Bioorthogonal Uncaging of the Active Metabolite of Irinotecan by Palladium-Functionalized Microdevices

Article abstract of DOI:10.1002/chem.201803725

SN-38, the active metabolite of irinotecan, is released upon liver hydrolysis to mediate potent antitumor activity. Systemic exposure to SN-38, however, also leads to serious side effects. To reduce systemic toxicity by controlling where and when SN-38 is generated, a new prodrug was specifically designed to be metabolically stable and undergo rapid palladium-mediated activation. Blocking the phenolic OH of SN-38 with a 2,6-bis(propargyloxy)benzyl group led to significant reduction of cytotoxic activity (up to 44-fold). Anticancer properties were swiftly restored in the presence of heterogeneous palladium (Pd) catalysts to kill colorectal cancer and glioma cells, proving the efficacy of this novel masking strategy for aromatic hydroxyls. Combination with a Pd-activated 5FU prodrug augmented the antiproliferative potency of the treatment, while displaying no activity in the absence of the Pd source, which illustrates the benefit of achieving controlled release of multiple approved therapeutics—sequentially or simultaneously—by the same bioorthogonal catalyst to increase anticancer activity.

Full text of DOI:10.1002/chem.201803725

A Journal of
Accepted Article
Title: Bioorthogonal Uncaging of the Active Metabolite of Irinotecan by
Palladium-Functionalized Microdevices
Authors: Catherine Adam, Ana M. Pérez-López, Lloyd Hamilton, Belén
Rubio-Ruiz, Thomas L. Bray, Dirk Sieger, Paul M. Brennan,
and Asier Unciti-Broceta
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201803725
Link to VoR: http://dx.doi.org/10.1002/chem.201803725
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10.1002/chem.201803725
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Bioorthogonal Uncaging of the Active Metabolite of Irinotecan by
Palladium-Functionalized Microdevices
Catherine Adam,[a] Ana M. Pérez-López,[a] Lloyd Hamilton,[b] Belén Rubio-Ruiz,[a] Thomas L. Bray,[a]
Dirk Sieger,[b],[a] Paul M. Brennan*[a],[c] and Asier Unciti-Broceta*[a]
Abstract: SN-38, the active metabolite of irinotecan, is released upon
liver hydrolysis to mediate potent antitumor activity. Systemic
exposure to SN-38, however, also leads to serious side effects. To
reduce systemic toxicity by controlling where and when SN-38 is
localize drug activity at the tumor site. While such approaches are
yet to demonstrate its utility in the clinic, they have the potential
to reduce systemic side effects and enhance treatment efficacy
by generating greater drug levels at the disease site than can be
safely achieved by standard chemotherapy.
generated,
a new prodrug was specifically designed to be
metabolically stable and undergo rapid Palladium-mediated activation.
Blocking the phenolic OH of SN-38 with a 2,6-bis(propargyloxy)benzyl
group led to significant reduction of cytotoxic activity (up to 44-fold).
Anticancer properties were swiftly restored in the presence of
heterogeneous Palladium (Pd) catalysts to kill colorectal cancer and
glioma cells, proving the efficacy of this novel masking strategy for
aromatic hydroxyls. Combination with a Pd-activated 5FU prodrug
augmented the antiproliferative potency of the treatment, while
displaying no activity in the absence of the Pd source, which illustrates
the benefit of achieving controlled release of multiple approved
therapeutics —sequentially or simultaneously— by the same
bioorthogonal catalyst to increase anticancer activity.
