Development and bioorthogonal activation of palladium-labile prodrugs of gemcitabine

Article abstract of DOI:10.1021/jm500531z

Bioorthogonal chemistry has become one of the main driving forces in current chemical biology, inspiring the search for novel biocompatible chemospecific reactions for the past decade. Alongside the well-established labeling strategies that originated the bioorthogonal paradigm, we have recently proposed the use of heterogeneous palladium chemistry and bioorthogonal Pd ⁰-labile prodrugs to develop spatially targeted therapies. Herein, we report the generation of biologically inert precursors of cytotoxic gemcitabine by introducing Pd⁰-cleavable groups in positions that are mechanistically relevant for gemcitabine's pharmacological activity. Cell viability studies in pancreatic cancer cells showed that carbamate functionalization of the 4-amino group of gemcitabine significantly reduced (>23-fold) the prodrugs' cytotoxicity. The N-propargyloxycarbonyl (N-Poc) promoiety displayed the highest sensitivity to heterogeneous palladium catalysis under biocompatible conditions, with a reaction half-life of less than 6 h. Zebrafish studies with allyl, propargyl, and benzyl carbamate-protected rhodamines confirmed N-Poc as the most suitable masking group for implementing in vivo bioorthogonal organometallic chemistry.

Full text of DOI:10.1021/jm500531z

Article
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Terms of Use
Development and Bioorthogonal Activation of Palladium-Labile
Prodrugs of Gemcitabine
Jason T. Weiss,† John C. Dawson,† Craig Fraser,† Witold Rybski,†,‡ Carmen Torres-Sanchez,§
́
Mark Bradley,∥ E. Elizabeth Patton,†,‡ Neil O. Carragher,† and Asier Unciti-Broceta*,†
‡
†Edinburgh Cancer Research UK Centre, and MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine,
University of Edinburgh, Crewe Road South, Edinburgh EH4 2XR, U.K.
§Wolfson School of Mechanical and Manufacturing Engineering, Loughborough University, Loughborough LE11 3TU, U.K.
∥School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, U.K.
S
* Supporting Information
ABSTRACT: Bioorthogonal chemistry has become one of the
main driving forces in current chemical biology, inspiring the
search for novel biocompatible chemospecific reactions for the
past decade. Alongside the well-established labeling strategies
that originated the bioorthogonal paradigm, we have recently
proposed the use of heterogeneous palladium chemistry and
bioorthogonal Pd0-labile prodrugs to develop spatially targeted
therapies. Herein, we report the generation of biologically inert
precursors of cytotoxic gemcitabine by introducing Pd0-
cleavable groups in positions that are mechanistically relevant
for gemcitabine’s pharmacological activity. Cell viability studies
in pancreatic cancer cells showed that carbamate functionaliza-
tion of the 4-amino group of gemcitabine significantly reduced
(>23-fold) the prodrugs’ cytotoxicity. The N-propargyloxycarbonyl (N-Poc) promoiety displayed the highest sensitivity to
heterogeneous palladium catalysis under biocompatible conditions, with a reaction half-life of less than 6 h. Zebrafish studies with
allyl, propargyl, and benzyl carbamate-protected rhodamines confirmed N-Poc as the most suitable masking group for
implementing in vivo bioorthogonal organometallic chemistry.
electromagnetic radiations6,7 or biocompatible heterogeneous
INTRODUCTION
■
catalysts.8
Bioorthogonal chemistry is an emerging field of research that
Technically speaking, for a bioorthogonal reaction to be
focuses on the development and application of selective
optimal to living systems, the corresponding nonbiotic reactive
chemical reactions susceptible to occurring in a biological
partners need to display (i) high chemical stability to enzymatic
processing and (ii) selective reciprocal reactivity in biocompat-
ible conditions (water, pH 7.4, isotonicity, and 37 °C).
Importantly, the reactants and the resulting product/s should
be nontoxic, unless this is the intended outcome of the process.
The Staudinger ligation and the copper-free azide alkyne
cycloaddition, originally developed by the Bertozzi group,9−11
are representative examples of such chemistry. In addition to
catalyst-free ligations, the use of transition metals to function-
alize biomolecules or activate fluorogenic probes in living cells
has recently emerged as a new alternative in the field.8,12,13
These biocompatible processes, also known as bioorthogonal
organometallic (BOOM) reactions,14 are mediated by non-
biotic transition metals such as copper(I),15 ruthenium-
(II),16−18 or palladium species8,14,18−23 and have expanded
environment without interfering with the normal function of its
components.1,2 These reactions are used as labeling methods to
study cell constituents in their native state, including proteins
and biomolecules that cannot be monitored by genetically
encoded reporters (e.g., glycans, lipids, DNA, etc.).1−4 Looking
beyond the well-established labeling strategies that originated
the bioorthogonal field, the induction of local chemotherapy by
biologically inert means could allow for the reduction of
adverse pharmacological effects in distant organs and tissues.
This therapeutic paradigm has so far reached the clinic with the
so-called photodynamic therapy, a method based on the local
generation of cytotoxic reactive oxygen species by spatially
controlled excitation (and subsequent energy transfer to the
molecular oxygen present in the tissue) of a nontoxic
systemically administered photosensitizer with a benign light
source.5 Following the same principle of bioorthogonality,
novel strategies are being currently investigated by several
groups to enable local conversion of chemically masked
prodrugs into cytotoxic small molecules using either harmless
Received: April 3, 2014
Published: May 27, 2014
© 2014 American Chemical Society
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Figure 1. Gemcitabine and its cytotoxic mode of action. Following cell entry, gemcitabine is successively phosphorylated by deoxycytidine kinase at
its 5′-OH position to generate cytotoxic metabolites.
a
Scheme 1. Synthesis of Gemcitabine Prodrugs
a
Reagents and conditions: (i) DBU (2.5 equiv), DMF, addition of alkylchloroformate (1.5 equiv) at 4 °C, then r.t. overnight (47−53%); (ii) TBS-Cl
(1.1 equiv), imidazole (3.5 equiv), DMF, r.t., 2.5 h (88%); (iii) pyridine (3.2 equiv), DMF or THF, addition of alkylchloroformate (1.5 equiv) at 4
°C, then r.t. overnight 12−48h (42−69%); (iv) TBAF (2.5 equiv), THF, r.t., overnight (62−92%).
the diversity of chemical groups and transformations suitable
for use bioorthogonally in cell culture.
exhibited cytotoxicity, demonstrating the feasibility of the
method.
To explore this paradigm further, herein we report a
comprehensive study on the development and evaluation of
novel Pd0-labile prodrugs of gemcitabine (deoxycytidine
analogue clinically used to treat several types of cancers24)
using two masking strategies: (i) carbonate formation at the 5′-
OH group of the sugar moiety and (ii) carbamate masking of
the 4-NH2 group of the cytosine base (Figure 1).
