Bioorthogonal prodrug activation driven by a strain-promoted 1,3-dipolar cycloaddition

Article abstract of DOI:10.1039/c4sc02574a

Due to the formation of hydrolysis-susceptible adducts, the 1,3-dipolar cycloaddition between an azide and strained trans-cyclooctene (TCO) has been disregarded in the field of bioorthogonal chemistry. We report a method which uses the instability of the adducts to our advantage in a prodrug activation strategy. The reaction of trans-cyclooctenol (TCO-OH) with a model prodrug resulted in a rapid 1,3-dipolar cycloaddition with second-order rates of 0.017 M^-1 s^-1 and 0.027 M^-1 s^-1 for the equatorial and axial isomers, respectively, resulting in release of the active compound. ¹H NMR studies showed that activation proceeded via a triazoline and imine, both of which are rapidly hydrolyzed to release the model drug. Cytotoxicity of a doxorubicin prodrug was restored in vitro upon activation with TCO-OH, while with cis-cyclooctenol (CCO-OH) no activation was observed. The data also demonstrates the potential of this reaction in organic synthesis as a mild orthogonal protecting group strategy for amino and hydroxyl groups.

Full text of DOI:10.1039/c4sc02574a

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Bioorthogonal prodrug activation driven by a
strain-promoted 1,3-dipolar cycloaddition†
Cite this: Chem. Sci., 2015, 6, 1212
Siddharth S. Matikonda,‡ Douglas L. Orsi,‡ Verena Staudacher,‡ Imogen A. Jenkins,
Franziska Fiedler, Jiayi Chen and Allan B. Gamble*
Due to the formation of hydrolysis-susceptible adducts, the 1,3-dipolar cycloaddition between an azide and
strained trans-cyclooctene (TCO) has been disregarded in the field of bioorthogonal chemistry. We report a
method which uses the instability of the adducts to our advantage in a prodrug activation strategy. The
reaction of trans-cyclooctenol (TCO-OH) with a model prodrug resulted in a rapid 1,3-dipolar
ꢀ1
ꢀ1
ꢀ1 ꢀ1
cycloaddition with second-order rates of 0.017 M
s
and 0.027 M
s
for the equatorial and axial
1
isomers, respectively, resulting in release of the active compound. H NMR studies showed that
activation proceeded via a triazoline and imine, both of which are rapidly hydrolyzed to release the
model drug. Cytotoxicity of a doxorubicin prodrug was restored in vitro upon activation with TCO-OH,
while with cis-cyclooctenol (CCO-OH) no activation was observed. The data also demonstrates the
potential of this reaction in organic synthesis as a mild orthogonal protecting group strategy for amino
and hydroxyl groups.
Received 24th August 2014
Accepted 7th November 2014
DOI: 10.1039/c4sc02574a
www.rsc.org/chemicalscience
The ability to deliver drugs only at their site of action is highly the prodrug, e.g. antibody-directed enzyme prodrug therapy
9,10
11–14
advantageous in cancer therapy. Unfortunately, the current (ADEPT)
and bioorthogonal chemistry,
could help over-
arsenal of drugs used to treat cancer are generally non-selective, come the low tumor-to-background ratios as well as avoid the
1–3
leading to toxic side effects. To combat toxicity, prodrug requirement of a cleavable (and potentially unstable) linker in
3
–5
activation strategies have been developed.
Activation, via the administered prodrug. While ADEPT has shown promise in
cleavage of a deactivating linker on the prodrug, is usually cancer therapy, anti-enzyme immune responses have hindered
10
triggered by an overexpressed enzyme or change in the tumor its in vivo application.
environment (e.g. pH). However, the changes are oen so subtle Over the past decade, bioorthogonal chemistry has found
that selective activation of small molecule prodrugs is low, with widespread utility in biological imaging. To date, however,
15
3
many linkers susceptible to non-specic, off-target hydrolysis.
there are only a handful of examples using bioorthogonal
11,16–20
Antibody–drug conjugates (ADCs) have the potential to avoid chemistry for in vitro prodrug activation strategies.
The
some of the problems associated with small molecule prodrugs best examples using click chemistry are those of Robillard who
6
17
and have been utilised effectively over the past few decades.
utilized the Staudinger reaction, and more recently, the trans-
11
However, there are still some inherent problems with ADCs cyclooctene (TCO)/tetrazine ligation for prodrug-activation.
