Click to release: Instantaneous doxorubicin elimination upon tetrazine ligation

Article abstract of DOI:10.1002/anie.201305969

Eliminated without a trace: The fastest click reaction, the highly selective inverse-electron-demand Diels-Alder reaction, has been modified to enable selective bioorthogonal release. Thus, the click reaction of a tetrazine with a drug-bound trans-cyclooctene caused the instantaneous release of the drug and CO₂ (see scheme). One possible application is the chemically triggered release, and thereby activation, of a drug from a tumor-bound antibody-drug conjugate. Copyright

Full text of DOI:10.1002/anie.201305969

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DOI: 10.1002/anie.201305969
Bioorthogonal Reactions
Click to Release: Instantaneous Doxorubicin Elimination upon
Tetrazine Ligation**
Ron M. Versteegen, Raffaella Rossin, Wolter ten Hoeve, Henk M. Janssen, and
Marc S. Robillard*
Bioorthogonal conjugation or click reactions are enjoying
widespread use in the fields of chemistry, chemical biology,
molecular diagnostics, and medicine, among others, where
they enable the selective manipulation of molecules, cells,
particles and surfaces, and the tagging and tracking of
biomolecules in vitro and in vivo.[1] These reactions include
the Staudinger ligation, the azide–cyclooctyne cycloaddition,
and the inverse-electron-demand Diels–Alder (inv-DA) reac-
tion.[1,2] Analogous to click reactions, cleavable linkers, such
as the redox-sensitive disulfide and diazo linkers and the
hydrazine-labile levulinoyl linker, have many potential uses in
biological media.[3] Although these linkers are selective, they
often lack the level of bioorthogonality and broad applic-
ability of the click reactions, which is especially relevant when
extending the application scope to living cells, animals, and
humans. This issue may be addressed by adapting a click
reaction to effect selective release, instead of, or in addition
to, selective conjugation. From this family of reactions, only
the Staudinger ligation[4] and the parent Staudinger reaction[5]
have been applied in selective cleavage. However, their
reactivity is low, and the phosphine reagents are prone to
oxidation. We set out to develop a new bioorthogonal and
rapid elimination reaction that in addition to a broad scope of
in vitro applications would allow effective chemical manip-
ulation in living systems, and eventually humans. One
potential application is antibody–drug-conjugate (ADC)
release, wherein the linker between the tumor-bound anti-
body and the drug is selectively cleaved through reaction with
a probe, which is administered in a second step. This approach
does not rely on the currently employed endogenous intra-
cellular ADC activation mechanisms (such as enzymatic
activation), and thus expands the scope of suitable ADC
targets to those that do not efficiently internalize and to
extracellular-matrix constituents.[6]
So far, only the fastest click reaction, the inv-DA reaction,
has shown sufficient potential for use in living systems under
clinically relevant conditions. In the context of tumor-
pretargeted radioimmunoimaging, we[7,8] and others[9,10]
have shown that the inv-DA reaction between antibody-
conjugated trans-cyclooctene (TCO) and a radiolabeled tet-
razine occurs effectively in mice at low equimolar concen-
trations (Figure 1a and Scheme 1a). The cycloaddition results
Figure 1. a) Tumor pretargeting with the inverse-electron-demand
Diels–Alder (inv-DA) cycloaddition in live mice (circle: radiolabeled
moiety).[7] b) Envisioned on-tumor antibody–drug-conjugate (ADC)
release and activation enabled by the new inv-DA-based elimination
reaction (hexagon: drug).
[*] R. Rossin, M. S. Robillard
Tagworks Pharmaceuticals
High Tech Campus 11, 5656 AE Eindhoven (The Netherlands)
E-mail: marc.robillard@tagworkspharma.com
in an intermediate that rearranges by expulsion of dinitrogen
in a retro-Diels–Alder cycloaddition to a 4,5-dihydropyrid-
azine 5, which may tautomerize to a 1,4-dihydropyridazine
6,[2,11,12] especially under aqueous conditions.[13] Depending on
the substituents, the dihydropyridazine can be converted into
an aromatic pyridazine 7 in the presence of an oxidant, such as
dioxygen.[12] Importantly, the conversion of dihydropyrid-
azines into a pyridazine by the elimination of a leaving group
from the vinyl position or through a double-bond shift has
also been shown.[12,14]
R. M. Versteegen, H. M. Janssen
SyMO-Chem
Den Dolech 2, 5612 AZ Eindhoven (The Netherlands)
W. ten Hoeve
Syncom
Kadijk 3, 9747 AT Groningen (The Netherlands)
[**] We thank L. H. J. Kleijn (SyMO-Chem) for assistance in synthesis of
the starting compounds, Dr. Johan Lub for insightful discussions,
and Hugo Knobel for HRMS measurements (Philips Research). This
research is supported by NanoNextNL (The Netherlands).