Aiming to modulate drug activity by an internal self-
renewable control element rather than an external radiation
source (e.g., light in photopharmacology7), we have recently
proposed the development of nonbiological transition metals
into biocompatible catalytic devices to allow the activation of
systemically administered prodrugs at the location determined
by the device.8 This bioorthogonally activated chemotherapeu-
tic strategy would be optimal to implement focalized treatment
of unresectable primary or metastatic tumors and could also
find application in the treatment of other disorders such as local
infections, Parkinson’s disease, cardiovascular disorders, etc. In
the seminal investigation of such an approach,8 we reported the
development of a selective metallo-substrate (5-fluoro-1-
propargyluracil) and a solid-phase palladium catalyst (Pd0-
resins) as a highly efficient bioorthogonal prodrug/activator
system. On the basis of their complementary chemical roles,
cytotoxic 5-fluorouracil was efficiently generated via extrac-
ellular BOOM catalysis in cell culture.8 Importantly, when each
of these reagents was cultured separately, neither of them
RESULTS AND DISCUSSION
■
Design and Synthesis of Pd0-Labile Gemcitabine
Prodrugs. Gemcitabine (marketed as Gemzar by Eli Lilly
and Co.) is an antimetabolite antineoplastic agent widely
employed, in combination or alone, to treat several difficult-to-
cure cancerous processes (e.g., nonsmall-cell lung carcinoma,
metastatic breast cancer, and ovarian cancer),24 including being
a first-line therapy in the treatment of pancreatic cancer.25 It
displays a narrow therapeutic index, with its most severe side
effects being myelosuppression, pulmonary toxicity, and renal
failure.24 Gemcitabine is intracellularly converted by deoxy-
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cytidine kinase into its therapeutically active metabolites, the 5′-
diphosphate and 5′-triphosphate derivatives (Figure 1).
Gemcitabine 5′-diphosphate inhibits ribonucleotide reductase,
a strictly conserved enzyme among all living organisms
responsible for regulating the total rate of DNA synthesis,
while the triphosphorylated derivative can become incorporated
into the DNA, thereby inhibiting nuclear replication.26
On the basis of gemcitabine’s mode of action, masking the
5′-OH group of its sugar moiety would chemically block the
generation of its cytotoxic metabolites (Figure 1). In a different
manner but similarly relevant, modification of the 4-amino
group of the cytosine base would result in a reduction of the
enzyme−substrate recognition. The absence of a free NH2
group in that position would hinder the formation of essential
hydrogen bonding interactions with the amino acid Asp133
located within the activation site of the enzyme, thus reducing
the efficacy of the phosphorylation process.27 Furthermore, the
main detoxification route of gemcitabine is via hydrolytic
deamination of the 4-NH2 group. This is mediated in the liver
by cytidine deaminase, which irreversibly converts gemcitabine
into its inactive metabolite difluoro-deoxyuridine.28 Conse-
quently, masking the 4-NH2 group would result in a doubly
beneficial effect to the bioorthogonal strategy: it would reduce
the cytotoxic properties of the resulting prodrug and protect
the circulating drug precursor from premature liver deactivation
before reaching the target tissue.
Allyl and propargyloxycarbonyl groups (Alloc and Poc,
respectively) have been widely employed as OH and NH2
protection strategies29−32 and have shown sensitivity to
palladium catalysis in biocompatible conditions.8,14,19 There-
fore, both groups were chosen to derivatize gemcitabine 1 into
palladium-labile prodrugs. Because of the requirement of an
additional hydrogen source to undergo palladium-mediated
cleavage, the carboxybenzyl group (Cbz) was used to generate
control prodrugs resistant to oxidative catalysis.29,33 Prodrugs
were synthesized using a semisynthetic strategy by functional-
ization of gemcitabine, 1 (Scheme 1). Treatment of 1 with allyl,
propargyl, or benzyl chloroformate in the presence of DBU
formed the corresponding carbonate prodrugs 2a,p,b in
moderate yields. To synthesize the 4-carbamate derivatives,
the primary alcohol in position 5′ was first silylated using TBS-
Cl and imidazole, followed by treatment with the correspond-
ing alkyl chloroformates. TBAF-mediated deprotection yielded
the final carbamate prodrugs 5a,p,b in moderate to good yields.
Bioorthogonality Study: Carbonate vs Carbamate
Gemcitabine Prodrugs. Although, in principle, the masking
of either the 5′-OH or the 4-NH2 group of gemcitabine could
both have a significant impact on the drug’s antiproliferative
properties, the limiting factor in the development of any
bioorthogonally activated prodrug approach is the stability of
the protecting group to enzymatic metabolism. To determine
the robustness of each deactivation strategy in cell culture,
dose−response studies with gemcitabine (1) and prodrugs
2a,p,b and 5a,p,b were carried out in two human cancer cell
lines. Pancreas adenocarcinoma BxPC-3 and Mia PaCa-2 cells
were chosen as cell models due to the clinical relevance of
gemcitabine as a first-line drug in pancreatic cancer therapy.24,25
As shown in Figure 2, carbamate-protected prodrugs 5a,p,b
showed significant reduction of cytotoxicity relative to that of
gemcitabine (EC50 (5a,p,b)/EC50 (1) > 23) in both cell lines,
verifying the efficacy of the deactivation strategy. On the
contrary, prodrugs 2a,p,b displayed cytotoxicity similar to that
of gemcitabine (1), suggesting that these prodrugs are rapidly
Figure 2. Study of the prodrugs’ bioorthogonality. Semilog dose−
response curves and calculated EC50 values of prodrugs 2a,p,b (in
gray) and 5a,p,b (in blue) in comparison to those of unmodified
gemcitabine (1, in red) in (a) BxPC-3 and (b) Mia PaCa-2 cells. Cell
viability was measured at day 4 using PrestoBlue reagent. Error bars:
SD from n = 3.
bioactivated inside cells. These results are in accordance with
the weaker nature of the carbonate bond32 and strongly
indicate that carbonate-based masking strategies are not
suitable for bioorthogonal applications. Consequently, only
carbamate prodrugs 5a,p,b were moved forward in this study.
Pd0-Mediated Conversion of Prodrugs 5a,p,b into
Gemcitabine. Polystyrene-supported reagents have been
employed for a wide range of cell-based applications and
have shown excellent biocompatibility both in vitro and in
vivo.8,34−36 As catalytically active Pd0 nanoparticles can be
readily generated and trapped in an amino-functionalized
polystyrene matrix,8,14 Pd0-functionalized resins (150 μm in
average diameter, Figure 3a) were prepared from NovaSyn TG
amino resin HL following the procedure previously reported8
and used as the heterogeneous palladium source (4.4% w/w in
Pd). To test the susceptibility of the different carbamates to
Pd0-mediated catalysis in a biocompatible environment,
compounds 5a,p,b (100 μM) and Pd0-resins (1 mg/mL,
[Pd0] = 400 μM) were dispersed in PBS (300 mOsm/kg, pH
7.4) and incubated at 37 °C for 24 h (Figure 3b). Reactions
were analyzed by HPLC at different time points (0, 6, and 24
h), with prodrugs 5a,p,b being retained for 2.56, 2.45, and 2.96
min, respectively, and the gemcitabine peak appearing at 0.93
min (Figure 3c−f). Treatment of Cbz-protected compound 5b
with the palladium source produced negligible levels of
gemcitabine (1) after 24 h (Figure 3f), which corresponds
with the need for an additional hydrogen source to achieve
reductive cleavage of Cbz groups.29,33 On the contrary,
incubation of compounds 5a and 5p with Pd0-resins led to
the generation of significant levels of gemcitabine (1) in less
than 24 h (Figure 3d,e). Interestingly, deprotection of 5p
proceeded in a faster and cleaner manner, with a reaction half-
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Figure 3. Palladium-mediated conversion assay in cell-free biocompatible conditions. (a) SEM image of Pd0-resins. Scale bar = 150 μm. (b) Pd0-
mediated carbamate cleavage of compounds 5a,p,b. (c) HPLC chromatogram of unmodified gemcitabine (1). (d−f) HPLC chromatograms (UV
detector 280 nm) of 100 μM PBS solutions of compounds 5a (left panel), 5p (central panel), and 5b (right panel) treated with Pd0-resins at 37 °C
for 0 h (top), 6 h (central), and 24 h (bottom). (g) Proposed mechanism for the Pd0-catalyzed cleavage of O/N-propargyl groups in water.
