme 3. Synthesis of Protease-Activated Prodrug Triggers
a
Fmoc-protected PABA (3) was synthesized from commercially available starting materials and loaded onto 2-chlorotrityl resin. Solid-phase peptide
synthesis (SPPS) of bis-alloc-aFK-PABA (5) and N-acetyl-GaFK(alloc)-PABA (8) was achieved with Fmoc-protected amino acids followed by
cleavage from resin with TFA. Alloc-D-Ala (4) used in synthesis of 5 is described in Supporting Information.
a
Scheme 4. Coupling of Peptide Trigger to Doxazolidine
a
Activation of 5 or 8 to the mixed carbonates 6 and 9 was followed by reaction with doxaz to produce the protected prodrugs 7 and 10.
Deprotection of the alloc groups was accomplished with tetrakis(triphenylphosphine)palladium(0) to liberate the free bases 1 and 2. Acidification
resulted in either the acetate or phosphate salts of 1 (1a or 1b, respectively) and 2 (2a or 2b, respectively).
6597
dx.doi.org/10.1021/jm300714p | J. Med. Chem. 2012, 55, 6595−6607

Journal of Medicinal Chemistry
Article
expressed proteases can take on a more active role in the
tumorigenic transformation and development, primarily through
the liberation and activation of extracellular matrix (ECM)
associated growth factors and cytokines.17−20 Of particular
interest is the plasmin system, in which the zymogen
plasminogen is converted to active plasmin at or near the cell
surface by surface-bound urokinase-type plasminogen activator
(uPA). Plasmin can then degrade the ECM and basement
membrane components directly or indirectly through activation
of a variety of other proteolytic proenzymes, including the matrix
metalloproteases (MMPs).21 Not surprisingly, high levels of
plasmin pathway components often correlate with poor
prognosis in a wide panel of tumor types (see review by
Ulisse22), including difficult to treat cancers such as lung,23−25
liver,26,27 and pancreas.28−30
7 was formed by coupling to doxaz (Scheme 4). This was a slow
and poor-yielding reaction, typically requiring approximately 7−
10 days to reach 85−90% completion by RP-HPLC and resulted
in only a 33% isolated yield following radial silica gel
chromatography. This reaction also yielded a small amount of
alloc-aFK(alloc)-PABC-Dox due to hydrolysis of the doxaz by
adventitious water and more rapid reaction of dox than doxaz.
For use as a control in future efficacy comparisons, this material
was also purified and deprotected.
Final deprotection of the alloc groups by palladium-catalyzed
allyl transfer to morpholine was accomplished with tetrakis-
(triphenylphosphine) Pd0 with subsequent acidification
(Scheme 4). Initially, this acidification was performed with
acetic acid to yield the bis-acetate salt of aFK-Doxaz (1a).
1
However, H NMR analysis of the crude reaction mixture
Given the widespread significance of proteolytic activity in
tumor progression, metastasis, and overall prognosis, coupled
with its relative absence for normal, static tissues, proteolytic
activity makes an attractive tumor-selective filter and tool for
prodrug activation. Many protease-activated prodrug strategies
have been designed and characterized for many different
cytotoxic chemotherapeutics, including dox. In 1999, de Groot
and colleagues described a prodrug for dox in which activation
was accomplished by plasmin cleavage of a tripeptide, followed
by self-elimination of a spacer unit.31 In animal models, this
prodrug exhibited antitumor efficacy with a significantly better
toxicity profile than dox.32 Here, using this design as a model, we
describe the synthesis and characterization of a similar prodrug
for protease-activated delivery of doxaz, a significantly more
active anthracycline.
indicated the presence of a side product representing
approximately 15% of the total anthracycline composition.
This impurity was tentatively assigned to be the diastereomer of
aFK-Doxaz arising from epimerization of the α-methyl group of
the N-terminal alanine residue based on chemical shift analysis.
All other peaks appeared as expected, and integration of the
duplicated alanine α-methyl signals summed to equal the
integration of the 5′-methyl peak on the daunosamine sugar
was consistent with the anticipated 1:1 molar ratio of the
respective moieties. A homonuclear gradient COSY experiment
also confirmed coupling between the suspected L-alanine α-
methyl and α-proton signals, consistent with our assignment of
the impurity as the diastereomer resulting from Ala epimeriza-
tion. RP-HPLC was able to distinguish the two diastereomers,
but poor chromatographic resolution prevented isolation of
sufficient quantities of spectroscopically pure materials. Mod-
ification of the proposed synthesis was therefore required to
reduce the abundance of the diastereomer in order to simplify
biological characterization.
RESULTS AND DISCUSSION
■
Strategy, Design, and Synthesis of aFK-Doxaz (1). Our
initial design and proposed synthesis of 1 paralleled that of the
plasmin-activated prodrug of dox published by de Groot et al.31
Enzyme recognition is afforded by the tripartate amino acid
enzyme recognition sequence D-alanine-L-phenylalanine-L-lysine
(aFK) coupled via an arylamide bond to the p-aminobenzyl
alcohol (PABA) derived Katzenellenbogen spacer,33 which is
coupled to the daunosamine sugar moiety of the anthracycline to
form the carbamate prodrug. Upon enzymatic cleavage at the
carboxyl end of the lysine, the liberated aniline undergoes a
spontaneous 1,6 elimination to release the iminoquinone
methide and, following decarboxylation of the carbamic acid,
release of the active therapeutic (Scheme 2).
We hypothesized that the epimerization was occurring as a
result of the acidification procedure following the final alloc-
deprotection reaction. Additional attempts to salt-out the
product by varying the temperature, time, and acidic reagent
failed to yield satisfactory results. Moreover, no gains in purity
were made during our exploration of various amino-protecting
t
groups, including Boc, Fmoc, and TFA. Nevertheless, while
additional synthetic routes were being explored, we successfully
isolated small quantities of prodrug by RP-HPLC purification to
yield the pure bis-phosphate salt of aFK-Doxaz (1b) and the bis-
acetate salt of aFK-Dox.
Biological Characterization of the Bis-phosphate Salt
of aFK-Doxaz (1b). Given that the challenges of the synthesis of
plasmin-activated doxaz prodrugs limit the efficiency and
scalability of the synthesis of aFK-Doxaz (1b), a limited
biological characterization of 1b was undertaken while new
synthetic routes were being explored. This characterization
focused on demonstrating that 1b could be hydrolyzed by
plasmin and proving that plasmin hydrolysis released doxaz and
not dox.
To test the ability of plasmin to cleave 1b, we incubated 50 μM
1b with 3.3 μg/mL plasmin in PBS at 37 °C. The reaction
progress was monitored by RP-HPLC; small samples were
removed from the reaction at various times and analyzed for the
presence of the anthracycline chromophore by absorbance at 480
nm. Because of the instability of doxaz under aqueous conditions,
doxaz is rarely, if ever, directly observed by RP-HPLC. Instead, its
formation was inferred by the presence of dox over the time
course. As expected, plasmin readily catalyzed the hydrolysis of
In de Groot’s synthesis, the tripeptide was prepared using
solution-phase chemistry and subsequently coupled to PABA.
Our synthetic strategy differed only in that we used solid-phase
peptide synthesis (SPPS) and built the peptide directly onto
resin-bound PABA (Scheme 3). Fmoc-PABA (3) was formed in
high yield by reaction of Fmoc-OSu with commercially available
PABA in dry dioxane and was subsequently etherified by loading
onto a 2-chlorotrityl chloride polystyrene resin. SPPS proceeded
by standard Fmoc chemistry using Fmoc-L-Lys(Nε-alloc), Fmoc-
L-Phe, and alloc-D-Ala (4), which was prepared from
commercially available D-Ala and allyl chloroformate. Coupling
of Fmoc-L-Lys to PABA was allowed to proceed for 16 h owing to
the poor nucleophilicity of the aniline, but standard 1 h coupling
times afforded high yields of the remaining carboxamide
couplings. Following cleavage from the resin with trifluoroacetic
acid to yield the free protected peptido-PABA (5) and
subsequent activation to the p-nitrophenyl carbonate of alloc-
aFK(alloc)-PABA (6), the bis-alloc protected carbamate prodrug
6598
dx.doi.org/10.1021/jm300714p | J. Med. Chem. 2012, 55, 6595−6607

Journal of Medicinal Chemistry
Article
1b (Figure 1), and in the absence of enzyme, 1b was fully stable
(data not shown).
Figure 2. Response of MCF-7 breast cancer cells (dark bars) and
MiaPaCa-2 pancreatic cancer cells (light bars) to dox, doxaz, aFK-Dox,
and 1b. The two prodrugs were tested in the absence or presence of
plasmin. Response is measured by the IC50 in units of log M, and the
values are the average of at least six measurements. The toxicity of aFK-
Dox toward MCF-7 cells was outside the concentration range of the
Figure 1. RP-HPLC analysis of an enzymatic reaction between 50 μM
1b and 3.3 μg/mL human plasmin. The reaction was performed at pH
7.4 in PBS at 37 °C. A second measurement using slightly different
reaction times is shown as diamonds with crosses (run 2). The data were
fit using nonlinear least-squares analysis with a single-exponential
association model.
a
experiment (>−6). The error bars represent the standard error. IC50
values for dox and doxaz are from ref 13.
The cell-free HPLC analysis of enzymatic hydrolysis, coupled
with the 1H NMR verification that doxaz was intact in the final
prodrug, suggests that plasmin was catalyzing the release of
doxaz, which then quickly hydrolyzed to dox before the analysis
of the reaction. In theory, the presence of a serine protease likely
has little to no effect on the half-life of the liberated doxaz.