6
including; (1) immunogenicity of the antibody, (2) difficulty in The fast kinetics of the tetrazine ligation provide in vivo
maintaining the balance between stability vs. cleavability of the potential, however, this is yet to be demonstrated for prodrug
antibody–drug linker, and (3) decreased potency of the drug as activation and we envisage a number of challenges that could be
the effective concentration is limited by the number of cell encountered in future studies. These may include; (1) reduced
5
surface receptors (ꢁ10 receptors per cell) and number of reactivity of the TCO-conjugate when it is present in a sterically
molecules that can be attached to the antibody. In addition, low demanding environment (e.g. attached to both the antibody and
target-to-background drug ratios of monoclonal antibodies cytotoxic drug), and (2) a low tumor-to-background ADC ratio
typically result in low tumor concentrations of the therapeutic leading to off-target release of the cytotoxic drug. Attachment of
7,8
agent. Pre-targeting strategies in which a non-toxic conjugate the tetrazine to the antibody would allow for pre-targeting with
is allowed to accumulate at the tumor before administration of a non-toxic conjugate, however, the doxorubicin–TCO prodrug
which would then need to be administered separately will be
exposed to potential isomerization back to the unreactive cis-
School of Pharmacy, University of Otago, Dunedin, 9054, New Zealand. E-mail: allan. cyclooctene (CCO), a problem which is avoided when the TCO is
gamble@otago.ac.nz
21
attached to the antibody via a suitably designed linker.
†
1
‡
Electronic supplementary information (ESI) available. See DOI:
0.1039/c4sc02574a
Inspired by the initial bioorthogonal activation strategies
and the in vivo challenges that they posed, we set out to develop
These authors contributed equally to the work.
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a new bioorthogonal prodrug activation based on a 1,3-dipolar these proof-of-concept experiments two azido-PABC analogues
click reaction between a TCO and an azide. Interestingly, this were synthesized (Scheme 2). Coumarin probes 8a and 8b,
click reaction has received much less attention than the strain- masking a 7-hydroxycoumarin and 7-amino-4-methylcoumarin,
promoted 1,3-dipolar cycloaddition of cyclooctynes and azides respectively, were selected to investigate the mechanism and
(
SPAAC) due to the instability of the formed adducts in aqueous rate of the 1,3-dipolar cycloaddition, triazoline/imine degrada-
15a,15c,22,23
conditions.
In 1992, Shea reported a reaction between a tion, and overall release of the drug from a carbonate and
24
3
strained TCO 2 and 2,4,6-trinitrophenylazide 1 in CDCl . Tri- carbamate linked drug. The previously reported doxorubicin
17
azoline 3 formed from this highly electron-decient system prodrug 9 was synthesized to examine in vitro bioorthogonal
could not be isolated. Instead a rearrangement in which activation, and stability/activation of the aryl azide linker in
diatomic nitrogen was released gave three isolated products; mouse serum. TCO 2 and trans-cyclooctenol (TCO-OH) 10 were
31
aldimine 4, ketimine 5 and aziridine 6 (Scheme 1).
synthesized using a modied procedure of Fox, in which the
We recognized that analogues of imine 4 or 5, when correctly CCO or cis-cyclooctenol (CCO-OH) was irradiated at 254 nm in
attached to an electron-decient self-eliminating linker (e.g. p- the presence of methyl benzoate (see ESI†). For TCO-OH 10 a
5,25
aminobenzyloxycarbonyl, PABC),
could have potential in mixture of diastereomers (1.42 : 1) was isolated and used in
targeted prodrug activation strategies (Fig. 1) or orthogonal preliminary studies. The major (equatorial) and minor (axial)
protection/deprotection chemistry. If an imine could be gener- isomers were subsequently separated for detailed kinetic
ated in situ by a 1,3-dipolar cycloaddition (step 1–3), acid-cata- analyses.
2
6
lyzed hydrolysis in the tumor microenvironment (pH ¼ 6.0–
Initially the cycloaddition of 8a/8b with TCO-OH 10 (Scheme
27,28
7.4)
would lead to a rapid 1,6-elimination and removal of the 3) was investigated using spectrouorometry (ex 360 nm, em
PABC protecting group from the prodrug to release the active 455 nm). Fluorescence of 7-hydroxycoumarin 13a or 7-amino-4-
2
5,26
drug (step 4 and 5).