life of <6 h (Figure 3e). These results are in accordance with
previous observations8,37,38 that have reported the efficient
cleavage of various propargylated compounds by palladium
species in biocompatible conditions. A potential mechanism for
this process and for the reported formation of nontoxic 1-
hydroxyacetone8,39 (6) as a reaction byproduct is proposed in
Figure 3g.
Pd0-Mediated Prodrug Activation in Cell Culture.
Bioorthogonal in situ generation of gemcitabine (1) from the
carbamate prodrugs was first investigated in standard cell
culture conditions with BxPC-3 cells using Pd0-resins as the
extracellular activating device. Prodrugs 5a,p,b and Pd0-resins
were incubated independently (negative controls) or in
Figure 4. Palladium-mediated conversion of prodrugs 5a,p,b into
combination (BOOM activation assay) and unmodified
gemcitabine (1) in pancreatic cancer BxPC-3 cells. Experiments: 0.1%
gemcitabine (1) used as the positive control. While neither
(v/v) DMSO (untreated cell control, in gray); 0.67 mg/mL of Pd0-
the prodrugs nor the Pd0-resins exhibited cytotoxicity, a
resins (negative control, in black); gemcitabine (100 nM, positive
combination of 5a and 5p with Pd0-resins displayed a strong
control, in red); prodrug 5a,p,b (100 nM, negative control, in blue);
and 0.67 mg/mL of Pd0-resins + prodrug 5a,p,b (100 nM, BOOM
activation, in green). Cell viability was measured at day 5 using
PrestoBlue reagent. Error bars: SD from n = 3.
toxigenic effect (Figure 4), confirming the in situ bioorthogonal
synthesis of cytotoxic gemcitabine (1). In the context of the
present study, “toxigenic effect” describes the gemcitabine levels
generated by each prodrug/Pd0-resin combination in cell
culture which in turn result in a cytotoxic phenotype. This
effect is indirectly quantified by determining the antiprolifer-
ative activity caused by each prodrug/Pd0-resin combination,
which is directly proportional to the concentration of cytotoxic
drug generated, and qualitatively proved by the verification of
its mode of action. Consistent with the anticipated resistance of
the N-Cbz functional group to palladium-mediated oxidative
cleavage, cells treated with prodrug 5b and Pd0-resins did not
produce any negative effect on cell viability.
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Dose−response studies were subsequently carried out to
compare the treatment of 5a and 5p/Pd0-resin combinations at
different concentrations in two cell lines: BxPC-3 and Mia
PaCa-2 cells. As shown in Figure 5a,b, reduction of cell viability
Real-time monitoring of BxPC-3 cell proliferation using an
IncuCyte ZOOM microscope was performed to compare the
effect of each prodrug/catalyst combination upon the kinetics
of tumor cell growth. Time-lapse imaging of the negative
controls 5a and 5p showed a standard growth curve up to 100%
cell confluence level by day 4−5 (light and dark blue curves,
respectively; Figure 6), while gemcitabine treatment efficiently
Figure 6. Real-time cell confluence study in pancreatic cancer BxPC-3
cells. The cell population was monitored for 120 h using an IncuCyte
ZOOM system in an incubator (5% CO2 and 37 °C). Drug/prodrug
concentration: 30 nM. Error bars: SD from n = 3.
suppressed cell proliferation within 24 h. In accordance with
the cell viability studies, the toxigenic effect mediated by either
prodrug 5a or 5p incubated with Pd0-resins (light and dark
green curves, respectively; Figure 6) were patently different
(see Supporting Information for a motion picture of the
IncuCyte experiment). The prodrug 5p/catalyst combination
inhibited cell proliferation with the same efficacy and kinetic
response as unmodified gemcitabine (1), proving that cytotoxic
levels of the drug were rapidly generated. Although treatment
with the 5a/catalyst combination markedly delayed BxPC-3 cell
growth relative to the negative controls, its antiproliferative
effect was significantly inferior to either the 5p/Pd0-resins
combination or unmodified gemcitabine (1), indicating
quantitatively lower levels of active drug being generated.
Study of Cytotoxic Mode of Action. Double-stranded
breaks caused by DNA damage induce phosphorylation of the
variant histone of the H2A protein family, γ-H2AX.
Phosphorylated γ-H2AX is responsible for recruiting and
localizing the DNA repair mechanism.40 Gemcitabine damages
DNA through partial DNA chain termination and by stalling of
replication forks.41 In order to analyze the toxigenic effect
caused by the prodrug/catalyst combination at the molecular
level, immunofluorescence studies were carried out to probe for
phosphorylated γ-H2AX as a marker of DNA damage in Mia
PaCa-2 cells (Figure 7). While the negative controls (Pd0-resins
or prodrug 5p separately incubated; Figure 7a,b) did not show
the presence of phosphorylated γ-H2AX in the cell nuclei, cells
treated with either gemcitabine (Figure 7c) or the Pd0-resins +
5p combination (Figure 7d) expressed significant levels of
phospho-γ-H2AX. This study further illustrates that the
cytotoxic activity generated from the prodrug/catalyst combi-
nation and unmodified gemcitabine is equivalent.
Figure 5. Bioorthogonally activated toxigenic effect in cancer cell
culture: (a) BxPC-3 and (b) Mia PaCa-2 cells. Dose−response study:
0.1% (v/v) DMSO (untreated cell control, in gray); 0.67 mg/mL Pd0-
resins (negative control, in black); gemcitabine (positive control, in
red); prodrug 5a,p (negative control, in blue); and 0.67 mg/mL of
Pd0-resins + prodrug 5a,p (BOOM activation, in green). Cell viability
was measured at day 5 using PrestoBlue reagent. Error bars: SD
from n = 3.
was observed with compounds 5a and 5p only when incubated
with Pd0-resins. Analysis of the toxigenic effect displayed by
each prodrug/catalyst combination clearly ranked the N-Poc-
protected prodrug 5p as the most effective gemcitabine-
generating precursor in the presence of Pd0-resins, exhibiting
analogous cytotoxic activity to that of the unmodified drug.