However, because of the inability of HPLC to differentiate
between release of dox and release of doxaz, we do not know
from the above experiment whether activation of 1b released
doxaz or dox. Since doxaz is so much more cytotoxic than dox
despite its short lifetime, a cell-based comparison of 1b and aFK-
Dox can distinguish a prodrug that releases doxaz from one that
releases dox. Two cell lines were chosen for this analysis: the
human breast carcinoma line MCF-7 and the human pancreatic
carcinoma line MiaPaCa-2. In a 3 h treatment, followed by 3−4
days of growth (until DMSO-only treatment lanes reached 80%
confluency), the dox and doxaz prodrugs produced dramatically
different growth inhibition profiles and IC50 values (Figure 2). In
both cell lines, 1b was more toxic than aFK-Dox when treated
with drug alone. This activity likely reflects the endogenous
activation of cell-expressed plasminogen to plasmin. Addition-
ally, 1b alone was equally or more toxic in both lines than the
combination of aFK-Dox and plasmin, and adding plasmin to 1b
increased this difference substantially, exhibiting greater toxicity
than treating with fully active dox alone. Taken together, these
data confirm that 1b releases doxaz, rather than dox, in a plasmin-
dependent manner. Further, considering the previously reported
preclinical advantages conferred by aFK-Dox,32 these results
suggest that further development of protease-activated doxaz
prodrugs may produce a potent, clinically attractive targeted
anticancer drug.
addition of the commercially available protected amino acid,
N-acetyl glycine, at the N-terminus of the peptide. Glycine
incorporation would negate the N-terminal amino acid’s α-
proton acidity by placing an achiral residue at this position and
eliminate the need for removal of two alloc groups in the final
deprotection step. Additionally, the presence of N-acetyl
protection of the amino terminus may provide resistance against
exopeptidase activity.34−36 Small-scale synthesis of GaFK-Doxaz
(2) yielded a single diastereomer (>95% purity by RP-HPLC)
without significant reduction of the yield as a result of the
additional deprotection, activation, and coupling reactions. We
therefore undertook a scaled synthesis of 2 and present a more
comprehensive biological characterization.
Analogous to the synthesis of 1b, N-Ac-GaFK(alloc)-PABA
(8) was prepared by SPPS (Scheme 3) and subsequently
transformed to the mixed carbonate (9, Scheme 4). Coupling of
9 with doxaz to form the protected peptidyl prodrug (10) was
improved by addition of 1-hydroxybenzotriazole (HOBt) to the
reaction mixture, resulting in complete consumption of the
starting anthracycline after 72 h, as measured by RP-HPLC.
Preparative HPLC purification gave 10 in 46% yield.
Deprotection of the lysine ε-amino group with tetrakis-
(triphenylphosphine)palladium(0) was carried out in dichloro-
methane acidified with acetic acid to yield the acetate salt of
AcGaFK-PABC-Doxaz (2a). 1H NMR of the crude salt revealed
a significant amount of contaminating triphenylphosphine oxide,
due to oxidation of the deprotection catalyst. This contaminant,
in early preparations, persisted through the final RP-HPLC
purification and was removed from subsequent preparations by
washing an aqueous solution of 2a with ethyl acetate. The crude
product was purified by preparative RP-HPLC from a C-18
column and isocratic conditions of 1:1 acetonitrile/20 mM
phosphate buffer, pH 4.6−4.8, to give the pure phosphate salt
(2b), which was formulated in PEG-400 in saline (10% v/v) with
a yield of 78%.
Strategy, Design, and Synthesis of GaFK-Doxaz (2).
Preliminary characterizations of 1b demonstrated that plasmin-
activated doxaz prodrugs have potential as effective anticancer
compounds and are significantly more efficacious than similar
dox prodrugs. However, formation of the alanine epimer
prevented isolation of sufficient quantities of pure material for
wider biological analyses. Modification of the deprotection
conditions proved unsatisfactory, but we hypothesized that the
epimerization might be circumvented altogether with the
Enzyme Kinetics of Activation. The rate and kinetics of
hydrolysis of 2b were measured by RP-HPLC, monitoring the
dox released from doxaz hydrolysis after enzyme-mediated
cleavage of 2b. In a time course of activation, 50 μM 2b was
6599
dx.doi.org/10.1021/jm300714p | J. Med. Chem. 2012, 55, 6595−6607

Journal of Medicinal Chemistry
Article
incubated at 37 °C with 1.2 μg/mL human plasmin in PBS. As
expected from our characterization of 1b, dox was quickly
produced and the prodrug nearly fully consumed by 2 h,
indicating release of doxaz, which then quickly degraded to dox
(Figure 3). In separate reactions containing no enzyme, there
activator by removing any small thiols that may have been
blocking the active site cysteine of cathepsin B,42−45 reactions
between 2b and the enzyme produced dox. The specific activity
for purified human cathepsin B on 2b was defined to be 1040 U/
mg (Table 1). Under the same conditions as cathepsin B, papain,
a cysteine protease derived from papaya and a representative
member of the papain family of proteases, showed a specific
activity of 890 U/mg (data not shown), suggesting that other
members of the papain family in humans may also function to
activate 2b.
Michaelis−Menten kinetic experiments for the two human
enzymes, plasmin and cathepsin B, were performed on substrate
ranges from 10 to 400 μM using the same buffer conditions as
above. Curve-fitting analyses (Table 1) demonstrated that the
enzymes were similar in Km (171 41 μM for plasmin compared
to 108 12 μM for cathepsin B), but the calculated kcat values
((2.45 0.46 μM 2b/s)/μM plasmin and (0.49 0.02 μM 2b/
s)/μM cathepsin B) revealed that plasmin was 5-fold better at
catalyzing the hydrolysis of 2b than was cathepsin B. Addition-
ally, plasmin was more than 3-fold more efficient than cathepsin
B. The relatively low catalytic activity of cathepsin B was not due
to significant deviation from an optimal condition, such as pH
values closer to 5; in activity assays in which the PBS-NAC buffer
was acidified with phosphoric acid to pH 5.5, cathepsin B was
only 10% as active against 2b as at pH 6.4 in standard PBS-NAC
(data not shown), suggesting that our results at pH 6.5 may be
indicative of the best activity of cathepsin B toward 2b. These
results indicate that serine proteases are better activators of 2b
than cysteine proteases but that tumors that have high levels of
cathepsin B present in their microenvironments are still likely to
be affected by 2b treatment, even in the absence of plasmin,
thereby broadening the spectrum of a potential proteolytically
activated doxaz-based therapeutic regimen.
Figure 3. RP-HPLC analysis of an enzymatic reaction between 50 μM
2b and 1.2 μg/mL human plasmin. The reaction was performed in PBS,
pH 7.4, at 37 °C. Repeated measurements with slightly different reaction
times are shown as either circles with included dots (run 2) or crosses
(run 3). The combined data were fit using nonlinear least-squares
analysis with a single-exponential association model.
was no observable increase in dox over the course of 24 h,
showing that 2b is stable in the absence of enzyme. To simplify
downstream analyses, we made measurements of the activity of
plasmin using 2b as a substrate. By use of the definition of 1 unit
(U) as the amount of enzyme that catalyzes 1 μM/min of 50 μM
2b, purified human plasmin was calculated to have a specific
activity of 1940 U/mg (Table 1). A similar serine protease,
Plasma Stability of 2b. The activity of proteases in the
bloodstream is hindered by the presence of α2-macroglobulin
and α2-antiplasmins; therefore, 2b should show little to no
products of hydrolysis when incubated with human plasma. To
demonstrate this, 50 μM 2b was incubated for 24 h at 37 °C in
plasma from healthy adult human donors. Periodically, samples
were removed and analyzed for the appearance of dox and
disappearance of 2b by RP-HPLC and absorbance at 480 nm. To
prevent damage to the analytical reverse phase column, the
proteins were first removed by precipitation with ethanol. The
results shown in Figure 4 indicate that 2b levels in the
supernatant do decrease with time, but a similar increase in
dox is not observed, accounting for only approximately 9% of the
total absorbance at 480 nm. Additionally, the loss of 480 nm
absorbance from the prodrug was not distributed to other,
unidentified red products but instead was lost from the total
absorbance of the sample. This suggests that 2b was incorporated
into the pellet upon ethanol precipitation, likely through
nonspecific binding to serum albumin, which is well-known as
a drug carrier.46,47 The lack of dox production indicates that, in
human plasma, 2b is very stable over 24 h and is unlikely to be
prematurely activated.
Table 1. Activity of Human Plasmin and Human Cathepsin B
Using 2b as a Substrate
a
plasmin (human)
1940
cathepsin B (human)
specific activity (U/mg)
Vmax (μM Dox/min)
1040
3.76 0.43
1.32 0.06
108.25 12.47
0.49 0.02
(4.59 0.33) × 103
K
M (μM)
turnover number (kcat, s−1
catalytic efficiency (M−1 s−1
171.10 41.25
2.45 0.46
(1.43 0.14) × 104
)
)
a
Plasmin reactions were performed in PBS, pH 7.4, and cathepsin B
reactions were performed in PBS containing 10 mM N-acetylcysteine
(PBS-NAC), pH 6.5. One unit is defined as the hydrolysis of 1 μM 2b
per min.
trypsin, was chosen as a comparison to measure the specificity of
the GaFK peptide toward plasmin. Reactions with bovine trypsin
also readily produced dox, and a specific activity for bovine
trypsin was calculated to be 4340 U/mg, indicating that serine
proteases other than plasmin can activate 2b.
While 2b was specifically designed for activation by plasmin,
there are members of other protease families that cut the C-
terminal side of Phe-Lys sequences, and the enzyme cathepsin B
is of particular interest because of its implication in the
progression and poor prognosis for a variety of human
cancers.37−39 A prodrug that delivers dox upon cathepsin B
cleavage following Phe-Lys has been described.40,41 In initial
experiments in PBS, human cathepsin B failed to demonstrate
any HPLC-detectable production of dox. However, when 10 mM
N-acetylcysteine was added to the PBS (PBS-NAC), which
served to both acidify the buffer (to pH 6.5) and act as an
Growth Inhibition of a Panel of Human Cancer Lines.
Initial growth inhibition tests with 2b were performed on
MiaPaCa-2 and MCF-7 human cancer cell lines. As with 1b, both
cell lines responded equally well to prodrug alone, with log IC50
values in the range of −6.6 to −6.7 (Table 2). When co-treated
with 0.2 U plasmin, the potency of the treatments increased by
approximately 10-fold in each cell line, confirming that plasmin
can mediate the release of doxaz from 2b. As in the HPLC activity
6600
dx.doi.org/10.1021/jm300714p | J. Med. Chem. 2012, 55, 6595−6607

Journal of Medicinal Chemistry
Article
tested with 2b alone and 2b + 0.2 U exogenous human plasmin.