Palladium (Pd) catalysts are one of the tools of choice currently
under investigation to release caged drugs in vitro and in vivo.[7-
11,17-20]
The selection of this metal is based on its high bio-
compatibility, its versatility to adopt different shapes and sizes and
its remarkable capabilities to catalyze N- and O-dealkylation
reactions on manifold types of substrates under physiological
conditions.[21-23] Our lab is currently investigating the application
of heterogeneous Pd catalysts as implantable devices to mediate
drug release with spatiotemporal control and in a catalytic fashion,
with the goal of improving the safety profile of chemotherapies
without having the short, limited life of other local therapy
modalities such as drug eluting devices (carmustine wafers)[24] or
brachytherapy.[25]
The camptothecin-derived topoisomerase I inhibitors topotecan
and irinotecan (Figure 1a) are anticancer drugs used in the
treatment of ovarian (topotecan), lung (both) and colon
(irinotecan) cancers. Irinotecan crosses the blood brain barrier[26]
and displays high cytotoxic activity against glioblastoma cells with
multi-drug resistance to other therapies.[27] In addition, several
Phase II clinical trials have reported that the combination of
bevacizumab and irinotecan shows promising activity in recurrent
malignant glioma with a modest increment in median survival.[28-
30] The side effects of irinotecan treatments are, however, a major
concern that limits its therapeutic dose and significantly impacts
patient’s quality of life. To circumvent this issue, the intratumoral
implantation of irinotecan-loaded drug-eluting beads has been
investigated in patients with recurrent glioblastoma[31] and
colorectal liver metastasis.[32] While these treatments are well
tolerated, the drug is fully cleared from the organism within hours
of implantation, thus reducing its anticancer effect.[31,32] Of note,
irinotecan is in fact an orally-bioavailable prodrug that requires
enzymatic conversion into its active metabolite SN-38 (1) to reach
its full cytotoxic potential (Figure 1a).[33] Although glioma cells
have been shown to partly metabolize irinotecan, the drug is
primarily metabolized into 1 in the liver, from where it distributes
throughout the organism. This further rationalizes the limited
effect of irinotecan-loaded drug-eluting devices in the treatment of
brain cancers.
Introduction
Bioorthogonal reactions are designed to take place in cells and
organisms without interfering with biological functions.[1] In the
pursuit of exploiting such processes in cancer therapy, reactions
and tools that were once exclusively used to synthesize drugs in
chemistry labs have been recently adapted to perform such tasks
in living systems.[2-20] Chemotherapeutics as doxorubicin,[2-7]
5FU,[8-11] gemcitabine,[17] floxuridine,[18] vorinostat[19] or nitric oxide
precursors[20] can be “manufactured” from inactive precursors in
biological environments through a variety of bio-independent
processes, including click-to-release reactions and bioorthogonal
organometallic catalysis. In combination with a suitable cancer
targeting strategy (e.g. antibody-based tumor targeting,[15]
enhanced permeability and retention effect,[6,11] intratumoral
implantation[7]), these highly selective reactions can facilitate the
spatially controlled release of one or more therapeutic agents to
[a]
Dr C. Adam, Dr A. M. Pérez-López, Dr B. Rubio-Ruiz, T. L. Bray, Dr
P. M. Brennan and Prof. A. Unciti-Broceta
Cancer Research UK Edinburgh Centre, MRC Institute of Genetics and
Molecular Medicine, University of Edinburgh (UK)
*E-mail: Asier.Unciti-Broceta@igmm.ed.ac.uk
[b]
L. Hamilton and Dr. D. Sieger
Building on previous success in the development and bio-
independent release of caged drugs with catalyst-loaded de-
vices,[7-11] herein we report the design, synthesis and screening
of a novel class of inactive camptothecin derivatives that are
selectively converted into cytotoxic 1 by bioorthogonal Palladium
chemistry.
Centre for Neurogeneration, The Chancellor's Building, University of
Edinburgh (UK)
[c]
Dr P. M. Brennan
Centre for Clinical Brain Sciences, University of Edinburgh (UK)
*E-mail: paul.brennan@ed.ac.uk
Supporting information for this article is given via a link at the end of the
document.
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10.1002/chem.201803725
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a
O
5
O
O
7
6
17
N
22
9
N
16
8
C
N
N
D
B
10
11
O
N
A
E
HO
O
2
3
O
O
N
21
N
15
13
14
N
N
20
12
O
18
HO
O
O
O
HO
HO
19
Topotecan
Camptothecin
Irinotecan
b
BIOLOGICAL
ACTIVATION
Carboxylesterase
metabolism
BIOORTHOGONAL
ACTIVATION
O
O
alkyl
N
N
HO
O
O
O
Palladium-catalyzed
uncaging
N
N
O
O
HO
HO
O-alkyl prodrugs
1
c
Figure 1. (a) Camptothecin and clinically approved derivatives topotecan and irinotecan. (b) Biological (enzymatic) activation of irinotecan to release the active
metabolite 1 and bioorthogonal (palladium-catalyzed) activation of alkylated prodrugs of 1. (c) Dose-response curves for HCT116, U-87 and U-251 cells after 5 d
treatment with 1, topotecan and irinotecan. Error bars: ± SEM, n = 3.