Importantly, neither the palladium source nor the prodrugs
exhibited antiproliferative activity when separately incubated at
any of the concentrations tested, thus supporting the
bioorthogonality of the strategy. It is important to note that
the combination of N-Alloc-protected prodrug 5a and Pd0-
resins resulted in significantly higher toxigenic effect against
BxPC-3 cells than that in Mia PaCa-2 cells. Since both cell lines
display similar sensitivity to unmodified gemcitabine, these
results show that the palladium-mediated cleavage of the N-
Alloc group of prodrug 5a is significantly affected by either the
cell type or the culture conditions (BxPC-3 cells are grown in
RPMI, while Mia PaCa-2 cells are cultured in DMEM
medium). On the contrary, the combined treatment of 5p
and Pd0-resins displayed comparable toxigenic effects in both
cell lines, indicating that the biological environment has little or
no influence on the N-Poc deprotection process, as would be
expected from a truly bioorthogonal reaction.
Pd0-Mediated Probe Activation in Zebrafish. We have
recently shown that catalytically functional Pd0-resins can be
implanted in the yolk sac of zebrafish embryos without
inducing toxicity or affecting the embryo development.8 To
compare the in vivo bioorthogonality and palladium sensitivity
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strategy would allow generating locally increased concen-
trations of a functionally active small molecule by palladium
heterogeneous catalysis in vivo.
CONCLUSIONS
■
The chemical protection of gemcitabine’s 4-amino group as a
carbamate led to a significant reduction of the drug’s cytotoxic
activity. Among the carbamate derivatives investigated, the N-
Poc group exhibited the highest sensitivity to palladium-
mediated cleavage at 37 °C in both buffered solution and cell
culture. This is noteworthy since, to date, our group14,19 and
others12,13,16−18,42−44 have favored the use of N-Alloc groups
over N-Poc to implement coupling and decoupling strategies45
for BOOM studies. Cell viability, time-lapse microscopy, and
DNA damage assays confirmed the bioorthogonal generation of
cytotoxic gemcitabine from the combined treatment of
extracellular Pd0-resins and the N-Poc-protected precursor in
pancreatic cancer cell culture. Unlike the N-Alloc- and N-Cbz-
protected derivatives, the bis-N,N′-Poc-rhodamine 1108
became locally activated by a Pd0-resin implanted in the yolk
sac of zebrafish embryos, further reinforcing its nomination as
the carbamate group of choice for developing bioorthogonal
studies based on heterogeneous palladium catalysis. Parallel
independent studies from Chen et al. (intracellular protein
activation by palladium-mediated homogeneous catalysis),
published just before the submission of this article,46 reported
analogous findings with the N-Poc group. Finally, the simplicity
of the masking strategy together with the biocompatibility and
efficacy of the palladium-mediated deprotection process
underlines the potential of this method to modulate the
pharmacodynamics and pharmacokinetics of amino-containing
drugs.
Figure 7. DNA damage study. Merge fluorescent images of Mia PaCa-
2 cells 24 h after treatment with (a) 0.67 mg/mL Pd0-resins (negative
control); (b) prodrug 5p (300 nM, negative control); (c) gemcitabine
(300 nM, positive control); and 0.67 mg/mL of Pd0-resins + prodrug
5p (300 nM, BOOM activation assay). Fluorescent labels: Hoechst
33342 for cell nuclei (blue), Alexa Fluor 594 phalloidin for F-actin
(red), and anti-phospho-histone γ-H2AX + Alexa Fluor 488 secondary
antibody for phosphorylated γ-H2AX (green).
of each of the carbamate protecting groups under study, N,N′-
bis(alkyloxycarbonyl) rhodamine derivatives 8a,p,b were
prepared and used as off-on fluorescent probes (Figure 8a).
Compounds 8a,p,b were synthesized from rhodamine 110, 7,
by reaction with the corresponding alkylchloroformate and a
base (triethylamine or pyridine) in dry DMF. The resulting
nonfluorescent rhodamine precursors were incubated with 2-
dpf zebrafish embryos containing either a nonfunctionalized
resin (nonactive resin) or a Pd0-resin (active) in the yolk sac for
24 h and subsequently analyzed by fluorescence microscopy.
Zebrafish embryos treated with Alloc-protected compound 8a
showed high levels of fluorescence emission regardless of the
presence or absence of palladium, particularly from the yolk sac
and the digestive system (Figure 8b,c, top panel). Because of
the high levels of biochemically generated fluorescence
background, local palladium-mediated activation of 8a within
the zebrafish yolk was not detectable, an indication of the low
bioorthogonality of the N-Alloc group in zebrafish. On the
contrary, compounds 8p and 8b showed improved biochemical
stability, with the fluorescent background signal mostly being
observed from the digestive system, which is in accordance with
our previous observations.8 As expected, Cbz-protected
compound 8b did not exhibit sensitivity to palladium catalysis
(Figure 8b,c, lower panel), while local palladium-mediated
generation of rhodamine 110 was clearly observed from 8p in
the zebrafish yolk sac containing a Pd0-resin (Figure 8b,c,
middle panel). As shown in Figure 8d, image analysis
demonstrated up to a 4-fold increase in fluorescence intensity
within close vicinity of the Pd0-resin (blue line). On the
contrary, apart from the autofluorescence intensity typically
observed from the nonfunctionalized resin, this inactive resin
did not lead to an increment of fluorescence levels in the area
surrounding it (red line). This study suggests that, from the
carbamate groups herein studied, only the N-Poc masking
EXPERIMENTAL PROCEDURES
■
Synthetic Procedures. General Methods. Chemicals and solvents
were purchased from Fisher Scientific, Sigma-Aldrich, or VWR
International Ltd. Gemcitabine HCl was purchased from Shandong
Boyuan Pharmaceutical Co. Ltd. NMR spectra were recorded at
ambient temperature on a 500 MHz Bruker Avance III spectrometer.
Chemical shifts are reported in parts per million (ppm) relative to the
solvent peak. Rf values were determined on Merck TLC Silica gel 60
F254 plates under a 254 nm UV source. Purification was carried out by
flash column chromatography using commercially available silica gel
(220−440 mesh, Sigma-Aldrich). All compounds used in the biological
experiments were >95% pure, as measured by HPLC using an
evaporative light scattering detector. Method: eluent A, water and
formic acid (0.1%); eluent B, acetonitrile and formic acid (0.1%); A/B
= 95:5 to 5:95 in 3 min, isocratic 1 min, 5:95 to 95:5 in 1 min, and
isocratic 1 min.
Synthesis of Pd0-Resins. Pd0-functionalized resins were prepared
from NovaSyn TG amino resin HL (0.39 mmol NH2/g) as previously
described.8
Synthesis of Carbonate-Protected Derivatives 2a,p,b. Gemcita-
bine HCl (150 mg, 0.5 mmol) was dissolved in dry DMF (3 mL) with
DBU (194 μL, 1.30 mmol) under N2 atmosphere. The mixture was
then cooled to 4 °C in an ice bath. Allyl, propargyl, or benzyl
chloroformate (0.75 mmol) was added dropwise to the mixture. The
mixture was stirred overnight and allowed to warm up to room
temperature (r.t.). The solvents were then removed in vacuo, the crude
redissolved with 25% isopropanol (IPA) in CHCl3 (20 mL), and
washed with H2O (20 mL). The aqueous layer was washed with 25%
IPA in CHCl3 (3 × 20 mL) and the combined organic layers dried
over MgSO4, filtered, and concentrated in vacuo.