As shown in Table 3, we tested four pancreatic cancer lines
Table 3. Growth Inhibition of a Panel of Cell Lines by 24 h
Treatment with 2b with or without 0.2 U of Human Plasmin,
a
Represented by the IC50 in log M Units
2b alone
2b + plasmin (0.2 U)
BxPC3
L3.3
−6.0 0.1
−6.6 0.1
−7.4 0.1
−8.40 0.07
−8.58 0.04
−8.50 0.04
−7.53 0.05
−7.93 0.05
L3.5
−6.65 0.04
−6.50 0.08
−5.80 0.03
−6.04 0.05
−6.3 0.3
L3.6PL
b
SHP77
b
NCI-RES/Adr
d
cancer cell mean
−8.1 0.5
−8.52 0.04
c
Figure 4. RP-HPLC analysis of the stability of 2b in human plasma over
48 h. 2b (50 μM) was incubated with undiluted human plasma from
healthy individuals, and the time points were analyzed for the presence
of all 480 nm absorbing species after precipitation of the protein fraction
with absolute ethanol. Only 2b (dark diamonds) and dox (light
diamonds) were present. The points represent the mean the standard
deviation of three measurements, and percentages are relative to the 2b
peak area present at the start of the experiment.
HEK-293T
−6.7 0.1
a
BxPC3, L3.3, L3.5, and L3.6PL are pancreatic cancer. SHP77 are
small-cell lung cancer, and NCI-RES/Adr are ovarian cancer cells. The
last two lines are dox-resistant. HEK-293T cells are non-tumor-derived
human embryonic kidney cells. Values for individual cell lines are the
mean standard error of at least 12 measurements. The values for
cancer cells were averaged and are shown in the “cancer cell mean”
b
c
row
the standard deviation. Doxorubicin resistant. Non-tumor-
d
derived. P < 0.0001 relative to 2b alone.
assays described above, co-treatment with bovine trypsin (0.2 U)
reduced growth to a similar extent as plasmin, demonstrating that
other serine proteases can easily activate 2b. This has potential to
be very beneficial, as many cancers have been found to have high
levels of trypsin or trypsin-like serine protease activity.48−51
Additionally, compared to treatment with 2b alone, treatment
with either cathepsin B or papain along with 2b resulted in
increased growth inhibition in both cell lines. However, this was
significantly less than the growth inhibition mediated by 2b
activated by either serine protease. These results further indicate
that serine proteases are more effective at activating 2b. When
proteolytic activity is inhibited with either 5 μM aprotinin to
inhibit serine proteases or Roche’s Complete protease inhibitor
cocktail (made to 1× according to the manufacturer’s
instructions), the reduction in cellular response to 2b is apparent
but not significant. This may be due to constant secretion of
active proteases by the cell, which effectively overwhelms the
limited amount of added inhibitor, since the potency of the
inhibitors was verified by their ability to prevent the increase in
potency of a 2b−plasmin co-treatment over 2b treatment alone
(data not shown). However, the inability of high concentrations
of protease inhibitors to protect cells could also could indicate
that there are alternative mechanisms of activation beyond those
probed in this study. These alternative mechanisms will be the
subject of future studies and characaterization of 2b.
(BxPC3, L3.3, L3.5, and L3.6PL), a small cell lung cancer line
(SHP77), and an ovarian cancer line (NCI-RES/Adr). The last
two lines, SHP77 and NCI-RES/Adr, are resistant to dox but are
sensitive to doxaz treatment.11 Finally, we tested a noncancer
derived, but uPA-rich, human cell line, HEK-293T. Among the
cancer cell panel, the average log IC50 was −6.3, with a range
from −5.8 in the SHP77 to −6.65 in the L3.5. The growth of the
nontumor HEK-293T line was inhibited to approximately the
same extent as the cancer lines with either treatment condition.
This is in line with observations that this cell line expresses a
particularly high level of uPA, a serine protease that converts
plasminogen into plasmin.52 Given that at least two different
serine protease enzymes can catalyze the hydrolysis of 2b, it is
plausible that other enzymes with related activity, such as uPA
and tissue plasminogen activator (tPA), could exhibit at least low
level catalysis of 2b. Since tumors often overexpress multiple
members of proteolytic pathways, while normal tissues tightly
control protease activity, the broad specificity of 2b may allow for
a more tunable therapeutic; an efficacious dose for a very uPA/
plasmin-heavy tumor may be different from an equivalently
efficacious dose for a cathepsin B-heavy tumor. The proteolytic
profile contributed by invading endothelial cells during active
angiogenesis may allow for further distinction between tumor
and normal tissues, thereby potentially allowing doses to be
reduced to achieve the same antitumor effect with fewer side
effects and tissue toxicities.
The results presented above indicate that 2b may be activated
by both serine and cysteine proteases; in a mixed protease
environment, as is likely present at a tumor, plasmin and related
serine proteases will play a larger role in activation of 2b than will
cysteine proteases. Therefore, a panel of cancer cell lines was
Cellular Uptake. The presumed activation scheme for 2b
utilizes external activation by secreted proteases, followed by
cellular uptake of the active doxaz cytotoxin. However, it is
a
Table 2. Growth Inhibition of MCF-7 and MiaPaCa-2 Cells by 24 h Treatment of 2b with Various Activators and Inhibitors
activation
inhibition
2b + plasmin
(0.2 U)
2b + trypsin
(0.2 U)
2b + papain
(0.2 U)
2b + cathepsin B
2b + aprotinin
(5 μM)
2b + Complete protease
2b alone
(0.2 U)
inhibition (1×)
MiaPaCa-2
MCF-7
−6.6 0.2
−6.7 0.1
−7.63 0.03
−7.57 0.02
−7.58 0.04
−7.5 0.1
−7.16 0.03
−7.22 0.05
−6.80 0.08
−6.92 0.09
−6.55 0.06
−6.53 0.04
−6.4 0.2
−6.6 0.3
a
IC50 values are reported as log M, and the value of U is defined as the hydrolysis of 1 μM 2b per min.
6601
dx.doi.org/10.1021/jm300714p | J. Med. Chem. 2012, 55, 6595−6607

Journal of Medicinal Chemistry
Article
possible that some fraction of 2b may cross the plasma
membrane and be activated internally. Although there is likely
to be far fewer intracellularly active proteases relative to the
potential activity of external proteases at a tumor site, several
cellular processes require proteolytic pathways, particularly
proteosome and lysosomal protein degradation pathways. To
test for cellular uptake of 2b, MCF-7 and MiaPaCa-2 were
treated with either 0.5 μM 2b alone or co-treatments of 0.5 μM
2b and 0.2 U plasmin for up to 5 h. The cells were then analyzed
by flow cytometry for the anthracycline fluorophore. Uptake of
2b alone is characterized by a fast initial increase in fluorescence,
up to 1.8-fold over background, followed by a slower increase to
approximately 2- to 2.5-fold over background for the rest of the
period (Figure 5), likely representing a baseline rate of hydrolysis
shown, this ratio of uptake difference, observed between dox-
sensitive SK-HEP-1 liver carcinoma cells and dox-resistant NCI-
RES/Adr (formerly MCF-7/Adr) cells, results in cytotoxic
responses that differ by about 1.3−1.5 orders of magnitude.13
While future studies will provide a better characterization, such as
a description of the fast but small initial increase in fluorescence
of cells in response to 2b without exogenous plasmin, these data
suggest that intact 2b is poorly taken up by cancer cells but that
upon activation, liberated doxaz readily crosses the membrane.
Therefore, it is unlikely that high levels of 2b activation will occur
by intracellular proteolytic pathways, reducing the likelihood for
side effects and toxicities to normal tissues and organs.
CONCLUSIONS
■
The aim of this research was to produce a high-potency, broad
spectrum antitumor prodrug to deliver our powerful cytotoxin
doxaz. Release of doxaz in this design is mediated by proteolytic
hydrolysis, as compared to our previous work with a CES2-
activated doxaz prodrug, which has shown promise but is likely a
narrow spectrum prodrug.15 Our first design of a proteolytically
released doxaz prodrug was based upon previously published
work on dox but was limited in its development potential by a
persistent epimerization of the terminal non-natural D-alanine.
This is unlikely to have any directly detrimental effects on
endopeptidase-specific hydrolysis of the Lys-p-aminobenzyla-
mide bond. However, the loss of the non-natural isomer at the N-
terminus may increase the susceptibility of the prodrug to
degradation by aminopeptidase activity.53 This could result in
poorer plasma stability of the intact prodrug, but perhaps more
importantly, it changes the peptidyl substrate. This could result
in unknown catalytic hydrolysis rates from our targeted enzymes,
which may change the profile of activating proteases and
potentially introduce a host of side effects that may otherwise not
be present in therapy with this prodrug. However, using the small
amounts of isomerically pure 1b, we were able to demonstrate
that additional efforts to improve the design and synthesis of a
protease-activated doxaz prodrug were warranted. Plasmin-
induced cleavage of 1b was fast and robust, and there was a
significant advantage in cytotoxic activity by using doxaz as the
warhead instead of dox. Therefore, having evidence that a doxaz
prodrug containing the aFK sequence was a promising core for a
protease-activated prodrug, we directed our efforts at producing
a more scalable version that avoided the epimerization problems
of 1b.
A more successful scalable design incorporated N-acetyl
glycine at the amino terminus of the peptidyl chain. 2b, unlike 1b,
exhibited no evidence of epimerization at any point in the
synthetic process and maintained good kinetics of activation by
plasmin and stability in human plasma. 2b was a good substrate
for plasmin but could also be hydrolyzed by the related serine
protease trypsin, as well as members of the papain family,
specifically papain and human cathepsin B. This multienzyme
characteristic of 2b adds to the spectrum of tumor profiles against
which it could be beneficial, particularly in the case of cathepsin
B, but also suggests that enzymes with trypsin-like serine
protease activity, such as the tumor-associated enzyme uPA, can
also contribute to activation. Antitumor activity alone was good
and was enhanced by the addition of exogenous enzymes.
Importantly, 2b was active against SHP77 and NCI-RES/Adr
cells, both of which are resistant to dox and sensitive to doxaz.
Finally, although most normal cells possess some proteolytic
activity, particularly in the lysosomal compartment, unactivated
2b is unlikely to be highly toxic because of its limited membrane
Figure 5. Uptake of 2b with or without plasmin into human cancer cells.