Results and Discussion
It has been recently reported that the blockade of the NH2 group
of doxorubicin with an o-(propargyloxy)benzyloxycarbonyl group
dramatically reduces the bioactivity of the resulting derivative,
while making it activatable by Pd chemistry.[7] Inspired on this
Rationale for prodrug design and synthesis of 2a-d.
Preliminary screening of the anticancer properties of 1, topotecan
and irinotecan against colorectal cancer HCT116 cells, and
glioma U-87 and U-251 cell lines, confirmed the superior potency
of 1 relative to irinotecan (Figure 1c). Topotecan also has lower
potency against the HCT116 cell line and, to a lesser extent, U-
251 when compared to 1 (Figure 1c), but the activity gap is not
as prominent as in irinotecan, which has greater than 80-fold
difference in EC50 (Table S1). Encouraged by the dramatic
change in activity between compounds that only differ by the
chemical group found around the C10 position of the
camptothecin scaffold (ring A, Figure 1a), it was rationalized that
the aromatic hydroxyl group of 1 was a convenient handle where
bulky masking groups could be incorporated and thereby reduce
prodrug-target interactions.
observation,
a
novel masking group —namely 2,6-
bis(propargyloxy)benzyl— incorporating two symmetrically-
placed Pd-sensitive triggers was designed to block the OH at C10
of 1 and thereby endow high metabolic stability and increased
sensitivity to Pd catalysis. Upon catalyst-mediated O-dealkylation
of either of the propargyl groups, the masking group will eliminate
spontaneously to release 1 (see activation mechanism in
Scheme 1b) Derivatives 2a and 2b, incorporating a propargyl and
a 4-propargyloxybenzyl group, respectively, were used as
positive controls. A 2,6-bis(methoxy)benzyl group was also tested
as a steric mimic of the 2,6-bis(propargyloxy)benzyl group that is
unable to be cleaved by Pd chemistry (negative control).
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Prodrugs 2a-d were prepared in a single Williamson ether
coupling of 1 and the corresponding alkyl halide in the presence
of a base (Scheme 1). Commercially available propargyl bromide
and 2,6-dimethylbenzyl chloride were used in the synthesis of 2a
and 2d, respectively, and benzyl chlorides 5 and 9 were used (see
Supp. Inf.) for the preparation of 2b and 2c. Of note, alkylation
rates greatly increased under microwave irradiation.
a
O
O
alkyl
(i) or (ii)
N
N
HO
O
O
O
N
N
O
HO
O
HO
Me
1
2a-d
2d
2a
2b
2c
alkyl
Me
=
O
O
O
b
Pd0
+ 2H2O
OH
O
O
+ H2O
+
1
SN-38
HO
SN-38
1,6-elimination
O
Pd-devices
OH
R=
H,
R
R
O
O
HO
HO
O
+ H2O
+
SN-38
1
SN-38
HO
+ 2 or 4H2O
O
OH
O
OH
Pd0
1,4-elimination
Scheme 1. a) Synthesis prodrugs 2a-d. Reagents and conditions: (i) Alkyl halide, K2CO3, DMF, rt, overnight; or (ii) Alkyl halide, K2CO3, MeCN, µw, 120 ºC, 2 h. b)
Proposed Pd-triggered uncaging mechanism of 2b,c.
Cytotoxicity study: 1 vs 2a-d. The antiproliferative properties of
compounds 2a-d were tested in cancer cell culture against
HCT116, U-87 and U-251 cells and compared to 1. In line with the
expectation that steric bulk around the phenol group at C10
reduces compounds’ capacity to interact with its target
(topoisomerase I),[34] it was found that the larger the alkyl group
the greater the reduction in cytotoxicity (Fig. 2). O-propargylation
(2a) of 1 slightly reduced cytotoxicity compared to the parent
compound (up to 3-fold reduction, Table 1). Incorporation of a 4-
propargyloxybenzyl group (2b) had a superior while still minor
effect (up to 9-fold reduction). In contrast, compound 2c
(containing the novel 2,6-bis(propargyloxy)benzyl group) led to a
much greater reduction in cytotoxicity across the three cell lines
(up to 44-fold reduction) and, thus, was selected for the next
phase of investigation. Control compound 2d also decreased the
experimental cytotoxicity in all cell lines (Fig. S1) by a similar
magnitude to 2c. EC50 values are shown in Table 1.