4-Amino-1-(2-deoxy-2,2-difluoro-5-O-[allyloxycarbon-yl]-β-D-er-
ythro-pentofuranosyl)pyrimidin-2(1H)-one] (2a). The crude was
purified by column chromatography using 8% MeOH in DCM (Rf
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Figure 8. Pd0-mediated carbamate cleavage of rhodamine precursors in zebrafish. (a) BOOM conversion of nonfluorescent lipophilic compounds
8a,p,b into highly fluorescent hydrophilic rhodamine 110. (b) 3-dpf zebrafish embryos (n = 5) containing a nonfunctionalized resin (indicated with
white arrows) after incubation with 5 μM of compounds 8a (top), 8p (middle), and 8b (bottom) for 24 h at 31 °C. (c) 3-dpf zebrafish embryos (n =
5) containing a Pd0-resin (indicated with purple arrows) after incubation with 5 μM of compounds 8a (top), 8p (middle), and 8b (bottom) for 24 h
at 31 °C. Fish were imaged by phase contrast and fluorescent microscopy (ex., 470/40; em., 525/50). (d) Analysis of fluorescence intensity/pixel
across a horizontal line of approximately 300 μm drawn in the yolk sac of the zebrafish embryos and encompassing both the resin and the area
surrounding it. Note: the red and blue lines represent the areas of fluorescence intensity profile measured over the inactive resin (b, middle right
panel) and the Pd0-resin (c, middle left panel), respectively.
1
0.29, 10% MeOH in DCM), which yielded a light green solid (90 mg,
52% yield). 1H NMR (500 MHz, DMSO) δ 7.50 (d, J = 7.5, 1H), 7.42
(d, J = 5.7, 2H), 6.44 (d, J = 6.3, 1H), 6.17 (s, 1H), 5.99−5.89 (m,
1H), 5.79 (d, J = 7.5, 1H), 5.38−5.23 (m, 2H), 4.63 (dt, J = 5.5, 1.3,
2H), 4.47−4.37 (m, 2H), 4.18 (s, 1H), 4.07−4.00 (m, 1H). 13C NMR
(126 MHz, DMSO) δ 165.63 (C), 154.48 (C), 154.07 (C), 141.16
(CH), 132.02 (CH), 122.60 (t, JC−F = 264.6, C), 118.56 (CH2), 94.87
(CH), 83.89 (CH), 77.22 (CH), 70.09 (t, JC−F = 25.2, CH), 68.16
(CH2), 66.31 (CH2). LRMS (ESI) m/z 346.0 [M − H]−. HRMS
(FAB) m/z [M − H]− calcd for C13H14O6N3F2, 346.0856; found,
346.0859.
4-Amino-1-(2-deoxy-2,2-difluoro-5-O-[propargyloxy-carbonyl]-β-
D-erythro-pentofuranosyl)pyrimidin-2(1H)-one] (2p). The crude was
purified by column chromatography using 8% MeOH in DCM (Rf
0.23, 10% MeOH in DCM), which yielded a pale yellow solid (81 mg,
47%). 1H NMR (500 MHz, DMSO) δ 7.65 (d, J = 7.5, 1H), 7.44 (d, J
= 13.0, 2H), 6.24 (t, J = 5.0, 1H), 5.81 (d, J = 7.5, 1H), 5.28−5.26 (m,
2H), 4.88 (s, 2H), 4.22−4.16 (m, 1H), 3.79−3.62 (m, 3H). 13C NMR
(126 MHz, DMSO) δ 165.71 (C), 154.41 (C), 152.63 (C), 141.43
(CH), 121.48 (t, JC−F = 262.1, C), 94.83 (CH), 83.76 (CH), 79.16
(CH), 78.11 (CH), 77.25 (C), 73.39 (t, JC−F = 26.5, CH), 59.18
(CH2), 56.37 (CH2). LRMS (ESI) m/z 379.9 [M + Cl]−. HRMS
(FAB) m/z [M − H]− calcd for C13H12O6N3F2, 344.0700; found,
344.0728.
yield). H NMR (500 MHz, DMSO) δ 7.49 (d, J = 7.5, 1H), 7.44−
7.33 (m, 7H), 6.44 (d, J = 6.2, 1H), 6.17 (s, 1H), 5.76 (d, J = 7.5, 1H),
5.17 (s, 2H), 4.52−4.35 (m, 2H), 4.18 (s, 1H), 4.03 (t, J = 6.1, 1H).
13C NMR (126 MHz, DMSO) δ 165.60 (C), 154.45 (C), 154.20 (C),
141.15 (CH), 135.04 (C), 128.50 (CH)2, 128.42 (CH), 128.17
(CH)2, 122.57 (t, JC−F = 258.3, C), 94.83 (CH), 83.86 (CH), 77.18
(CH), 70.10 (t, JC−F = 23.4, CH), 69.22 (CH2), 66.39 (CH2). LRMS
(ESI) m/z 398.0 [M + H]+. HRMS (FAB) m/z [M − H]− calcd for
C17H16O6N3F2, 396.1013; found, 396.1016.
Synthesis of Carbamate-Protected Derivatives 5a,p,b. Synthesis
of Silylated Derivative 3. Gemcitabine HCl (500 mg, 1.67 mmol) was
dissolved in dry DMF (5 mL) with imidazole (398 mg, 5.85 mmol)
and TBS-Cl (301 mg, 2.0 mmol) stirred at r.t. overnight. The mixture
was then concentrated in vacuo, redissolved in EtOAc (50 mL), and
washed with H2O (50 mL). The aqueous layer was washed twice with
EtOAc and, subsequently, the organic layers combined, washed with
brine (150 mL), and dried over MgSO4. The crude was purified by
column chromatography (eluent 8% MeOH in DCM) to yield
1
intermediate 3 as a white solid 552 mg (88%). H NMR (500 MHz,
DMSO) δ 7.63 (d, J = 7.5, 1H), 7.38 (s, 2H), 6.31 (d, J = 5.1, 1H),
6.14 (t, J = 7.7, 1H), 5.76 (d, J = 7.5, 1H), 4.19−4.06 (m, 1H), 3.95 (d,
J = 11.8, 1H), 3.90−3.77 (m, 2H), 0.90 (s, 9H), 0.09 (d, J = 2.1, 6H).
13C NMR (126 MHz, DMSO) δ 165.58 (C), 154.56 (C), 139.91
(CH), 122.95 (t, JC−F = 258.3, C), 94.51 (CH), 83.45 (t, JC−F = 31.5,
CH), 79.76 (CH), 68.23 (t, JC−F = 22.7, CH), 60.72 (CH2), 25.71
(CH3), 17.99 (C), −5.52(CH3)3, −5.63 (CH3)2. MS (ESI) m/z 378.0
[M + H]+.