While simple aryl azides can form stable methylcoumarin 13b was quenched by the azido-PABC linker
29
triazolines in organic solvents, we hypothesized that, in an (probe 8a/8b), which upon addition of TCO-OH 10 (10-fold
aqueous environment, the reaction of a TCO and an aryl azide excess) resulted in activation of 8a/8b (Fig. 2). By 6 h 80–90% of
bearing an electron-decient PABC group would favour in situ coumarin had been released from both carbonate 8a and
triazoline rearrangement via alkyl migration towards the carbamate 8b. In the absence of TCO-OH 10, a low level of
24
desired imine rather than aziridine formation.
carbonate hydrolysis in 8a was observed, however, no back-
Herein we report the rst in vitro example of a bioorthogonal ground hydrolysis was evident for the carbamate analogue 8b.
prodrug activation strategy utilizing the 1,3-dipolar cycloaddi- Reaction of 8a with CCO-OH (10-fold excess) had no effect on
tion of an azide and a strained trans-cyclooctenol (TCO-OH). the release of 13a, with only background hydrolysis measured
Our strategy demonstrates greater potential for bioorthogonally (10% at 24 h). As amides, esters and ethers of 13a/13b are non-
2
6,32–34
activated azide prodrugs in vivo as it holds two key advantages uorescent (ex 360 nm, em 455 nm),
over the use of the Staudinger reaction for prodrug activation;
(
the intermediates
11b and 12b would not exhibit any uorescence at the measured
1) activation is 1–2 orders of magnitude faster with potential excitation and emission wavelengths, hence the measured
for future improvements (not possible with the Staudinger uorescence is the result of released coumarin 13a/13b.
17
30
reaction), and (2) TCO can be modied so that it is more Therefore the results from our spectrouorometry studies imply
metabolically stable (i.e. no deactivation by isomerization to the that the reaction proceeds via triazoline 11b followed by an
21
cis-isomer) than the oxidation prone and serum reactive imine intermediate such as 12b.
30
phosphine. Our results also demonstrate the potential use of
To conrm that the reaction we observed in the initial
the cycloaddition in synthetic chemistry as an orthogonal spectrouorometry studies proceeds via the triazoline and
1
protection/deprotection strategy of –NH
groups.
2
and –OH functional aldimine, and not some alternate mechanism, a series of H
NMR experiments in CDCl and CD CN/D O were performed
3
3
2
In order for our strategy to succeed we rst needed to with 8a (Fig. 3 and Fig. S11–S14†). Reaction of TCO-OH 10 with
determine if the labile aldimine was favored over the stable 8a would have given a complex mixture of regio- and stereo-
aziridine, therefore enabling release of the active drug. For isomers 11b and 12b, therefore, TCO 2 was used (Scheme 3,
conc. 8a: 6.7 mM, TCO 2: 18.7 mM). This allowed us to monitor
1
for the less complex intermediates 11a and 12a in the H NMR
3
spectrum. Initially we examined the reaction in CDCl and
observed that approx. 50% of probe 8a had been consumed
within 3 hours and converted to the triazoline 11a. At 24 hours,
trace amounts of the imine 12a were observed, and by 5 days
1
2a was clearly visible in the reaction mixture. The isomers of
imine 12a (3 : 1 ratio) were identied via the imine proton
N]CH) observed as two doublets at d 7.75 (major, J ¼ 5.2 Hz)
and 7.77 (minor, J ¼ 4.4 Hz). Interestingly, no detectable levels
of the released coumarin 13a were observed in CDCl , even aer
days. This indicates that unlike the example of Shea (no tri-
(
3
5
24
24
Scheme 1 1,3-dipolar cycloaddition reported by Shea.
azoline detected), our benzyloxycarbonyl triazoline 11a and
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Fig. 1 Proposed general strategy for prodrug activation via a 1,3-dipolar cycloaddition. The targeting ligand could be an antibody, peptide or
small molecule.
ꢂ
Scheme 2 Conditions: (i) NaNO
2
, 5 M HCl then NaN
3
, 0 C, 73%; (ii) 4-
Fig. 2 Release of 7-hydroxycoumarin 13a from 8a and 7-amino-4-
methylcoumarin 13b from 8b in PBS : MeCN (1 : 1), measured by
fluorescence (ex 360, em 455). Error represented as ꢃSD (n ¼ 3).