An amount of 250 000 cells per well of either MCF-7 (A) or MiaPaCa-2
(B) was treated with 0.5 μM 2b alone (dark diamonds) or 0.5 μM 2b
with 0.2 U human plasmin (light diamonds) for various time periods and
analyzed by flow cytometry for the presence of fluorescence due to the
anthracycline fluorophore and compared to the fluorescence of
untreated cells. Data representative of repeated measurements (at
different time points) are shown here, with variation of approximately
10−20% between the experiments.
of 2b to release the highly permeable doxaz by cell-secreted
proteases. When exogenous plasmin is added, the cellular
fluorescence is much greater, approaching 7-fold over back-
ground by 4−5 h, presumably reflecting the increased abundance
of cell-permeable doxaz. Although the difference in cellular
fluorescence between the co-treatment and treatment with only
2b is not particularly dramatic (approximately 3-fold), this level
can have a demonstrable difference in toxicity. As previously
6602
dx.doi.org/10.1021/jm300714p | J. Med. Chem. 2012, 55, 6595−6607

Journal of Medicinal Chemistry
Article
was diluted with 100 mM sodium phosphate buffer, pH 8.5, for a final
concentration of 0.4 mg/mL dox−HCl as a mixture of precipitated and
solubilized material. The free base of dox was extracted from the solution
by two washes with equal volumes of chloroform, leaving almost no red
color in the aqueous fraction. The chloroform was dried with anhydrous
sodium sulfate, filtered, and removed by rotary evaporation to yield a red
solid. The solid was dried thoroughly under high vacuum (10−2 Torr)
for at least 2.5 h to yield pure dox free base in a final yield of 98%, as
indicated by optical density at 480 nm and a molar extinction coefficient
of 11 500 M−1 cm−1 in 75% DMSO/25% water.
Doxazolidine. Dox free base was dissolved to a final concentration
of 5−10 mg/mL in deuteriochloroform, and 1.1−2 equiv of prilled
paraformaldehyde (Aldrich, Milwaukee, WI) was added. The reaction
mixture was monitored by 1H NMR, with complete consumption of dox
occurring after 2−3 days, as evidenced by the appearance of the doxaz−
methylene AX pattern at 4.31 and 4.68 ppm and doxf AX pattern at 4.21
and 4.73 ppm in the 1H NMR spectrum. Loss of dox was evident by the
dox-specific aromatic triplet pattern for the proton at the 2-position,
which appears at 7.81 ppm (compared to 7.78 for doxaz and 7.70 for
doxf). The amount of paraformaldehyde added determines the doxaz/
doxf ratio, since doxaz reacts with an additional equiv of formaldehyde to
form doxf. Complete assignments of the 1H NMR spectra for doxaz and
doxf have been published.10 The reaction mixture was then filtered and
the deuteriochloroform removed by rotary evaporation. The doxaz/doxf
mixture was used without further purification, since doxf readily
hydrolyzes to produces 2 equiv of doxaz.
Fmoc-p-aminobenzyl Alcohol (Fmoc-PABA, 3). N-
(Fluorenylmethyloxycarbonyloxy)succinimide (N-Fmoc-N-hydroxy-
succinimide, 6.0 g, 17.8 mmol) in p-dioxane (60 mL) was added
dropwise to p-aminobenzyl alcohol (PABA, 2.5 g, 20.5 mmol) in p-
dioxane (30 mL). After the mixture was stirred for 48 h, 90 mL of
deionized H2O was added and the desired product immediately
precipitated. The product was isolated by filtration and washed 4 times
with deionized H2O (60 mL). The isolated Fmoc-PABA (5.9 g, 95%)
showed the following spectral properties: 1H NMR (CDCl3) δ 4.26 (t,
1H, J = 7 Hz, Fmoc-CH), 4.53 (d, 2H, J = 7 Hz, Fmoc-CH2), 4.63 (s, 2H,
PABA-CH2), 6.62 (bs, 1H, NH), 7.30 (t, 2H, J = 7 Hz, Fmoc), 7.34 (d,
2H, J = 7 Hz, PABA), 7.36 (b, 2H, PABA), 7.40 (t, 2H, J = 7 Hz, Fmoc),
7.60 (d, 2H, J = 7 Hz, Fmoc), 7.77 ppm (d, 2H, J = 7 Hz, Fmoc); ESI-
MS, observed m/z = 384.1014; calculated m/z for (M + K+) = 384.0996.
Loading of Fmoc-PABA to 2-Chlorotrityl Resin. Fmoc-PABA (2
equiv) was added to 1.0 g of 2-chlorotrityl chloride polystyrene resin in
10 mL of dry THF followed by 4 equiv of dry pyridine. The solution was
stirred and heated at 60 °C in an oil bath for 16 h under argon. The
solution was filtered through a coarse sintered glass frit, and the resin was
washed with DCM/MeOH/DIEA, 17:2:1 mL (3×), followed by 10 mL
of DCM (2×), 10 mL of DMF (2×), 10 mL of DCM (3×). The resin
was dried under high vacuum (10−2 Torr) overnight and then checked
for loading level according to previously published methods.54
Solid Phase Peptide Synthesis. The peptide was synthesized by
the solid phase method using the Fmoc strategy starting with preloaded
Fmoc-PABA. The peptide was prepared on a 0.25 mmol scale by single
amino acid couplings using a 4-fold excess of N-acetyl-Gly and the
Fmoc-protected amino acids D-Ala (Chem-Impex, International, Wood
Dale, IL), L-Phe, and L-Lys-alloc. Activation was done with 2-(1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HBTU)/N-hydroxybenzotriazole (HOBT), and the synthesis was
performed on an ABI 433A peptide synthesizer (Applied Biosystems,
Carlsbad, CA). Owing to the poor nucleophilicity of the aniline nitrogen
of PABA, the coupling of the first amino acid, Fmoc-Lys(alloc), required
an extended coupling time by modifying the software instructions to
vortex the reaction for 60 s, then allow it to sit for 10 min. This cycle was
repeated 88 times. Fmoc groups were removed by sequential treatment
(3×) with 20% piperidine/DMF, and all other amino acid couplings
followed standard conditions, to yield Ac-Gly-D-Ala-L-Phe-L-Lys(alloc)-
PABA (Ac-GaFK(alloc)-PABA) coupled to the polystyrene resin.
Cleavage of Ac-GaFK(alloc)-PABA (8) from the Resin and
Activation to Ac-GaFK(alloc)-PABA-pNP Carbonate (9). The resin
was placed into a medium-grain fritted funnel and washed twice with
DCM (20 mL). Pyridine/MeOH (10% v/v, 20 mL) was added to the
permeability. Therefore, the majority of the release of doxaz is
expected to occur externally because of secreted proteases, the
expression of which has been consistently linked to highly
invasive, angiogenic tumor environments.21,22
MATERIALS AND METHODS
■
General. Clinical samples of doxorubicin hydrochloride formulated
with lactose were gifted by FeRx (Aurora, CO). Unless otherwise noted,
all reagents and amino acids for solid-phase peptide synthesis were
purchased from Nova Biosciences (La Jolla, CA) and other chemicals
and reagents were acquired from Aldrich (Milwaukee, WI). Analytical
HPLC was performed on Agilent 1050/1100 hybrid instruments
equipped with a 1050 series autoinjector, a 1100 series UV/visible diode
array detector, and a 1046A fluorescence detector (Santa Clara, CA). An
Agilent Zorbax octadecylsilyl (C18) reverse phase column (4.6 mm i.d.
× 150 mm, 5 μm) was used for chromatography. Unless specifically
indicated, elution was performed at 1 mL/min and room temperature
with gradients of acetonitrile and 20 mM sodium phosphate buffer, pH
4.6, containing 0.02% sodium azide. Method 1 had acetonitrile
percentages of 20% from 0 to 1.5 min, 30% at 5 min, 80% from 15 to
20 min, and 20% at 24 min. Method 2 consisted of acetonitrile
percentages: 20% to 40% from 0 to 5 min, 50% from 8 to 9 min, and 20%
at 11 min. The presence of anthracycline-containing molecules was
monitored by absorbance at 480 nm, and retention times are noted
individually. All NMR spectra were taken at 400 or 500 MHz on a Varian
Unity INOVA spectrometer (Palo Alto, CA) or a Bruker-Avance III 300
MHz spectrometer (Billerica, MA) in deuterated solvents from
Cambridge Isotope Laboratories, Inc. (Andover, MA). Chemical shifts
are reported in δ values of ppm and were standardized by the residual
solvent peak in MestReNova NMR software (Mestrelab Research,
Santiago de Compostela, Spain). The purity of all characterized
compounds was verified to be at least 95% by using a combination of
HPLC (monitoring wavelengths of 230, 260, 280, 300, 400, and 480
nm) and NMR. UV−vis spectroscopy was performed on a Hewlett-
Packard/Agilent 8452A diode array instrument. Electrospray mass
spectra were obtained with a Perkin-Elmer Sciex API III+ (Waltham,
MA) or ABI Pulsar QqT high resolution instrument (Foster City, CA),
equipped with an ion-spray source at atmospheric pressure. Purification
by radial chromatography was done with a Harrison Research model
7924T Chromatotron (Palo Alto, CA). Analysis by flow cytometry was
performed with a Becton-Dickinson Biosciences FACscan (San Jose,
CA), and a PowerWave X plate reader (BIO-TEK Instruments,
Winooski, VT) was used for analyzing 96-well plate growth inhibition
assays.
General Tissue Culture. All tissue culture reagents were acquired
from Gibco Life Sciences (Grand Island, NY) and plates from Sarstedt,
Inc. (Newton, NC) or Corning, Inc. (Corning, NY) unless otherwise
noted. All cells were acquired from the American Type Culture
Collection (Rockville, MD) except NCI/RES-Adr (a gift from William
Wells, Michigan State University, East Lansing, MI), L3.3, L3.5, and
L3.6PL (all gifts of Wells Messersmith, University of Colorado,
Anschutz Medical Campus, Denver, CO). NCI/RES-Adr and SHP77
were maintained in RPMI-1640, supplemented with 10% (v/v) fetal calf
serum and 1% (v/v) pencillin−streptomycin stock solution. MCF-7,
MiaPaCa2, BxPC3, L3.3, L3.5, L3.6PL, and the nontumor-derived
HEK-293T were grown in Dulbecco’s modified essential medium
(DMEM) containing the same above supplements in the same
concentrations. All of the above cells were grown in a humidified
incubator under an atmosphere of 5% CO2/95% air at 37 °C.