4-Amino-1-(2-deoxy-2,2-difluoro-5-O-[benzyloxy-carbonyl]-β-D-
erythro-pentofuranosyl)pyrimidin-2(1H)-one] (2b). The crude was
purified by column chromatography using 6% MeOH in DCM (Rf
0.05, 4% MeOH in DCM), which yielded a white solid (70 mg, 53%
5401
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Journal of Medicinal Chemistry
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Synthesis of Intermediate 4a. Compound 3 (100 mg, 0.27 mmol)
was dissolved in dry THF (2.5 mL) with pyridine (61 μL, 0.75 mmol)
and the mixture cooled to 4 °C in an ice bath. Allyl chloroformate (55
μL, 0.52 mmol) was added dropwise to the mixture and stirred for 1 h.
The solvent was removed in vacuo and purified via flash
chromatography (eluent: 3% MeOH in DCM) to yield compound
4a as a white solid.
4-(Allyloxycarbonylamino)-1-(2-deoxy-2,2-difluoro-5-O-[tert-
butyl(dimethyl)silyl]-β-D-erythro-pentofuranosyl)pyrimidin-2(1H)-
one] (4a). Eighty-three milligrams, 69% yield (Rf 0.53, 10% MeOH in
DCM). 1H NMR (500 MHz, MeOD) δ 8.29 (d, J = 7.7, 1H), 7.32 (d,
J = 7.6, 1H), 6.25 (t, J = 6.7, 1H), 6.04−5.94 (m, 1H), 5.39 (dq, J =
17.2, 1.5, 1H), 5.27 (dq, J = 10.5, 2.6, 1.3, 1H), 4.70 (dt, J = 5.6, 1.4,
2H), 4.33−4.24 (m, J = 12.5, 8.8, 1H), 4.10 (d, J = 12.0, 1H), 4.03−
3.89 (m, 2H), 0.97 (s, 9H), 0.17 (s, 6H). 13C NMR (126 MHz,
MeOD) δ 165.38 (C), 157.33 (C), 154.35 (C), 144.81 (CH), 133.24
(CH), 123.87 (t, JC−F = 259.7, C), 118.88 (CH2), 96.95 (CH), 86.22
(t, JC−F = 32.8, CH), 82.46 (CH), 69.56 (t, JC−F = 23.9, CH), 67.56
(CH2), 61.68 (CH2), 26.37 (CH3)3, 19.26 (C), −5.35 (CH3), −5.45
(CH3). MS (ESI) m/z 462.0 [M + H]+.
Synthesis of Intermediates 4p,b. Compound 3 (100 mg, 0.27
mmol) was dissolved in dry DMF (2.5 mL) with pyridine (61 μL, 0.75
mmol) and the mixture cooled to 4 °C in an ice bath. Propargyl (63
μL, 0.65 mmol) or benzyl chloroformate (56 μL, 0.39 mmol) was
added dropwise to the mixture. The mixture was stirred at r.t.
overnight (for 4b) or for 48 h (for 4p). The solvents were removed
and the mixture redissolved with EtOAc (30 mL) and washed with
H2O (30 mL). The aqueous layer was washed twice more with EtOAc
(30 mL each) and the combined organic layers dried over MgSO4,
solids filtered off, and concentrated in vacuo. The crude was purified
via flash chromatography (eluent: 3% MeOH in DCM) to yield
compounds 4p,b as white solids.
8.5, 3.1, 1H), 3.84−3.61 (m, 2H). 13C NMR (126 MHz, DMSO) δ
163.40 (C), 153.99 (C), 152.82 (C), 144.45 (CH), 132.39 (CH),
122.94 (t, JC−F = 258.3, C), 118.04 (CH2), 94.82 (CH), 84.07 (t, JC−F
= 32.8, CH), 80.98 (CH), 68.37 (t, JC−F = 22.7, CH), 65.64 (CH2),
58.79 (CH2). LRMS (ESI) m/z 346.0 [M − H]−. HRMS (FAB) m/z
[M − H]− calcd for C13H14O6N3F2, 346.0856; found, 346.0853.
4-(Propargyloxycarbonylamino)-1-(2-deoxy-2,2-difluoro-β-D-er-
ythro-pentofuranosyl)pyrimidin-2(1H)-one (5p). Fifteen milligrams
1
(62% yield). Rf 0.41 (10% MeOH in DCM). H NMR (500 MHz,
DMSO) δ 11.06 (s, 1H), 8.25 (d, J = 7.6, 1H), 7.08 (d, J = 7.6, 1H),
6.30 (d, J = 6.5, 1H), 6.16 (t, J = 7.4, 1H), 5.29 (t, J = 5.4, 1H), 4.80
(d, J = 2.3, 2H), 4.25−4.13 (m, 1H), 3.89 (dt, J = 8.5, 3.1, 1H), 3.84−
3.63 (m, 2H), 3.62 (t, J = 2.3, 1H). 13C NMR (126 MHz, DMSO) δ
163.26 (C), 153.93 (C), 152.37 (C), 144.61 (CH), 122.92 (C), 94.80
(CH), 84.09 (CH), 81.01 (CH), 78.21 (CH), 78.18 (C) 68.37 (CH),
58.78 (CH2), 53.01 (CH2). LRMS (ESI) m/z 344.0 [M − H]−.
HRMS (FAB) m/z [M − H]− calcd for C13H12O6N3F2, 344.0700;
found, 344.0719.
4-(Benzyloxycarbonylamino)-1-(2-deoxy-2,2-difluoro-β-D-eryth-
ro-pentofuranosyl)pyrimidin-2(1H)-one (5b). Fifty-one milligrams,
92% yield. Rf 0.42 (10% MeOH in DCM). 1H NMR (500 MHz,
DMSO) δ 10.98 (s, 1H), 8.23 (d, J = 7.6, 1H), 7.45−7.32 (m, 5H),
7.11 (d, J = 7.6, 1H), 6.31 (d, J = 6.5, 1H), 6.16 (t, J = 7.4, 1H), 5.29
(t, J = 5.5, 1H), 5.20 (s, 2H), 4.25−4.13 (m, 1H), 3.88 (dt, J = 8.5, 3.1,
1H), 3.84−3.62 (m, 2H). 13C NMR (126 MHz, DMSO) δ 163.38
(C), 153.99 (C), 153.00 (C), 144.46 (CH), 135.81 (C), 128.48
(CH)2, 128.19 (CH), 127.96 (CH)2, 122.93 (t, JC−F = 259.6, C), 94.86
(CH), 84.08 (t, JC−F = 30.2, CH), 80.98 (CH), 68.37 (t, JC−F = 22.7,
CH), 66.67 (CH2), 58.78 (CH2). LRMS (ESI) m/z 396.1 [M − H]−.
HRMS (FAB) m/z [M − H]− calcd for C17H16O6N3F2, 396.1013;
found, 396.1016.