ꢂ
nitrophenyl chloroformate, pyridine, DCM, 25 C, 57%; (iii) 7-hydroxy-
N, DMF, 25 C, 38%; (iv) 7-amino-4-methylcoumarin,
triphosgene, toluene, reflux, then 4-azidobenzyl alcohol, 25 C, 30%;
ꢂ
coumarin, Et
3
ꢂ
ꢂ
(
v) doxorubicin$HCl, Et
3
N, 4 A˚ molecular sieves, DMF, 25 C, 69%.
In a slightly more protic environment, CD CN/D O (9 : 1),
3
2
imine 12a are stable in organic solvent. If residual acid was not
removed from the CDCl (dependent on storage time of CDCl
coumarin 13a was rapidly released, with trace amounts imme-
diately observed upon mixing (time ¼ 0 h). At 47 hours none of
the initial probe 8a remained, and the major peaks observed
were due to the release of coumarin 13a and the imine hydro-
lysis product, aldehyde 14a. In this solvent system the internal
standard masked the area where the major and minor imine
protons were expected, but the absence of two doublets corre-
sponding to the aromatic protons of the imine 12a, and the
presence of the aldehyde peak at d 9.58 (d, J ¼ 1.2 Hz) indicated
that the imine was rapidly hydrolyzed in the presence of water.
The NMR studies indicate that water or acid is essential for
protonation and conversion of the triazoline 11a/b and imine
3
3
bottle) by ltration through a short plug of basic alumina, direct
conversion of probe 8a to the coumarin 13a occurred, with no
1
evidence of the intermediate imine 12a in the H NMR
spectrum.
12a/b intermediates to the PABC-analogue. As expected, the
PABC-analogue is not observed as it undergoes a rapid 1,6-
elimination to release coumarin 13a and aldehyde 14a/b.
The ketamine and aziridine intermediates analogous to that
reported for Shea's trinitrophenylazide derivatives 5 and 6
24
(
Scheme 1) were expected in small quantities, however, apart
from possible trace amounts (see aromatic region of 6.5–7.6
Scheme 3 Model prodrug activation strategy.
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1
Fig. 3 H NMR experiments in: (a) CDCl
3
(filtered through basic alumina) and (b) CD
3
CN/D
2
O (9 : 1); monitoring 1,3-dipolar cycloaddition
between TCO 2 and probe 8a. Legend: key structural proton shifts illustrated; #: coumarin probe 8a; *: triazoline 11a; -: aldimine 12a; C: 7-
hydroxycoumarin 13a; $: exocyclic aldehyde 14a; !: TCO 2; I.S.: Internal Standard, 4-iodonitrobenzene.
1
ꢀ3
ꢀ1 ꢀ1 15a
ppm in Fig. S14†), they were not observed in the H NMR Staudinger ligation (10
M
s ). Comparison of our reac-
2
4
spectra. This indicates that the thermodynamically favored tion rates to those of Shea demonstrate that with the highly
aldimine 12a dominates during the triazoline degradation. electron-decient tri-nitro substituted aromatic ring, a rate of
ꢀ1
ꢀ1
Based on our data from the spectrouorometry and NMR 0.687 M
s
3
in CDCl is achieved. While our rate for the
mechanistic studies, the reaction favors the synthesis of the cycloaddition is an order of magnitude slower than that of Shea,
desired and labile aldimine ($90% conversion), making it a synthesis of optimized linkers which have been substituted with
strong candidate for orthogonal protecting group chemistry and electron-withdrawing functional groups should enable us to
bioorthogonal prodrug activation.
reach or improve on these rates, particularly in an aqueous
The uorescent release and NMR studies show that TCO 2 environment.
and TCO-OH 10 activation of 8a/8b was responsible for the in
While the 1,3-dipolar cycloaddition is considered the key
situ generation of an aldimine with rapid release of coumarin rate-determining step in our approach, the triazoline and imine
13a/13b in protic solvents. However, the experiments did not degradation, and the nal 1,6-elimination also contribute to the
give an idea as to the speed for the initial 1,3-dipolar cycload- overall rate of drug/probe release. Therefore, our next goal was
dition in protic solvents. Using RP-HPLC we were able to to investigate the rate of triazoline and imine degradation.