We consequently envisioned that the versatile dihydro-
pyridazine motif could be enlisted to provoke the release of
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201305969.
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a suitably positioned TCO-bound substance, such as a drug
(Figure 1b and Scheme 1b). In particular, we hypothesized
that the 1,4-dihydropyridazine product 10 derived from
a tetrazine and a TCO 8 containing a carbamate-linked
drug at the allylic position could be prone to undergo
conversion into a conjugated pyridazine 11 by the formation
of an exocyclic double bond
benzylisocyanate or doxorubicin (via 4-nitrophenylcarbonate
activation of 16) into compounds 8 (Scheme 1). We also
prepared three tetrazines spanning a wide range in electron
density: 3,6-bis(2-pyridyl)-1,2,4,5-tetrazine (2), 3-(2-pyridyl)-
6-methyl-1,2,4,5-tetrazine (3), and 3,6-dimethyl-1,2,4,5-tetra-
zine (4), which were stable in phosphate-buffered saline
with the elimination of CO2
and an NH2-substituted drug
14. The pyridazine 11 might
subsequently rearrange to the
favored aromatic pyridazine
12. The key features of 10
resemble those of the well-
established self-immolative p-
aminobenzyloxycarbonyl
(PABC) linker used in
ADCs;[15] in a similar fashion,
the shift of the electron lone
pair of NH into the ring may
enable an electronic-cascade-
based release of the drug
(Scheme 1b). We also consid-
ered the less likely possibility
that 14 could be expelled
from the 4,5-tautomer 9 by
carbamic acid elimination
(decarboxylation)
initiated
by removal of the H-4
proton, again to afford 11;
analogously, 14 could be
expelled from 13. Such a pro-
cess is similar to that observed
with b-eliminative linkers
used in drug-linked macro-
molecules and hydrogels, but
would require a sufficiently
acidic H-4 atom.[16]
To test our hypothesis, we
prepared model compounds
(E)-cyclooct-2-en-1-yl ben-
zylamine carbamate (8a,e-
Bn) and the doxorubicin con-
jugates (E)-cyclooct-2-en-1-yl
doxorubicin carbamate (8a,e-
Dox; Scheme 1b–d). The
anticancer drug doxorubicin
was selected as a model for
future ADC applications with
potent toxins. (Z)-Cyclooct-2-
enol (15) was isomerized to
(E)-cyclooct-2-enol (16) by
photolysis[17] to afford two
isomers with the hydroxy
group
positioned
either
Scheme 1. a) Inv-DA reaction between TCO 1 and tetrazines 2–4. b) Synthesis of TCO 8 and potential
release mechanisms from inv-DA product 9, following the reaction of 8 with tetrazines 2–4; not all possible
tautomer conversions and stereoisomers are shown. c) Equatorial and axial isomers of (E)-cyclooct-4-enol
1 and (E)-cyclooct-2-en-1-yl carbamates 8. d) Doxorubicin (14-Dox) release from TCO conjugate 8a-Dox. a:
axially (isomer 16a) or equa-
torially (isomer 16e).[18] (E)-
Cyclooct-2-enol (16) was sub-
sequently converted with axially substituted trans-cyclooctene; e: equatorially substituted trans-cyclooctene.
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Table 1: Tetrazine stability and reactivity (second-order rate constants) towards TCO constructs, as
measured by UV/Vis spectroscopy at 540 nm (n=3).