Synthesis of Nonfluorescent Probes 8a,p. Probes 8a14 and 8p8
were prepared from rhodamine 110 chloride, 7, as previously
described.14,8
4-(Propargyloxycarbonylamino)-1-(2-deoxy-2,2-difluoro-5-O-
[tert-butyl(dimethyl)silyl]-β-D-erythro-pentofuranosyl)pyrimidin-
2(1H)-one] (4p). Fifty-three milligrams, 44% yield. (Rf 0.6, 10%
Synthesis of Nonfluorescent Probe 8b. Under a nitrogen
atmosphere, rhodamine 110 chloride (150 mg, 0.41 mmol) was
dissolved in dry DMF (4 mL). Separately, benzyl chloroformate (350
μL, 2.45 mmol) and pyridine (298 μL, 3.69 mmol) were dissolved in
DMF (1 mL) and added dropwise to the mixture. The reaction
mixture was stirred at r.t. for 48 h, solvents removed in vacuo, and the
resulting crude resuspended in 25% isopropanol in CHCl3 (30 mL)
and washed with H2O. The aqueous layer was then washed 5 times
with 25% isopropanol in CHCl3 (30 mL). The combined organic
layers were dried over anhydrous MgSO4 and the solids filtered off and
concentrated in vacuo. The crude was purified via flash chromatog-
raphy (hexane/ethyl acetate 2:1) to yield bis-N,N′-benzyloxycarbonyl-
rhodamine 110 (8b) as a white solid (31 mg, 13%). Rf 0.23 (hexane/
ethyl acetate 2:1); 1H NMR (500 MHz, DMSO) δ 10.12 (s, 2H), 8.01
(d, J = 7.6, 1H), 7.78 (t, J = 7.1, 1H), 7.72 (t, J = 7.3, 1H), 7.58 (d, J =
1.7, 2H), 7.45−7.33 (m, 10H), 7.27 (d, J = 7.6, 1H), 7.20−7.12 (m,
2H), 6.70 (d, J = 8.7, 2H), 5.18 (s, 4H). 13C NMR (126 MHz,
DMSO) δ 168.66 (C), 153.25 (C)2, 152.51 (C)2, 150.94 (C), 141.43
(C)2, 136.31 (C)2, 135.71 (CH), 130.21 (CH), 128.53 (CH)2, 128.47
(CH)4 128.19 (CH)4, 128.14 (CH)2 125.71 (C), 124.77 (CH),
123.95 (CH), 114.46 (CH)2, 112.48 (C)2, 105.10 (CH)2, 81.94 (C),
66.07 (CH2)2. MS (ESI) m/z 597.2 [M − H]−.
1
MeOH in DCM) H NMR (500 MHz, MeOD) δ 8.30 (d, J = 7.6,
1H), 7.29 (d, J = 7.6, 1H), 6.25 (t, J = 6.7, 1H), 4.84 (d, J = 2.5, 2H),
4.33−4.24 (m, 1H), 4.10 (d, J = 12.0, 1H), 4.03−3.90 (m, 2H), 2.99
(t, J = 2.3, 1H), 0.98 (s, 9H), 0.17 (s, 6H). 13C NMR (126 MHz,
MeOD) δ 165.28 (C), 157.29 (C), 154.57 (C), 144.97 (CH), 123.88
(t, JC−F = 259.6, C), 96.93 (CH), 86.15 (t, JC−F = 32.8, CH), 82.49
(CH), 77.70 (C), 76.92 (CH), 69.57 (t, JC−F = 22.7, CH), 61.69
(CH2), 54.34 (CH2), 26.36 (CH3)3, 19.26 (C), −5.36 (CH3), −5.46
(CH3). MS (ESI) m/z 460.2 [M + H]+.
4-(Benzyloxycarbonylamino)-1-(2-deoxy-2,2-difluoro-5-O-[tert-
butyl(dimethyl)silyl]-β-D-erythro-pentofuranosyl)pyrimidin-2(1H)-
one] (4b). Fifty-six milligrams, 42% yield (Rf 0.5, 10% MeOH in
1
DCM) which. H NMR (500 MHz, CDCl3) δ 8.11 (d, J = 7.5, 1H),
7.40−7.32 (m, 5H), 7.25−7.20 (m, 1H), 6.40−6.32 (m, 1H), 5.22 (s,
2H), 4.41−4.31 (m, 1H), 4.09−3.85 (m, 3H), 0.93 (s, 9H), 0.12 (s,
6H). 13C NMR (126 MHz, DMSO) δ 162.90 (C), 154.93 (C), 152.35
(C), 144.42 (CH), 135.02 (C), 128.85 (CH)2, 128.43 (CH)2, 122.32
(t, JC−F = 258.3, C), 95.54 (CH), 84.56 (t, JC−F = 32.8, CH), 81.62
(CH), 69.31 (t, JC−F = 26.5, CH), 68.22 (CH2), 60.69 (CH2), 25.97
(CH3)3, 18.5 (C), −5.32 (CH3), −5.41 (CH3). MS (ESI) m/z 534.1
[M + Na]+.
Synthesis of Carbamate-Protected Derivatives 5a,p,b. Com-
pounds 4a,p,b (48 mg, 0.1 mmol, 31 mg, 0.07 mmol, 73 mg, 0.14
mmol respectively) and TBAF solution (1.5 equiv for 4a,b and 2.5
equiv for 4p) were dissolved in anhydrous THF (3 mL) and stirred at
room temperature for 3 h (for 5b) or overnight (for 5a,p). The
resulting mixture was concentrated in vacuo and purified via flash
chromatography (eluent: 6% MeOH in DCM) to yield compounds
5a,p,b as white solids.
Biological Studies. General Methods. Cell lines were grown in
culture media supplemented with serum (10% FBS) and L-glutamine
(2 mM) and incubated in a tissue culture incubator at 37 °C and 5%
CO2. Human pancreas adenocarcinoma BxPC-3 cells (a kind gift from
Dr. Mark Duxbury) were cultured in Roswell Park Memorial Institute
(RPMI) media. Human pancreatic carcinoma Mia PaCa-2 cells (a kind
gift from Dr. Simon Wilkinson) were cultured in Dulbecco’s modified
Eagle’s media (DMEM).
Cell Viability Studies. Cells were seeded in a 96 well plate format at
the appropriate cell concentration (2,500 cells/well for BxPC-3 cells
and 1,000 cells/well for MiaPaCa-2 cells) and incubated for 48 h
before treatment. Each well was then replaced with fresh media
containing compound 1 or 2a,p,b/5a,p,b and incubated for 4 days.
Untreated cells were incubated with DMSO (0.1% v/v). PrestoBlue
4-(Allyloxycarbonylamino)-1-(2-deoxy-2,2-difluoro-β-D-erythro-
pentofuranosyl)pyrimidin-2(1H)-one [5a]. Thirty-two milligrams
1
(92% yield). Rf 0.27 (10% MeOH in DCM). H NMR (500 MHz,
DMSO) δ 10.96 (s, 1H), 8.23 (d, J = 7.6, 1H), 7.10 (d, J = 7.6, 1H),
6.31 (d, J = 6.5, 1H), 6.16 (t, J = 7.5, 1H), 6.00−5.89 (m, 1H), 5.42−
5.21 (m, 3H), 4.64 (d, J = 5.3, 2H), 4.24−4.13 (m, 1H), 3.88 (dt, J =
5402
dx.doi.org/10.1021/jm500531z | J. Med. Chem. 2014, 57, 5395−5404

Journal of Medicinal Chemistry
Article
cell viability reagent (10% v/v) was added to each well and the plate
incubated for 1 h. Fluorescence emission was detected using a
PerkinElmer Victor2 multilabel reader (excitation filter at 540 nm and
emissions filter at 590 nm). All conditions were normalized to the
untreated cells (100%) and curves fitted using GraphPad Prism using a
sigmoidal variable slope curve.
compliance with the Animals (Scientific Procedures) Act 1986 and
approved by the University of Edinburgh Ethics Committee.