measure the pseudo rst-order rate of the 1,3-dipolar cycload- Based on the spectrouorometry results (Fig. 2) we did not
ꢂ
dition in CH CN : PBS (1 : 1, 37 C) via the disappearance of expect the rates of the triazoline rearrangement and subsequent
3
probe 8a at 254 nm and calculate the second-order rate constant imine hydrolysis to be inuenced by the nature of the para-
(see Fig. S6 and S7†). In the presence of the TCO-OH major-10 substituted benzyloxy linker (carbonate vs. carbamate drug/
(
1
0
equatorial isomer) or minor-10 (axial isomer), the rate of the probe), thus we selected the more stable carbamate probe 8b to
ꢀ1
ꢀ1
,3-dipolar cycloaddition was measured as 0.017 M
s
ꢃ
investigate these steps (ESI Section 4†). To enable us to directly
ꢀ
1
ꢀ1
.003 and 0.027 M
s
ꢃ 0.006, respectively. The faster rate for measure release of the 7-amino-4-methylcoumarin 13b from the
minor-10 can be attributed to the higher energy in the axially triazoline intermediate, the 1,3-dipolar cycloaddition of 8b with
substituted ring, and will be important for future design of TCO major-10 was monitored by H NMR spectroscopy in
activating molecules. For comparison, the rate of the 1,3-dipolar CD CN and DMSO-d (Fig. S3†). Aer 19 h, H NMR analysis
31
1
1
3
6
cycloaddition between probe 8b and TCO major-10 was calcu- indicated that all of probe 8b had been consumed and con-
ꢀ
1
ꢀ1
lated as 0.020 M
that the presence of a carbamate linker does not affect the rate no imine, aziridine or 7-amino-4-methylcoumarin 13b
of cycloaddition (Fig. S8†). observed. An aliquot of the NMR sample was then diluted (1000-
s
ꢃ 0.0002 (CH
3
CN : PBS, 1 : 1), indicating verted to the triazoline intermediate 11b (diastereomers), with
Promisingly, even without modications to the aromatic ring fold) into PBS and the rate of triazoline and imine degradation
of the PABC linker or the TCO-OH ring, the rates are comparable was measured by the appearance of 13b on the spectrouo-
0
ꢀ3
ꢀ1
to the rst generation of SPAAC reactions (10 –10
M
rometer (Fig. S4† ex 360, em 455). By 1 h 88% of 13b had been
ꢀ
1
15a,15d
s
)
and are an order of magnitude faster than the released, and over the rst 40 min, the degradation and release
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process (three steps in total) appeared to follow pseudo rst- Table 1 IC50 values for doxorubicin 15, dox-prodrug 9, and activation
ꢂ
of 9 with cis- or trans-cyclooctenol 10, incubated for 72 h at 37 C
order kinetics with a half-life of 19 min (Fig. S5†). While we
cannot delineate the rates of the three processes, the kinetics
suggest that one of the steps, either triazoline degradation or
a,b,c
Compound
IC50
(mM)
imine hydrolysis is rate-limiting in a polar protic solvent. The Doxorubicin 15
increase in entropy and loss of CO
provides a signicant Dox-prodrug 9
9 + cis-cyclooctenol (100 mM)
0.71 (0.66–0.77)
49.9 (42.5–58.5)
55.0 (38.0–79.5)
0.96 (0.91–1.01)
1.47 (1.36–1.60)
1.34 (1.27–1.42)
4.98 (4.49–5.52)
2
35
thermodynamic driving force for the 1,6-elimination, thus
this is not expected to be rate-determining at a pH of 7.4 (t1/2
d
9
9
9
+ trans-cyclooctenol 10 (100 mM)
+ major-10 (100 mM)
+ minor-10 (100 mM)
¼
36
26
1
a
7 s), and is independent on the pK of the leaving group.
Our mechanistic studies with probe 8a and 8b demonstrate 9 + major-10 (10 mM)
that there are likely two steps which are rate determining in our
prodrug activation strategy, the 1,3-dipolar cycloaddition and
a
b
IC50 ¼ concentration required to kill 50% of cells. 95% condence
c
interval (n $ 6) is shown in parenthesis. Cell survival at 100 mm of
either the triazoline or imine degradation. However, as we CCO-OH, TCO-OH 10, major-10, minor-10 was 98%, 101%, 97% and
d
92%, respectively.
Mixture of major and minor diastereomers
progress to in vitro and in vivo pre-targeting studies, the rates of
triazoline and imine degradation become less signicant. Both
intermediates would be xed to the tumor cell surface, unable
to diffuse away from the tumor and exhibit off-target effects,
thus demonstrating the importance for selective and rapid
reactivity in the initial 1,3-dipolar cycloaddition. This will in
effect determine how much drug is released at the pre-targeted
tumor.