Table 1. Calculated EC50 values (nM) for 1 and 2a-d in colorectal and glioma
cell lines. n = 3.
Compound
HCT116
U-87
U-251
1
1.9
5.1
16
84
104
94
63
123
1466
603
5.8
12
22
205
92
2a
2b
2c
2d
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Figure 2. Dose response curves for HCT116, U-87 and U-251 cells after 5 d
treatment with 1 or 2a-c. Error bars: ± SEM, n = 3.
Prodrug Activation Studies: In Vitro and in Cell Culture.
Prodrug 2c was incubated with 30 µm Tentagel resins loaded with
Pd(0) nanoparticles (Pd-microdevices, 2 % w/w in Pd)[7] under
physiological conditions to assess its sensitivity to Pd catalysis.
Pd-microdevices were suspended in PBS with 10% v/v serum
containing either 2c, 1 (+ve control) or 2d (–ve control) and
incubated for 2 d at 37 ºC. Naked beads (used as purchased prior
to Pd loading) were used as a metal-free control. Reactions were
monitored by fluorescence spectroscopy. The characteristic
emission spectrum of 1 (λmax= 547 nm, which is absent in 2c and
2d) facilitated the study of drug release by fluorescence analysis.
As shown in Figure 3b,c, fluorescence signal from uncaged
compound 1 rapidly increased over time when 2c was incubated
with Pd-microdevices, being patently visible after 4 h. In contrast,
incubation of 2c alone (Figure S2) or with naked beads (Figure
3d) under the same conditions did not increase the signal at 547
nm (= no formation of 1). Similarly, incubation of the control
compound 2d (Figure S3) with Pd-resins did not result in the
release of 1. A decrease in the overall fluorescence intensity was
observed both in the presence of Pd-microdevices and naked
beads, presumably due to partial compound sequestration by the
device. Incubation of unmodified 1 with Pd-microdevices or naked
beads resulted in identical fluorescence spectra (Figure 3e),
indicating that Pd does not have any effect on the structural
integrity of the active drug 1.
Figure 3. (a) Palladium catalyzed conversion of 2c into 1. (b,c) Rate of
transformation of 2c (100 µM) into 1 in the presence of Pd(0) (1 mg/mL Pd-
devices) in PBS + 10% serum at 37 ºC. (b) Monitoring of changes in
fluorescence spectra over time. (c) Increase of fluorescence intensity at 547 nm
over time (d) Control experiment: fluorescence spectra of 2c (100 µM) after 2 d
in biocompatible conditions in the presence of naked beads. Black line
represents the fluorescent spectra of 1 (100 µM). (e) Control experiment:
fluorescence spectra of 1 after 2 d in biocompatible conditions in the presence
of Pd-devices (dotted line) or naked beads (solid line).
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controls) or in combination (activation assay) and compared to
treatment with unmodified 1 (+ve control) across a range of
concentrations. As expected,[7-11] the devices (which are larger
than cells and remain in the extracellular space) displayed no
toxic effect. In contrast, the Pd-devices/2c combination led to
potent antiproliferative effect in all cell lines, slightly lower than
that mediated by direct treatment with 1. Due to the high potency
of the active metabolite, nanomolar prodrug concentrations were
sufficient to achieve high toxicity, while 2c did not display
cytotoxicity in the absence of the Pd source. As expected, the
antiproliferative properties of 2d were not improved in the
presence of Pd-microdevices (see Figure S7).
Multi prodrug activation study: Co-treatment with Pro-5FU.