Synthesis. The synthesis of the final compound, 2, is described
below, while the synthesis of the initial design, 1, can be found in
Supporting Information. The water miscible solvents, tetrahydrofuran
(THF) and DMSO, were made dry by distillation from Na0/
benzophenone and storage over 4 Å molecular sieves, respectively.
Doxorubicin Free Base from Clinical Samples. Lyophilized
pellets of expired clinical samples of doxorubicin HCl (20−50 mg)
containing 100−250 mg of lactose monohydrate (Bedford Laboratories,
Bedford, OH) were dissolved in methanol to a final concentration of 2
mg/mL and combined in a separatory funnel. The methanol solution
6603
dx.doi.org/10.1021/jm300714p | J. Med. Chem. 2012, 55, 6595−6607

Journal of Medicinal Chemistry
Article
receiving flask to protect the peptide after cleavage. TFA/DCM (2% v/v,
10 mL) was then added and swirled for 1 min. The mixture was then
gently vacuum filtered without allowing the resin to proceed to dryness.
This step was repeated 3 times followed by a 20 mL DCM wash. The
filtrate contained the peptide and pyridinium trifluoroacetate salt and
was rotary-evaporated to ∼10% of the original volume. Approximately
40 mL of deionized water was added to the flask, and the solution was
allowed to cool in an ice bath for at least 10 min. The precipitate was
collected on a fritted funnel and washed with 10 mL of deionized water
(3×), followed by 10 mL of diethyl ether (2×). An oven-dried round-
bottom flask equipped with a magnetic stir bar was cooled under argon
and charged with 25.0 mL of dry THF and 8 (376.6 mg, 0.577 mmol).
To the stirring suspension, p-nitrophenyl chloroformate (127.9 mg, 1.1
equiv) was added followed immediately by pyridine (51.33 μL, 1.1
equiv). Over the course of 20 min, all solids went into solution, after
which the reaction progress was monitored by RP-HPLC (method 1)
until complete, as evidenced by the consumption of 8 (eluting at 13.4
min), which typically required 18−24 h. The reaction mixture was then
diluted with 250 mL of ethyl acetate and washed once with 150 mL of
deionized water, followed by extractions with 150 mL of saturated
sodium bicarbonate until the aqueous layer no longer turned yellow
(typically five extractions). The organic layer was dried over sodium
sulfate and concentrated by rotary evaporation to yield 400 mg (85%) of
a pale yellow solid that was used without further purification. Ac-
GaFK(alloc)-PABA-pNP was characterized by the following spectro-
scopic data with NMR assignments made from 1H NMR and
homonuclear COSY (DMSO-d6, 500 MHz): δ 0.94 (d, 3H, J = 7.0
Hz, Ala-CH3), 1.32−1.27 (m, 1H, Lys-γ′), 1.40−1.33 (m, 1H, Lys-γ),
1.49−1.40 (m, 2H, Lys-δ), 1.70−1.61 (m, 1H, Lys-β′), 1.77−1.71 (m,
1H, Lys-β), 1.82 (s, 3H, N-acetyl), 2.75 (dd, 1H, J = 13.7, 10.7 Hz, Phe-
β), 2.98 (apparent q, 2H, J = 6.3 Hz, Lys-ε), 3.09 (dd, 1H, J = 13.7, 3.5
Hz, Phe-β′), 3.62 and 3.66 (AB of ABX pattern, 2H, JAB = 16.5, JAX = 5.8,
JBX = 5.7 Hz, Gly-α), 4.24 (apparent p, 1H, J = 7.0 Hz, Ala-α), 4.35
(apparent q, 1H, J = 7.7 Hz, Lys-α), 4.43 (d, 2H, J = 5.3 Hz, alloc), 4.56
(ddd, 1H, J = 10.7, 8.6, 3.5 Hz, Phe-α), 5.14 (dd, 1H, J = 10.6, 3.0 Hz,
alloc), 5.25 (s, 2H, Bn), 5.88 (ddt, 1H, J = 17.0, 10.6, 5.3 Hz, alloc),
7.18−7.12 (m, 1H, p-Ph), 7.29−7.18 (m, 5H, o,m-Ph and Lys-ε-NH),
7.42 (d, 2H, J = 8.5 Hz, PABC-H2,6), 7.60−7.53 (AA′XX′ pattern, 2H,
PNP-H2,6), 7.67 (d, 2H, J = 8.5 Hz, PABC-H3,5), 8.00 (d, 1H, J = 7.3 Hz,
Ala-α-NH), 8.04 (X of ABX, 1H, JAX = 5.8, JBX = 5.7 Hz, Gly-α-NH),
8.22 (d, 1H, J = 7.4 Hz, Phe-α-NH), 8.24 (d, 1H, J = 6.2 Hz, Lys-α-NH),
8.36−8.28 (AA′XX′ pattern, 2H, PNP-H3,5), 10.05 (s, 1H, PABC-NH)
ppm.
1H, Lys-β), 1.88 (s, 3H, Ac), 2.1 (dd, 1H, J = 14, 4 Hz, 8), 2.17 (broad m,
1H, 2′), 2.41 (d, 1H, J = 14, Hz, 8), 3.01 (d, 1H, J = 18 Hz, 10), 2.98 (dd,
1H, J = 9, 5 Hz, Phe-β), 3.09 (m, 2H, Lys-ε), 3.23 (m, 1H, Phe-β), 3.23
(d, 1H, J = 18 Hz, 10), 3.98 (dd, 1H, J = 5, 1 Hz, 4′), 4.03 (s, 3H, 4-
OMe), 4.10 (m, 1H, Ala-α), 4.08−4.12 (m, 2H, 3′ and 5′), 4.45 (dd, 1H,
J = 9, 5 Hz, Lys-α), 4.49 (broad, 2H, 3‴), 4.55 (dd, 1H, J = 9, 6 Hz, Phe-
α), 4.70 (s, 2H, 14), 4.90 (d, 1H, J = 4 Hz, OCH2N), 4.96 (broad, 1H,
OCH2N), 5.06 and 5.8 (broad AB pattern, 2H, J = 11 Hz, Bn), 5.14 (d,
1H, J = 10 Hz, 1‴c ROESY), 5.23 (d, 1H, J = 18 Hz, 1‴t ROESY), 5.26
(m, 1H, 7), 5.39 (t, 1H, J = 5 Hz, 1′), 5.85 (ddt, 1H J = 16, 10, 5 Hz, 2‴),
7.15 (m, 1H, Phe-p-Ph), 7.16 (m, 2H, Phe-o-Ph), 7.22 (m, 2H, Phe-m-
Ph), 7.26 (d, 2H, J = 8 Hz, 2″ ROESY), 7.37 (d, 1H, J = 8 Hz, 3), 7.56 (d,
2H, J = 8 Hz, 3″), 7.74 (t, 1H, J = 8 Hz, 2), 7.99 (d, 1H, J = 8 Hz, 1); 13C
NMR chemical shifts from HSQC δ 15.75 (5′-Me), 16.19 (Ala-Me),
22.25 (Ac), 22.54 (Lys-γ), 29.11 (Lys-δ), 29.27 (2′), 30.49 (Lys -β),
34.09 (10), 36.73 (Phe-β), 35.84 (8), 40.33 (Lys-ε), 42.79 (Gly-α),
49.83 (Ala-α), 53.68 (Lys-α), 55.16 (Phe-α), 56.59 (4-OMe), 65.05
(14), 65.33 (3′ and 5′), 65.61 (3‴), 66.79 (Bn), 68.95 (7), 77.60 (4′),
78.83 (O-CH2-N), 99.59 (1′), 117.29 (1‴), 118.97 (3), 119.76 (1),
120.11 (3″), 126.88 (p-Phe), 128.81 (o-Phe), 128.79 (2″), 128.52 (m-
Phe), 132.79 (2‴), 135.54 ppm (2); some unprotonated carbons from
HMBC δ 121.7 (4a), 133.8 (6a), 161.5 (4), 170.5 (Lys-α CO), 171.8
(Ac-CO), 170.8 (Gly-CO), 171.9 (Phe-CO), 173.7 (Ala-CO), 213.9
ppm (13); MS-ESI+, observed MH+ 1234.4832; calculated MH+
1234.4827.
Deprotection To Form GaFK-Doxaz, Phosphate Salt (2b).
Compound 10 (5.3 mg, 0.0043 mmol) was dissolved in 600 μL of 5:1
DCM/AcOH. To this, tetrakis(triphenylphosphine)Pd0 (4.96 mg, 1
equiv, Strem Chemicals, Newburyport, MA) was added. The reaction
was left in the dark for 80 min, at which point the DCM was removed by
rotary evaporation. The acidic solution was diluted with 6 mL of 20 mM
phosphate buffer, pH 4.6, and extracted 3 times with 6−7 mL of ethyl
acetate to remove any triphenylphosphine oxide produced during the
deprotection, spinning briefly at 1500g to produce a clean interface. The
desired product was purified from the extracted aqueous solution by
preparative HPLC on a C-18 Dyanmax 100 Å, 25 cm semipreparatory
column (10 mm i.d.) run isocratically with 50% acetonitrile, 50% 20 mM
phosphate buffer, pH 4.6, at 3 mL/min. The product eluted at 10 min.