Next we examined the bioorthogonal potential of our
strategy via the activation of our doxorubicin-based prodrug 9
with TCO-OH 10 (Scheme 4) in a model murine melanoma cell
line (Table 1 and Fig. S16†). TCO-OH 10 was used in place of
TCO 2, due to its better solubility, low volatility, and potential to
attach a targeting ligand in the future.
(
1.42 : 1).
where the activation and release occurs, but suspect that it is
occurring both inside and outside the cells. However, from the
experiments we do know that release of 15 is promoted by the
1
,3-dipolar cycloaddition, and that in our proposed in vivo tar-
geting strategy (Fig. 1) release of 15 would be expected to occur
outside the cell followed by diffusion of the cytotoxic drug into
the closely located tumor cells.
Importantly, with only a 5-fold difference, improving the rate
of the cycloaddition (via modications to the aromatic ring of
the azido-PABC linker
higher release of 15 at even lower activation concentrations of
TCO-OH. Assuming one to six TCOs can be attached to each
monoclonal antibody which then binds to approximately 10
cell surface receptors, we estimate that a concentration of 0.4 to
2
(
signicantly less toxic than the parent drug 15 (70-fold) and
could be administered in relatively high doses, it will be
important to improve the rate of cycloaddition for our future
targeted in vivo studies, particularly when the number of acti-
vating TCO molecules on targeting ligands and therefore the
24,37
and TCO-OH 10 ring) could lead to
Against the melanoma cell line (Table 1 and Fig. S16†),
cytotoxicity of prodrug 9 was low (IC50: 49.9 mM) compared to
the parent drug 15 (IC50: 0.71 mM). This indicates that the azido-
PABC linker deactivates 15 and is stable in vitro. Combining 9
with TCO-OH 10 (100 mM), itself non-toxic to cells at this
concentration (Table 1 and Fig. S15†), restored activity of the
prodrug via release of 15 (IC : 0.96 mM). CCO-OH, also non-
7
5
6
.5 mM TCO would be present on the tumor cell surface
assuming cell volume of 400 femto litre). While prodrug 9 is
38
5
0
toxic at 100 mM, had no effect on release of 15 from 9 (IC : 55.0
5
0
mM), showing that bioorthogonal activation only occurs in the
presence of TCO-OH 10. Utilizing major-10 and minor-10 for
activation resulted in no signicant difference in IC50. Next we
examined the cytotoxicity of 9 when activated with a more bio-
logically relevant concentration of TCO-OH major-10 (10 mM).
To our excitement, an IC50 of 4.98 mM was obtained. While the
IC50 is approximately 5-fold lower than activation with 100 mM
TCO-OH 10, we envisage that release of 15 at a targeted and
doxorubicin-sensitive tumor (IC50 in low nanomolar range)
could still result in tumor cell death when prodrug 9, or
analogues thereof, are present at the tumor in the low micro-
molar range. From the current experiments we do not know
7
concentration on the cell surface is limited. A faster rate for the
cycloaddition will also help to overcome the relatively rapid
clearance rates observed in mice (t1/2 ¼ minutes) for small
39
molecules such as doxorubicin and its prodrugs, without the
requirement for larger and continuous dosing.
With proof-of-concept demonstrated in vitro, we next evalu-
ated the stability and activation of prodrug 9 in mouse serum so
as to determine its suitability for in vivo studies (ESI Section 8†).
While aryl azides can be prone to thiol reduction,
expected this to be minimal for prodrug 9 as the most cell-
40–42
we
relevant species (GSH and cysteine) require very high levels to
40,41
carry out azide reduction.
To conrm azide stability we
examined the degradation of 9 (100 mM) in 50% mouse ser-
um:PBS and PBS only (Table S1 and S2†). Prodrug 9 (t
min) was relatively stable in mouse serum (at 37 C), with 95%,
R
¼ 8.7
ꢂ
68%, and 56% intact at 4, 24 and 53 h, respectively (Table S1†).
The amount of doxorubicin 15 (tR ¼ 4.2 min) released in the
absence of TCO-OH 10 was also measured, and at 53 h, only 6%
was observed (Table S2†), indicating that most of the prodrug 9
Scheme 4 In vitro activation of prodrug 9.