Irinotecan is typically administered in combination with 5FU for
the treatment of colorectal cancer.[35] As our lab had previously
developed a Pd-labile prodrug of 5FU, a.k.a. Pro-5FU (Figure
5),[8] we were intrigued about the prospect of combining 2c and
Pro-5FU in the presence of Pd to simultaneously release two
clinically-used synergistic drugs by the same bioorthogonal
triggering mechanism. Such an approach could serve to boost
treatment efficacy without compromising safety, an optimal
strategy for fighting difficult-to-treat cancers. Following the
protocol described before, colorectal cancer HCT116 cells were
incubated with 2c and Pro-5FU in the presence and absence of
Pd-microdevices. The experiment was also performed in the U-
87 cell line, since the therapeutic effect mediated by the 2c/Pd-
devices combination was suboptimal (Figure 4). As shown in
Figure 5, no signs of toxicity were observed from the combination
of Pro-5FU (30 or 100 µM) and 2c (10 or 100 nM) in either cell
line (see Figure S8 for co-treatment dose-response curves).
Notably, in the presence of the activating device, the combined
treatment of the prodrugs elicited superior cytotoxic activity than
the 2c/Pd-devices or direct treatment with 1 (Figure 5); evidence
that both drugs were concomitantly released by Pd chemistry.
Preliminary assessment on the most effective timing of
administration of the combination partners against U-87 cells
suggested that compound 2c should be administered first (see
Figure S9). The potential of manufacturing multiple synergistic
drugs at a desired location is one of the most clinically relevant
features of this strategy.
Figure 4. Pd-catalyzed uncaging of 2c in cancer cell culture. Experiments: 0.1%
(v/v) DMSO (control, grey); 1 mg/mL of Pd-devices (–ve control, black stripes);
1 (+ve control, orange); 2c (–ve control, green); 1 mg/mL of Pd-devices + 2c
(activation assay, green with black stripes). Cell viability was measured at day
5 using PrestoBlue. Error bars: ± SEM, n = 3.
The bioorthogonal release of cytotoxic 1 from inactive 2c by
extracellular Pd-microdevices was then tested in cancer cell
culture in HCT116, U-87 and U-251 cells (Figure 4). Experiments
were conducted under standard cell culture conditions (media
supplemented with serum, 5% CO2, 37 °C). Cells were
independently treated with Pd-microdevices (1 mg/mL) or 2c (–ve
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combinations by the same bioorthogonal method. This study
showcases the versatile applicability of using heterogeneous
metal catalysts as a way to control the release of therapeutics at
desired locations. By intratumoral implantation of the Pd-
devices,[7] otherwise toxic therapeutic agents such as 1 and 5FU
could be generated at the tumor site to mediate focal
chemotherapy and thereby reduce systemic side effects.
Experimental Section
General. Reactions requiring anhydrous conditions were carried out under
nitrogen in oven-dried glassware. Dry solvents and reagents were
purchased from Acros, Fluorochem, Sigma-Aldrich or VWR and used as
received. Irinotecan was purchased from Carbosynth, and topotecan from
ABCR. Both were used as purchased. NMR spectra were recorded on a
Bruker 500 MHz spectrometer at 300 K and referenced relative to the
solvent residual peaks with chemical shifts (δ) reported in ppm. Coupling
constants (J) are reported in Hertz. High resolution mass spectra were
measured in a Bruker MicrOTOF II. Analytical and semi-preparative TLC
were performed using Merck TLC Silica Gel 60 F254 plates and visualized
by UV light and flash column chromatography using silica gel (220−440
mesh, Sigma-Aldrich). All compounds used in the biological experiments
were >95% pure by UPLC, as measured using a C18 Column, 60 ºC;
monitoring at 210 nm; eluent A, water with TFA (0.1%); eluent B,
acetonitrile. Method 1 @0.4 mL/min: A/B = 95:5 isocratic 0.2 min, 95:5 to
5:95 in 2.3 min, 5:95 isocratic 0.5 min, 5:95 to 95:5 in 0.01 min, 95:5
isocratic 0.49 min. Method 2 @0.2 mL/min: A/B = 95:5 isocratic 0.5 min,
95:5 to 5:95 in 10 min, 5:95 isocratic 0.49 min, 5:95 to 95:5 in 0.01 min.
Stock solutions (100 mM) were prepared in DMSO diluted to 5 µM in water.
General method for the synthesis of 2a-d. Compound 1 (1 eq.) was pre-
stirred with K2CO3 (1.5 eq.) in DMF or MeCN (20 mL/mmol) before adding
the corresponding alkyl halide (1.2 eq.). Reactions were then stirred at
ambient temperature overnight or heated under microwave irradiation for
2 h at 120 ºC. Solvent was removed under reduced pressure and the crude
prodrugs purified by semi-preparative TLC.