The eluent was lyophilized to yield a red solid consisting of a mixture of
the final material and sodium phosphate in 95% overall yield from 9. The
prodrug was dissolved away from the majority of the sodium phosphate
by five washes with 50 μL of 9:1 DMSO/water to yield an acidic water−
DMSO solution of 2b. Cold storage (>3 weeks at −20 °C) of 2b in this
form eventually resulted in multiple red products by HPLC, presumably
due to a low level reactivity of DMSO with the small percentage of
deprotonated Lys-ε-NH2. To prevent this, following removal of the
majority of the phosphate salts, the DMSO was evaporated by SpeedVac
(10−2 Torr) and the pure final material dissolved in saline (0.9% NaCl)
containing 10% PEG-400. To increase the stability of the product, 5 mM
sodium phosphate, pH 4.6, was added to this solution. Cold storage of
this formulation has shown good stability for at least 2 months. The
overall yield of the formulated final 2b was 78% from 9 and was
characterized by the following spectroscopic data with NMR assign-
ments made from 1H NMR, homonuclear COSY, ROESY, HSQC, and
HMBC spectra: 1H NMR at 50 °C (500 MHz, DMSO-d6/D2O
phosphate, pH 4) δ 1.01 (d, 3H, J = 7 Hz, Ala-Me), 1.17 (d, 3H, J = 7 Hz,
5′-Me), 1.24−1.42 (broad m, 2H, Lys-δ), 1.55 (p, 2H, J = 8 Hz, Lys-γ),
1.6−1.76 (broad m, 2H, Lys-β), 1.84 (m, 2H, 2′), 1.83 (s, 3H, Ac), 2.13
(dd, 1H, J = 14, 5 Hz, 8), 2.18 (dd, 1H, J = 14, 3 Hz, 8), 2.75 (m, 2H, Lys-
ε), 2.89 and 2.91 (AB pattern, 2H, J = 18 Hz, 10), 2.79 (m, 1H, Phe-β),
3.04 (m, 1H, Phe-β), 3.97 (dd, 1H, J = 6, 2 Hz, 4′), 4.14 (q, J = 6 Hz, 1H,
Ala-α), 3.93 (s, 3H, 4-OMe), 4.04 (q, J = 6 Hz, 1H, 3′), 4.24 (m, 1H, 5′),
4.26 (m, 1H, Lys-α), 4.42 (dd, 1H, J = 11, 2 Hz, Phe-α), 4.54 and 4.55
(AB pattern, J = 20 Hz, 2H, 14), 4.82 and 4.83 (AB pattern, 2H, J = 3 Hz,
OCH2N), 4.95 (dd, J = 5, 3 Hz, 1H, 7), 4.99 and 5.01 (AB pattern, 2H, J
= 13 Hz, Bn), 5.22 (t, 1H, J = 4 Hz, 1′), 7.10 (broad m, 1H, Phe-p-Ph),
7.16 (broad m, 4H, Phe-o- and m-Ph), 7.25 (d, 2H, J = 7 Hz, 2″ ROESY),
7.37 (d, 1H, J = 8 Hz, 3), 7.51 (broad, 2H, 3″), 7.56 (m, 1H, 2), 7.84 (m,
2H, 1 and 3); 13C NMR chemical shifts from HSQC δ 16.15 (5′-Me),
17.76 (Ala-Me), 22.50 (Ac), 26.57 (Lys-γ), 22.57 (Lys-δ), 29.27 (2′),
Ac-GaFK(alloc)-PABC-Doxaz (10). An oven-dried round-bottom
flask equipped with a magnetic stir bar was cooled under argon and
charged with 9 (126.13 mg, 0.1161 mmol), 1-hydroxybenzotriazole
(23.61 mg, 1 equiv), and 500 μL of dry DMSO. To this, doxaz (85.68
mg, 1 equiv) dissolved in 500 μL of dry DMSO was added, and the
mixture was left to stir under argon in the dark and monitored by HPLC
(method 1) until complete, as evidenced by the disappearance of
carbonate starting material (approximately 36 h). The reaction mixture
was then diluted with an additional 2 mL of DMSO. A 15 mL medium
fritted sintered glass funnel was charged with 10 mL of cold PBS, pH 7.4.
The red reaction mixture was added dropwise, and the resulting red
precipitate was washed four times with 10 mL of PBS and then two
washes with 10 mL of deionized water. The precipitate was washed from
the filter with a mixture of 10:1 chloroform/methanol and concentrated
by rotary evaporation. The majority of the crude red solid, which was a
mixture containing a 46% yield of 10, was moved forward without any
additional purification. However, to facilitate characterization of 10, the
red solid was purified by preparative silica gel TLC, spotted in and eluted
with 10:1 chloroform/methanol. The desired product (the major band)
was excised, suspended, and vortexed in the elution solvent and
centrifuged to remove the silica gel. The solvent was removed by rotary
evaporation to produce solid, pure 10 that was characterized by the
1
following spectroscopic data with NMR assignments made from H
1
NMR, homonuclear COSY, ROESY, HSQC, and HMBC spectra: H
NMR at 55 °C (500 MHz, CDCl3) δ 1.20 (d, 3H, J = 7 Hz, Ala-Me), 1.31
(d, 3H, J = 5 Hz, 5′-Me), 1.32 (m, 2H, Lys-γ), 1.44 (m, 1H, Lys-δ), 1.50
(broad m, 1H, Lys-δ), 1.73 (m, 1H, Lys-β), 1.75 (m, 1H, 2′), 1.87 (m,
6604
dx.doi.org/10.1021/jm300714p | J. Med. Chem. 2012, 55, 6595−6607

Journal of Medicinal Chemistry
Article
30.99 (Lys-β), 32.47 (10), 37.15 (Phe-β), 36.95 (8), 48.82 (Lys-ε),
42.37 (Gly-α), 48.86 (Ala-α), 49.7 (3′), 53.99 (Lys-α), 54.58 (Phe-α),
56.91 (4-OMe), 63.86 (14), 64.20 (5′), 66.40 (Bn), 69.85 (7), 77.45
(4′), 78.25 (O-CH2-N), 99.49 (1′), 11.46 (1 and 3), 120.03 (3″), 129.37
(Phe-p- and o-Ph), 128.61 (2″), 128.52 (Phe-m-Ph), 135.54 ppm (2);
some unprotonated carbons from HMBC δ 75.58 (9), 119.78 (12a),
135.18 (4a), 134.56 (6a), 161.39 (4), 161.42 (12), 171.73 (Ac-CO),
170.20 (Gly-CO), 171.9 (Phe-CO), 173.15 (Ala-CO), 214.12 ppm
(13); MS-ESI+, observed MH+ 1150.4590; calculated MH+ 1150.4615.
In Vitro Assays. HPLC Activity Assays. Assays to determine the
activities of various enzymes toward protease-cleavable doxaz prodrugs
were performed as a two-pronged measurement: a hydrolysis time
course to assess the activity of each enzyme stock on 2b and a
Michaelis−Menten kinetic analysis to assign kinetic values for the
activity of human-derived enzymes on 2b. Time course experiments
were conducted at 37 °C by incubating 50 μM prodrug with varying
concentrations of either plasmin (EMD Biosciences, La Jolla, CA) or
trypsin (Research Products International, Mt. Prospect, IL) in PBS.
Reactions with cathepsin B (Enzo Biosciences, Ann Arbor, MI) and
papain (Sigma, Milwaukee, WI) were performed by first diluting the
enzymes 2-fold in PBS-NAC (PBS with 10 mM N-acetylcysteine),
followed by reaction with 50 μM 2b in either PBS-NAC, pH 6.5, or PBS-
NAC, pH 5.5, at 37 °C. At various times, 50 μL of the reaction mixture
was added to an equal volume of absolute ethanol to quench the
reaction. The collected samples were stored at −20 °C for at least 2 h
and then centrifuged at 15000g for 5 min to precipitate the protein. The
supernatant was analyzed by RP-HPLC (method 2), monitoring the loss
of the starting material (elution at 8−9 min) and accumulation of dox
(elution at 4−5 min). The fraction dox was converted into micromolar
concentration and fit with a one-phase exponential association curve by
nonlinear least-squared analysis (GraphPad Prism 5.0, GraphPad
Software, Inc., La Jolla, CA). To calculate an activity for each enzyme,
only those points that produced less than 20% dox during the reaction
were analyzed to limit the analysis to those points in which the reaction
rate was approximately linear with time. The selected points were fit
with linear least-squared analysis, and the slope of the line was used to
calculate the activity of the enzymes on 2b. One unit was defined as the
amount of enzyme required to hydrolyze 1 μM prodrug per min under
these conditions (37 °C and 50 μM prodrug in buffer, either PBS or
PBS-NAC).
Michaelis−Menten kinetic analyses were carried out for human
plasmin and human cathepsin B, using 2b as a substrate and the buffer
conditions described above. A range of concentrations from 10 to 400
μM was made in buffer and enzyme to a final volume of 60 μL. The final
enzyme amounts were 0.2 U plasmin and 0.08 U cathepsin B. The
reactions were run at 37 °C for either 4 min (plasmin) or 20 min
(cathepsin B) and were then quenched and precipitated with 60 μL of
absolute ethanol and stored at −20 °C for a minimum of 2 h. The
samples were centrifuged for 5 min at 15000g, and the supernatants were
analyzed by RP-HPLC as above. Rates of reaction, in μM/min, were
calculated and fit with nonlinear least-squares analysis. Each reaction was
performed in triplicate.
Human Plasma Stability. To assay for the stability of the prodrug
in blood plasma, 2b was diluted with human plasma collected from
healthy individuals (a gift from Somalogic, Inc., Boulder, CO) to a final
drug concentration of 50 μM. The reaction was incubated at 37 °C for
24 h. Aliquots were taken at 0.25, 1, 3, 8, 12, and 24 h, quenched with an
equal volume of ethanol, and incubated at −20 °C overnight to
precipitate the proteins. Precipitated protein was removed by
centrifugation in a desktop centrifuge for 5 min at 15000g. The
supernatant was analyzed by RP-HPLC (plasma method: acetonitrile
from 20% to 40% over 5 min, to 70% at 10 min, to 80% at 13 min,
isocratic for 2 min, then back to 20% by 17 min). The percent
compositions of 2b (eluting at 8−9 min) and dox (eluting at 6.5−7.0
min) were determined from their relative absorbances at 480 nm, taking
into account all other unknown products that appeared during the
reaction and absorbed at 480 nm. The experiment was repeated in
triplicate.
cancer cell lines. The cells were seeded into 96-well plates at a density of
1000 cells/well and allowed to adhere overnight. The medium was
replaced with 90 μL of serum-free medium containing one or more of
plasmin (0.2 U/well), trypsin (0.2 U/well), cathepsin B (0.2 U/well),
aprotinin (5 μM, Research Products International, Mt. Prospect, IL), or
Complete Mini protease inhibitors (1× according to package
instructions, Roche Applied Science, Indianapolis, IN). 2b (10 μL)
was added to the wells for final concentrations between 2000 and 1 nM,
and controls received only vehicle (either DMSO or saline with 10%
PEG). The cells were treated with 2b and activator/inhibitor for 24 h, at
which point the treatment medium was replaced with Complete
medium and the cells were allowed to grow for 5 days or until the control
wells reached 80% confluency, whichever was shorter. The cells were
then fixed with 5% formalin in PBS for 10 min and stained with crystal
violet (0.01% w/v in water) for 20−30 min. The plates were rinsed and
air-dried, and the stain was redissolved either in a mixture of ethanol,
water, and methanol (6:3:1) or in 2-propanol/water (1:1) with 2%
sodium dodecylsulfate (SDS). Cell density was measured by absorbance
at 582 nm. Percent optical density, relative to control wells, was fit to a
variable slope sigmoidal dose−response curve by nonlinear regression
analysis in GraphPad Prism 5.0. These experiments indicated that pure
DMSO formulations or saline/PEG/phosphate formulations of 2b
resulted in identical growth inhibition.