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degradation was not due to azide reduction and subsequent 1,6- pre-targeting studies, we have demonstrated, for the rst time, a
elimination of 15. Activation of 9 in serum via the addition of strain promoted 1,3-dipolar cycloaddition with potential use in
TCO-OH major-10 (500 mM) resulted in rapid release of 15 (51% prodrug activation, imaging applications and orthogonal pro-
in 4 h, Table S2†). The amount of 15 which had been released tecting group strategies. The reaction proceeds with a second-
following activation decreased over time (4.7% at 48 h, Table order rate equivalent to the rst generation SPAAC and is 1–2
S2†), and correlated to an increase in a new peak (t
R
¼ 7.6 min, orders of magnitude faster than the Staudinger reaction
Fig. S23†). The decrease in 15 and appearance of the new peak enabling rapid orthogonal deprotection of azido-PABC pro-
was not observed in the control (PBS only) activation experi- tected amino and hydroxyl groups under mild conditions.
ment, with 79% of 15 observed at 48 h (Fig. S20, S24 and Table While azido-PABC carbonate-linked drugs are unlikely to be
S2†), indicating that free doxorubicin is slowly metabolized in stable in a biological milieu, the potential of azido-PABC
the serum or binds more strongly than prodrug 9 to serum carbamate drugs for bioorthogonal prodrug activation is
protein. The peak at 7.6 min was very weak in the serum demonstrated in a melanoma cell line, with the model doxo-
stability assay, conrming that the product is a metabolite of 15 rubicin prodrug 9 remaining deactivated until reaction with
and not prodrug 9. We are also aware that TCO-OH 10 could TCO-OH 10, upon which it regains the cytotoxicity of the parent
undergo slow isomerization back to the unreactive CCO-OH, drug 15. The prodrug also demonstrates good stability in mouse
however, future in vivo studies will use strategically designed serum over biologically relevant circulation times for cytotoxic
linkers attached to targeting ligands (e.g. antibodies) that are drugs (minutes to hours) and due to the rapid reactivity
8,21
known to stabilize the strained TCO-OH in vivo.
observed in serum:PBS, the azido-prodrug 9 does not appear to
Importantly, our serum studies are in contrast to the other bind strongly to, or react with, serum proteins like the cyclo-
potential azide prodrug activation strategy, the Staudinger octynes and triphenylphosphines of the in vivo SPAAC and
1
4,30,43
ligation, in which a byproduct of the phosphine formed in the Staudinger ligation pre-targeting studies.
30
serum resulted in severely reduced reaction efficiency. Exam-
We are currently investigating ways to improve the activation
rate of our prodrugs for future in vivo studies with modications
14
43
ining the in vivo SPAAC reported by Bertozzi and Robillard
indicated that strong covalent and non-covalent interactions to to the PABC linkers and TCO molecules (e.g. electron-with-
24
37
serum proteins could potentially reduce the effective concen- drawing functionalities such as nitro and uorine groups ).
tration, and therefore reactivity, of our prodrug 9 at the pre- Additionally, we are examining other linkers with azide triggers
targeted tumor. This could be detrimental to any pre-targeting that can be used as stable alternatives to the carbonate linker,
strategy as the strong serum binding in the case of cyclooctyne expanding the scope of our activation strategy to both alcoholic
resulted in even lower levels of in vivo reactivity than the slug- and phenolic drugs.
14
gish Staudinger ligation. While both groups reported that
their studied cyclooctynes (the reagent added aer tumor pre-
targeting) suffered from reduced in vivo reaction rates because
of strong covalent and non-covalent interactions with serum
Acknowledgements
This research was funded in part by a University of Otago
Research Grant (UORG) and a contract from the Health
Research Council (HRC) of New Zealand (A.B.G). The authors
would also like to thank Sarah Katzemich for her contributions
to the synthesis of the 7-hydroxycoumarin probe.
14,43
proteins,
the reactivity of our reagents did not appear to be
hindered in the presence of the mouse serum proteins (we
observed 51% of doxorubicin 15 being released in 4 h, Table
S2†). The rate was slightly faster in serum:PBS than in PBS alone
(34% release of doxorubicin 15 in 4 h, Table S2†), providing
further evidence that in vivo serum protein binding of prodrug 9
should be minimized (lower binding affinity than the cyclo-
Notes and references
14,43
octynes used in SPAAC).
Promisingly, the second order rate
of 1,3-dipolar cycloaddition for prodrug 9 (0.1 mM) with TCO
major-10 (10 mM) measured under pseudo rst-order reaction
conditions in 50% serum:PBS (Fig. S25†) was calculated to be
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