ASSOCIATED CONTENT
* Supporting Information
■
S
Pd0-Mediated Deprotection of 5a,p,b in Biocompatible Con-
ditions. Compounds 5a, 5p, and 5b (100 μM) were dissolved in PBS
(1 mL) with 1 mg of Pd0-resins and shaken at 1400 rpm and 37 °C in
a Thermomixer. Reaction crudes were monitored at 0, 6, 24, and 48 h
by analytical HPLC (Agilent) using an UV detector at 280 nm to avoid
the detection of PBS salts. Eluent A, water and formic acid (0.1%);
eluent B, acetonitrile and formic acid (0.1%); A/B = 95:5 to 5:95 in 3
min, isocratic 1 min, 5:95 to 95:5 in 1 min, and isocratic 1 min.
Pd0-Mediated Dealkylation of 5a,p,b in Cell Culture. BxPC-3 and
MiaPaCa-2 cells were plated as described before. Each well was then
replaced with fresh media containing Pd0-resins (0.67 mg/mL) with
DMSO (0.1% v/v); 5a,p,b (3, 10, 30, 100, and 300 nM) with DMSO
(0.1% v/v); gemcitabine (3, 10, 30, 100, and 300 nM) with DMSO
(0.1% v/v); or a combination of 0.67 mg/mL of Pd0-resins + 5a,p,b
(3, 10, 30, 100, and 300 nM) with DMSO (0.1% v/v). Untreated cells
were incubated with DMSO (0.1% v/v).
NMR spectra of compounds 2a,p,b, 5a,p,b, and 8a,p,b and 5-
day time-lapse motion picture of pancreatic BxPC-3 cell
proliferation under treatment with 30 nM 5a; Pd0-resins + 30
nM 5a; 30 nM of 5p; and Pd0-resins + 30 nM of 5p. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Phone: 00 44 1317773584. E-mail: Asier.Unciti-Broceta@
igmm.ed.ac.uk.
Notes
The opinions expressed in this publication are those of the
authors and do not necessarily represent those of MSD, nor its
affiliates.
Cell Viability Assay. Cells were incubated with drugs for 5 days.
PrestoBlue cell viability reagent (10% v/v) was added to each well and
the plate incubated for 60 min. Fluorescence emission was detected
and results normalized as described above.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Time-Lapse Imaging Study. BxPC-3 cell growth studies were
carried out at a single dose of gemcitabine (1) or 5a,p (30 nM). Each
well was imaged every 3 h over 5 d under standard incubation
conditions using an IncuCyte ZOOM microscope (placed inside the
incubator). Image-based analysis of cell confluence and Supporting
Information movie 1 were produced using the IncuCyte software.
DNA Damage Study. Mia PaCa-2 cells were seeded in an 8-well
chamber slide with a density of 2,000 cells/well and incubated for 48 h
prior to treatment. Each well was then replaced with fresh media
containing 0.1% DMSO (v/v) and the following treatments: Pd0-resins
(0.67 mg/mL); 5p (300 nM); 1 (300 nM); or a combination of Pd0-
resin + 5p (300 nM). The combination of 5p and Pd0 resins were
incubated for 24 h at 37 °C prior to treatment with cells. Following 24
h of treatment, cells were fixed with paraformaldehyde (4%, 20 min)
and permeabilized cells (Triton X-100, 0.1%) subsequently treated
with rabbit monoclonal antibody against Phospho-Histone H2AX
(Ser139) (1:400, Cell Signaling Technologies, cat. no. 9718) for 1 h at
room temperature. This was followed by 1 h of incubation at room
temperature with secondary Alexa Fluor 488 linked antibody (1:1000,
goat antirabbit, IgG, Life Technologies), Hoechst 33342 (1:8000, Life
Technologies), and Alexa Fluor 594 Phalloidin (1:500, Life
Technologies). Cells seeded in the chamber slide were imaged using
an Olympus FV1000 microscope and merged using the ImageJ
software (National Institutes of Health).
Zebrafish Studies. Wild type zebrafish embryos were collected from
AB-TPL breeding pairs and reared at 28 °C in E3 embryo medium.
Twenty-four hours postfertilization, embryos were treated with the
anesthetic tricaine and pierced in the yolk with a fine needle. Either
nonfunctionalized resin or a Pd0-resin was then rapidly inserted into
the yolk. Embryos that lost significant yolk in the procedure were
removed from the experiment. Embryos were then gently transferred
to fresh E3 medium and returned to 28 °C to ensure the yolk wound
was closed. The corresponding probe (8a,p,b) was added to the
embryo medium (final concentration 5 μM) and fish incubated for
additional 24 h at 31 °C. Fish were imaged using fluorescent
microscopy (Olympus Scan-R). The fold change in fluorescence with
the Pd0 resin + 8p in comparison to that with the inactive resin + 8p
were quantified using ImageJ software. A line was drawn horizontally
over the yolk sac of the embryo encompassing both the resin beads
and the area surrounding them (Figure 8b,c), and the pixel intensities
along the lines were calculated (Figure 8d). Experiments were
repeated at least twice with n = 4/per condition. Zebrafish husbandry
and experiments were performed under Home Office License in
J.T.W. is grateful to the College of Medicine and Veterinary
Medicine and the University of Edinburgh for a Darwin
International Scholarship and an Edinburgh Global Research
Scholarship. N.O.C. and A.U.B. thank RCUK and IGMM,
respectively, for an Academic Fellowship. W.R., C.F., and E.E.P.
are funded by the MRC. We are grateful to the Edinburgh
Cancer Research UK Centre for funding this research through
the CRUK Development Fund. This work has been partly
funded by a Heriot Watt University − IGMM pilot project and
the MSD Scottish Life Sciences Fund. We also thank Dr. Mark
Duxbury for his helpful comments regarding the clinical
applications of the prodrug approach.
ABBREVIATIONS USED
■
BOOM, bioorthogonal organometallic; Alloc, allyloxycarbonyl;
Poc, propargyloxycarbonyl; Cbz, carboxybenzyl; TBS-Cl, tert-
butyldimethylsilyl chloride; TBAF, tetrabutylammonium fluo-
ride; dpf, days postfertilization; TLC, thin layer chromatog-
raphy; DBU, 1,8-diazabicycloundec-7-ene; DMF, dimethylfor-
mamide; DCM, dichloromethane; r.t., room temperature;
CD3OD, deuterated methanol; RPMI, Roswell Park Memorial
Institute; DMEM, Dulbecco’s modified Eagle’s media; DMSO,
dimethyl sulfoxide
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