Following initial tests in MiaPaCa-2 and MCF-7 cells, tests with 2b
alone or with plasmin (0.2 U/well) were performed on an array of
pancreatic cancer lines (BxPC3, L3.3, L3.5, and L3.6PL), a small-cell
lung cancer line (SHP77), and an ovarian cancer line (NCI-RES/Adr),
as well as a nontumor derived cell line (HEK-293T). All experiments
were performed identically as described above.
Cellular Uptake. MCF-7 or MiaPaCa-2 cells were plated into six-
well plates at a density of 250 000 cells/well and allowed to adhere
overnight. The medium was then replaced with serum free medium, and
0.5 μM 2b or 0.5 μM 2b + 0.2 U plasmin was added (final
concentrations). The cells were incubated for up to 5 h, then trypsinized
and analyzed for the presence of the anthracycline fluorophore by flow
cytometry (FL3 channel). The mean fluorescence values were
converted to fold-over background, in which background was defined
as cells receiving no 2b.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic methods for 1b and NMR spectra for 10 and 2b. This
material is available free of charge via the Internet at http://pubs.
acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 303-492-6193. Fax: 303-492-5849. E-mail: tad.koch@
colorado.edu.
■
Present Address
†GSK Biologics, 553 Old Corvalis Road, Hamilton, MT 59840.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge Wells Messersmith and John Arcaroli
at the University of Colorado, Anschutz Medical Campus, for the
pancreatic cell lines L3.3, L3.5, and L3.6PL. Somalogic, Inc. of
Boulder, CO, provided the gift of the human plasma. We thank
Rich Shoemaker for help with NMR protocols; Glen Post, Jordan
Nafie, and Nick Taylor for synthetic work; and Kristen Barthel
for careful reading of the manuscript. We are thankful for
Bioscience Discovery Evaluation Grants 07-00018 and 11BGF-
34 from the State of Colorado and the University of Colorado
Office of Technology Transfer and NIH Grants R41 CA12424,
R21 CA143549, and R21 CA141101 for funding this research.
IC50 Assays. Initial characterization of 2b was performed in
MiaPaCa-2 human pancreatic carcinoma and MCF-7 human breast
6605
dx.doi.org/10.1021/jm300714p | J. Med. Chem. 2012, 55, 6595−6607

Journal of Medicinal Chemistry
Article
carboxylesterase as the primary enzyme for activation of a doxazolidine
carbamate prodrug. J. Med. Chem. 2008, 51, 298−304.
(15) Barthel, B. L.; Zhang, Z.; Rudnicki, D. L.; Coldren, C. D.;
Polinkovsky, M.; Sun, H.; Koch, G. G.; Chan, D. C.; Koch, T. H.
Preclinical efficacy of a carboxylesterase 2-activated prodrug of
doxazolidine. J. Med. Chem. 2009, 52, 7678−7688.
(16) Cecchin, E.; Corona, G.; Masier, S.; Biason, P.; Cattarossi, G.;
Frustaci, S.; Buonadonna, A.; Colussi, A.; Toffoli, G. Carboxylesterase
isoform 2 mRNA expression in peripheral blood mononuclear cells is a
predictive marker of the irinotecan to SN38 activation step in colorectal
cancer patients. Clin. Cancer Res. 2005, 11, 6901−6907.
(17) Duffy, M. J.; O’Grady, P.; Devaney, D.; O’Siorain, L.; Fennelly, J.
J.; Lijnen, H. R. Tissue-type plasminogen activator, a new prognostic
marker in breast cancer. Cancer Res. 1988, 48, 1348−1349.
(18) Giannelli, G.; Falk-Marzillier, J.; Schiraldi, O.; Stetler-Stevenson,
W. G.; Quaranta, V. Induction of cell migration by matrix metal-
loprotease-2 cleavage of laminin-5. Science 1997, 277, 225−228.
(19) Bemis, L. T.; Schedin, P. Reproductive state of rat mammary gland
stroma modulates human breast cancer cell migration and invasion.
Cancer Res. 2000, 60, 3414−3418.
ABBREVIATIONS USED
■
aFK, D-Ala-L-Phe-L-Lys; aFK-Dox, N-(N-(D-alanyl-L-phenyl-
alanyl-L-lysyl)-p-aminobenzyloxycarbonyl)doxorubicin; aFK-
Doxaz, N-(N-(D -alanyl-L -phenylalanyl-L -lysyl)-p-
aminobenzyloxycarbonyl)doxazolidine; alloc, allyloxycarbonyl;
CES2/hiCE, carboxylesterase 2; dox, doxorubicin; doxaz,
doxazolidine; doxf, doxoform; ECM, extracellular matrix;
Fmoc, 9-fluorenylmethyloxycarbonyl; Fmoc-OSu, N-Fmoc-N-
hydroxysuccinimide; GaFK-Doxaz, N-(N-(N-acetylglycyl-D-
alanyl-L-phenylalanyl-L-lysyl)-p-aminobenzyloxycarbonyl)-
doxazolidine; HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetra-
methyluronium hexafluorophosphate; HOBt, 1-hydroxybenzo-
triazole; IC50, inhibitory concentration 50%; MDR, multidrug
resistant; MMP, matrix metalloprotease; NAC, N-acetylcysteine;
P170GP, P-170 glycoprotein; PABA, p-aminobenzyl alcohol;
PABC, p-aminobenzyloxycarbonyl; PBS, phosphate buffered
saline; PEG, polyethylene glycol; pNP, p-nitrophenyl; PPD,
pentyl PABC-Doxaz; RP-HPLC, reverse phase high performance
liquid chromatography; SPPS, solid-phase peptide synthesis;
TFA, trifluoroacetic acid; tPA, tissue plasminogen activator; U,
unit; uPA, urokinase plasminogen activator
(20) Choong, P. F.; Nadesapillai, A. P. Urokinase plasminogen
activator system: a multifunctional role in tumor progression and
metastasis. Clin. Orthop. Relat. Res. 2003, S46−58.
(21) Lijnen, H. R. Plasmin and matrix metalloproteinases in vascular
remodeling. Thromb. Haemostasis 2001, 86, 324−333.
REFERENCES
■
(22) Ulisse, S.; Baldini, E.; Sorrenti, S.; D’Armiento, M. The urokinase
plasminogen activator system: a target for anti-cancer therapy. Curr.
Cancer Drug Targets 2009, 9, 32−71.
(1) Saltiel, E.; McGuire, W. Doxorubicin (adriamycin) cardiomyop-
athy. West. J. Med. 1983, 139, 332−341.
(2) Takemura, G.; Fujiwara, H. Doxorubicin-induced cardiomyopathy
from the cardiotoxic mechanisms to management. Prog. Cardiovasc. Dis.
2007, 49, 330−352.
(23) Cobos, E.; Jumper, C.; Lox, C. Pretreatment determination of the
serum urokinase plasminogen activator and its soluble receptor in
advanced small-cell lung cancer or non-small-cell lung cancer. Clin. Appl.
Thromb./Hemostasis 2003, 9, 241−246.
(24) Werle, B.; Kotzsch, M.; Lah, T. T.; Kos, J.; Gabrijelcic-Geiger, D.;
Spiess, E.; Schirren, J.; Ebert, W.; Fiehn, W.; Luther, T.; Magdolen, V.;
Schmitt, M.; Harbeck, N. Cathepsin B, plasminogenactivator-inhibitor
(PAI-1) and plasminogenactivator-receptor (uPAR) are prognostic
factors for patients with non-small cell lung cancer. Anticancer Res. 2004,
24, 4147−4161.
(25) D’Amico, T. A.; Brooks, K. R.; Joshi, M. B.; Conlon, D.; Herndon,
J.; Petersen, R. P.; Harpole, D. H. Serum protein expression predicts
recurrence in patients with early-stage lung cancer after resection. Ann.
Thorac. Surg. 2006, 81, 1982−1987 ; discussion 1987.
(26) De Petro, G.; Tavian, D.; Copeta, A.; Portolani, N.; Giulini, S. M.;
Barlati, S. Expression of urokinase-type plasminogen activator (u-PA),
u-PA receptor, and tissue-type PA messenger RNAs in human
hepatocellular carcinoma. Cancer Res. 1998, 58, 2234−2239.
(27) Zheng, Q.; Tang, Z. Y.; Xue, Q.; Shi, D. R.; Song, H. Y.; Tang, H.
B. Invasion and metastasis of hepatocellular carcinoma in relation to
urokinase-type plasminogen activator, its receptor and inhibitor. J.
Cancer Res. Clin. Oncol. 2000, 126, 641−646.
(28) Takeuchi, Y.; Nakao, A.; Harada, A.; Nonami, T.; Fukatsu, T.;
Takagi, H. Expression of plasminogen activators and their inhibitors in
human pancreatic carcinoma: immunohistochemical study. Am. J.
Gastroenterol. 1993, 88, 1928−1933.
(29) Cantero, D.; Friess, H.; Deflorin, J.; Zimmermann, A.; Brundler,
M. A.; Riesle, E.; Korc, M.; Buchler, M. W. Enhanced expression of
urokinase plasminogen activator and its receptor in pancreatic
carcinoma. Br. J. Cancer 1997, 75, 388−395.