Figure 5. Combined Pd-catalyzed activation of 2c and Pro-5FU in cancer cell
culture. Experiments: 0.1% (v/v) DMSO (control, grey); 1 mg/mL of Pd-devices
(–ve control, black stripes); 1 mg/mL of Pd-devices + 2c (+ve control activation,
green with black stripes); 1 + 5FU (+ve control, yellow); 2c + Pro-5FU (–ve
control, light blue); 1 mg/mL of Pd-devices + 2c + Pro-5FU (combined activation
assay, blue stripes). Cell viability was measured at day 5 using PrestoBlue.
Error bars: ± SEM, n = 3.
Synthesis and characterization of 2c. 38 mg scale, MeCN, µw, purified
4% MeOH/ CH2Cl2 to yield a yellow solid (23 mg, 39%). 1H NMR (500 MHz,
DMSO-d6) δ 8.07 (d, J = 9.2 Hz, 1H), 7.68 (d, J = 2.7 Hz, 1H), 7.53 (dd, J
= 9.2, 2.7 Hz, 1H), 7.41 (t, J = 8.4 Hz, 1H), 7.29 (s, 1H), 6.87 (d, J = 8.4
Hz, 2H), 6.49 (s, 1H), 5.44 (d, J = 1.4 Hz, 2H), 5.33 (s, 2H), 5.26 (s, 2H),
4.88 (d, J = 2.4 Hz, 4H), 3.56 (t, J = 2.4 Hz, 2H), 3.24 (q, J = 7.3, 2H), 1.88
(m, 2H), 1.33 (t, J = 7.4 Hz, 3H), 0.89 (t, J = 7.3 Hz, 3H); 13C NMR (126
MHz, DMSO-d6) δ 173.0, 158.4, 157.6, 156.4, 150.5, 150.0, 146.8, 145.1,
144.5, 131.9, 130.9, 130.3, 128.8, 128.3, 123.1, 118.6, 113.4, 106.8, 105.1,
96.5, 79.6, 78.9, 72.9, 65.7, 56.7, 50.0, 30.7, 22.8, 14.0, 8.2; HRMS (ESI+)
m/z [M+H]+ calcd for C35H31N2O7: 591.2126 found: 591.2149. Purity: 95%
(UPLC, method 2).
Conclusions
In this manuscript we have reported the first inactive precursor of
the potent topoisomerase I inhibitor SN-38 (1) that is specifically
released by Pd chemistry. Prodrug 2c was successfully masked
with a novel alkyl group, 2,6-bis(propargyloxy)benzyl, which
incorporates two Pd-labile propargyl groups at the ortho positions
of the benzyl group to increase steric hindrance and augment the
rate of cleavage in the presence of Pd catalysts. 2c showed low
inherent cytotoxicity to colorectal cancer and glioma cells. In the
presence of extracellular Pd-microdevices, the Pd-labile group at
the C10 position of the prodrug was efficiently cleaved to release
the cytotoxic compound 1 both in vitro and in cancer cell culture.
While previous attempts to make multiple bioactive agents in cell
culture have been reported,[36] herein it is shown for the first time
the concomitant uncaging of two drugs used in clinical
Conflict of Interests
The authors declare that compounds 2b,c are protected under
patent application PCT/GB2017/051379.
Acknowledgements
We are grateful to the CRUK (Pioneer Award) and the EPSRC
(EP/N021134/1) for funding. BR-R and TLB thanks the EC
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10.1002/chem.201803725
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Catherine Adam, Ana M. Pérez-López,
Belén Rubio-Ruiz, Thomas L. Bray,
Lloyd Hamilton, Dirk Sieger, Paul M.
Brennan,* and Asier Unciti-Broceta*
2c
IRINOTECAN
BIOLOGICAL
ACTIVATION
BIOORTHOGONAL
ACTIVATION
Page No. – Page No.
Bioorthogonal Uncaging of the Active
Metabolite of Irinotecan by Palladium-
Functionalized Microdevices
Carboxylesterases
Pd nanoparticles
SN-38
nM potency against cancer cell lines
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