(30) Shin, S. J.; Kim, K. O.; Kim, M. K.; Lee, K. H.; Hyun, M. S.; Kim,
K. J.; Choi, J. H.; Song, H. S. Expression of E-cadherin and uPA and their
association with the prognosis of pancreatic cancer. Jpn. J. Clin. Oncol.
2005, 35, 342−348.
(3) Nielsen, D.; Maare, C.; Skovsgaard, T. Cellular resistance to
anthracyclines. Gen. Pharmacol. 1996, 27, 251−255.
(4) Myers, C. E.; McGuire, W. P.; Liss, R. H.; Ifrim, I.; Grotzinger, K.;
Young, R. C. Adriamycin: the role of lipid peroxidation in cardiac
toxicity and tumor response. Science 1977, 197, 165−167.
(5) Siveski-Iliskovic, N.; Hill, M.; Chow, D. A.; Singal, P. K. Probucol
protects against adriamycin cardiomyopathy without interfering with its
antitumor effect. Circulation 1995, 91, 10−15.
(6) Weiss, R. B. The anthracyclines: Will we ever find a better
doxorubicin? Semin. Oncol. 1992, 19, 670−686.
(7) Krown, S. E.; Northfelt, D. W.; Osoba, D.; Stewart, J. S. Use of
liposomal anthracyclines in Kaposi’s sarcoma. Semin. Oncol. 2004, 31,
36−52.
(8) Robert, N. J.; Vogel, C. L.; Henderson, I. C.; Sparano, J. A.; Moore,
M. R.; Silverman, P.; Overmoyer, B. A.; Shapiro, C. L.; Park, J. W.;
Colbern, G. T.; Winer, E. P.; Gabizon, A. A. The role of the liposomal
anthracyclines and other systemic therapies in the management of
advanced breast cancer. Semin. Oncol. 2004, 31, 106−146.
(9) Lorusso, D.; Di Stefano, A.; Carone, V.; Fagotti, A.; Pisconti, S.;
Scambia, G. Pegylated liposomal doxorubicin-related palmar-plantar
erythrodysesthesia (“hand−foot” syndrome). Ann. Oncol. 2007, 18,
1159−1164.
(10) Post, G. C.; Barthel, B. L.; Burkhart, D. J.; Hagadorn, J. R.; Koch,
T. H. Doxazolidine, a proposed active metabolite of doxorubicin that
cross-links DNA. J. Med. Chem. 2005, 48, 7648−7657.
(11) Kalet, B. T.; McBryde, M. B.; Espinosa, J. M.; Koch, T. H.
Doxazolidine induction of apoptosis by a topoisomerase II independent
mechanism. J. Med. Chem. 2007, 50, 4493−4500.
(12) Fenick, D. J.; Taatjes, D. J.; Koch, T. H. Doxoform and
daunoform: anthracycline−formaldehyde conjugates toxic to resistant
tumor cells. J. Med. Chem. 1997, 40, 2452−2461.
(13) Burkhart, D. J.; Barthel, B. L.; Post, G. C.; Kalet, B. T.; Nafie, J. W.;
Shoemaker, R. K.; Koch, T. H. Design, synthesis, and preliminary
evaluation of doxazolidine carbamates as prodrugs activated by
carboxylesterases. J. Med. Chem. 2006, 49, 7002−7012.
(14) Barthel, B. L.; Torres, R. C.; Hyatt, J. L.; Edwards, C. C.; Hatfield,
M. J.; Potter, P. M.; Koch, T. H. Identification of human intestinal
(31) de Groot, F. M. H.; de Bart, A. C. W.; Verheijen, J. H.; Scheeren,
H. W. Synthesis and biological evaluation of novel prodrugs of
anthracyclines for selective activation by the tumor-associated protease
plasmin. J. Med. Chem. 1999, 42, 5277−5283.
(32) Devy, L.; de Groot, F. H. M.; Blacher, S.; Hajitou, A.; Beusker, P.;
Scheeren, W.; Foldart, J.-M.; Noel, A. Plasmin-activated doxorubicin
6606
dx.doi.org/10.1021/jm300714p | J. Med. Chem. 2012, 55, 6595−6607

Journal of Medicinal Chemistry
Article
prodrugs containing a spacer reduce tumor growth and angiogenesis
without systemic toxicity. FASEB J. 2004, 18, 565−567.
(33) Carl, P. L.; Chakravarty, P. K.; Katzenellenbogen, J. A. A novel
connector linkage applicable in prodrug design. J. Med. Chem. 1981, 24,
479−480.
(34) Low, T. L.; Goldstein, A. L. The chemistry and biology of
thymosin. II. Amino acid sequence analysis of thymosin alpha1 and
polypeptide beta1. J. Biol. Chem. 1979, 254, 987−995.
(35) Abiko, T.; Sekino, H. Synthesis of an immunologically active
fragment analog of prothymosin alpha with enhanced enzymatic
stability. Chem. Pharm. Bull. (Tokyo) 1991, 39, 752−756.
(36) Nguyen, L. T.; Chau, J. K.; Perry, N. A.; de Boer, L.; Zaat, S. A.;
Vogel, H. J. Serum stabilities of short tryptophan- and arginine-rich
antimicrobial peptide analogs. PLoS One 2010, 5.
(matriptase/MT-SP1) is highly overexpressed in cervical carcinoma.
Cancer 2003, 98, 1898−1904.
(52) Nolan, C.; Hall, L. S.; Barlow, G. H.; Tribby, I. I. E. Plasminogen
activator from human embryonic kidney cell cultures. Evidence for a
proactivator. Biochim. Biophys. Acta 1977, 496, 384−400.
(53) Carl, P. L.; Chakravarty, P. K.; Katzenellenbogen, J. A.; Weber, M.
J. Protease-activated “prodrugs” for cancer chemotherapy. Proc. Natl.
Acad. Sci. U.S.A. 1980, 77, 2224−2228.
(54) Gude, M.; Ryf, J.; White, P. An accurate method for quantitation
of Fmoc-derivitized solid phase supports. Lett. Pept. Sci. 2002, 9, 203−
206.
(37) Kos, J.; Werle, B.; Lah, T.; Brunner, N. Cysteine proteinases and
their inhibitors in extracellular fluids: markers for diagnosis and
prognosis in cancer. Int. J. Biol. Markers 2000, 15, 84−89.
(38) Jedeszko, C.; Sloane, B. F. Cysteine cathepsins in human cancer.
Biol. Chem. 2004, 385, 1017−1027.
(39) Joyce, J. A.; Baruch, A.; Chehade, K.; Meyer-Morse, N.; Giraudo,
E.; Tsai, F. Y.; Greenbaum, D. C.; Hager, J. H.; Bogyo, M.; Hanahan, D.
Cathepsin cysteine proteases are effectors of invasive growth and
angiogenesis during multistage tumorigenesis. Cancer Cell 2004, 5,
443−453.
(40) Dubowchik, G. M.; Firestone, R. A.; Padilla, L.; Willner, D.;
Hofstead, S. J.; Mosure, K.; Knipe, J. O.; Lasch, S. J.; Trail, P. A.
Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin
from internalizing immunoconjugates: model studies of enzymatic drug
release and antigen-specific in vitro anticancer activity. Bioconjugate
Chem. 2002, 13, 855−869.
(41) Schmid, B.; Chung, D. E.; Warnecke, A.; Fichtner, I.; Kratz, F.
Albumin-binding prodrugs of camptothecin and doxorubicin with an
Ala-Leu-Ala-Leu-linker that are cleaved by cathepsin B: synthesis and
antitumor efficacy. Bioconjugate Chem. 2007, 18, 702−716.
(42) Misaka, E.; Tappel, A. L. Inhibition studies of cathepsins A, B, C
and D from rat liver lysosomes. Comp. Biochem. Physiol., Part B 1971, 38,
651−662.
(43) Hirao, T.; Hara, K.; Takahashi, K. Purification and character-
ization of cathepsin B from monkey skeletal muscle. J. Biochem. 1984, 95,
871−879.
(44) Agarwal, S. K.; Choudhury, S. D.; Lamsal, M.; Khan, M. Y.
Catalytic and physico-chemical characteristics of goat spleen cathepsin
B. Biochem. Mol. Biol. Int. 1997, 42, 1215−1226.
(45) Lechuga, C. G.; Hernandez-Nazara, Z. H.; Hernandez, E.;
Bustamante, M.; Desierto, G.; Cotty, A.; Dharker, N.; Choe, M.;
Rojkind, M. PI3K is involved in PDGF-beta receptor upregulation post-
PDGF-BB treatment in mouse HSC. Am. J. Physiol.: Gastrointest. Liver
Physiol. 2006, 291, G1051−1061.
(46) Fehske, K. J.; Muller, W. E.; Wollert, U. The location of drug
binding sites in human serum albumin. Biochem. Pharmacol. 1981, 30,
687−692.
(47) Kratochwil, N. A.; Huber, W.; Muller, F.; Kansy, M.; Gerber, P. R.
Predicting plasma protein binding of drugs: a new approach. Biochem.
Pharmacol. 2002, 64, 1355−1374.
(48) Koivunen, E.; Ristimaki, A.; Itkonen, O.; Osman, S.; Vuento, M.;
Stenman, U. H. Tumor-associated trypsin participates in cancer cell-
mediated degradation of extracellular matrix. Cancer Res. 1991, 51,
2107−2112.
(49) Miszczuk-Jamska, B.; Merten, M.; Guy-Crotte, O.; Amouric, M.;
Clemente, F.; Schoumacher, R. A.; Figarella, C. Characterization of
trypsinogens 1 and 2 in two human pancreatic adenocarcinoma cell
lines; CFPAC-1 and CAPAN-1. FEBS Lett. 1991, 294, 175−178.
(50) Yamamoto, H.; Iku, S.; Itoh, F.; Tang, X.; Hosokawa, M.; Imai, K.
Association of trypsin expression with recurrence and poor prognosis in
human esophageal squamous cell carcinoma. Cancer 2001, 91, 1324−
1331.
(51) Santin, A. D.; Cane, S.; Bellone, S.; Bignotti, E.; Palmieri, M.; De
Las Casas, L. E.; Anfossi, S.; Roman, J. J.; O’Brien, T.; Pecorelli, S. The
novel serine protease tumor-associated differentially expressed gene-15
6607
dx.doi.org/10.1021/jm300714p | J. Med. Chem. 2012, 55, 6595−6607