pyridazine reported by Fox and co-
workers.[13] In parallel, the initial
4,5-tautomer was converted into
a product with equal mass, accord-
ing to GC–MS (see Figure S5) and
Probe
Tetrazine stability at 378C
Tetrazine reactivity with TCOs in MeCN at 208C:
k2 [mÀ1 sÀ1
8a-Bn
]
in PBS:
t1/2 [h]
in serum: proportion 8e-Bn
1a
intact at 4 h [%]
HPLC–MS/PDA
(PDA = photo-
diode array; Figure 3a). This prod-
uct was identified as the 1,4-tauto-
mer 10a/13a on the basis, for exam-
ple, of the D2O-exchangeable NH
signal at d = 7.05 ppm (see Sec-
2
3
4
9.6Æ0.13
75.1Æ4.2
0.37Æ0.10
57.7Æ5.0
5.94Æ0.24
0.54Æ0.06
1140Æ85
141.5Æ14.80 96.3Æ1.9
–
–
–
–
14.4Æ0.35
100Æ2.0
(PBS) at 378C with half-lives greater than 9 h (Table 1). The
reactivity of 2 towards the reference TCO (E)-cyclooct-4-enol
1a (Scheme 1a,c) was 1140mÀ1 sÀ1 in MeCN at 208C, which
corresponds to at least the rate constant of k2 = 7.9 ꢀ
104 mÀ1 sÀ1 in water at 208C found for 1a and a slightly less
reactive 4-amido-2-pyridyl derivative of 2.[8] The reactivity of
2 towards axial and equatorial 8a,e-Bn was 57.70 and
0.37mÀ1 sÀ1, respectively, in MeCN at 208C (Table 1).
Although the axial isomer would be expected to be more
reactive,[8] the large 156-fold difference is probably also due to
steric hindrance of the approach of the tetrazine by the
equatorial benzylcarbamate moiety. The 20-fold lower reac-
tivity of 8a-Bn as compared to that of 1a is presumably
a result of the same steric effect. We selected 8a-Bn for
subsequent studies, measured its reactivity with tetrazines 3
and 4, and found an expected approximately 10- and 100-fold
decrease relative to its reactivity with 2 owing to the increased
electron density in these tetrazines (Table 1). Carbamate 8a-
Bn appeared to be as stable (t1/2 @ 20 days in PBS at 208C) as
the (E)-cyclooct-4-enol-derived carbamates that are widely
used in click reactions.[1,2]
tion S6 in the Supporting Information).[19] Overall, the
reaction yielded about 60% of both benzylamine (14-Bn)
and the elimination product 12, and 40% of the 1,4-tautomer
10a/13a, which did not eliminate benzylamine (Figure 3a; see
Figure S4 for a plot of product formation for this NMR
spectroscopic experiment). When the reaction was repeated
in 33% [D3]MeCN in deuterated PBS, we observed an
expected faster reaction and 64% free benzylamine, thus
demonstrating maintained efficacy of the pyridazine elimi-
nation under aqueous conditions (see Section S6 in the
Supporting Information).
In contrast to the reaction with 4, no significant release of
benzylamine was observed for the reaction of 8a-Bn with 2 in
dry [D3]MeCN or in 33% [D3]MeCN in deuterated PBS after
18 h. 1H NMR spectroscopic analysis in [D3]MeCN did reveal
the rapid formation of the 4,5-tautomer of the inv-DA adduct,
but this structure appeared to be stable. The results of the
reaction between 8a-Bn and tetrazine 3 fell in between those
of the reactions of 2 and 4, with 45% benzylamine released
after 18 h in 33% [D3]MeCN in deuterated PBS. Despite the
complexity arising from the unsymmetrically substituted
tetrazine 3 and the corresponding regioisomers of the inv-
We next used 1H NMR spectroscopy to monitor the
reaction between TCO 8a-
Bn and tetrazine 4 in dry
[D3]MeCN at 208C. Within
minutes,
a rapid inv-DA
reaction led to the complete
conversion of tetrazine 4 and
the formation of the initial
4,5-tautomer of the inv-DA
adduct (9a,Figure 2; see also
Figure S3 in the Supporting
Information). To our delight,
the amount of the 4,5-tauto-
mer started to decline after
approximately 10 min in
conjunction with the forma-
tion of free benzylamine (14-
Bn) and the non-aromatic
elimination product 11, char-
acterized by the alkylidene
proton at 6.55 ppm. Inter-
mediate 11 spontaneously
rearranged to aromatic 12,
the signals for which
matched the characteristic
signals of a similar (E)-cyclo-
Figure 2. 1H NMR spectroscopic analysis of the reaction of tetrazine 4 with TCO 8a-Bn in [D3]MeCN at 208C.
1
2
3
~
&
Legend: ꢀ: tetrazine 4; : TCO 8a-Bn; +: 4,5-inv-DA adduct 9a (R ,R =CH3; R =Bn); : non-aromatic
1
2
^
*
elimination product 11; : aromatic elimination product 12; : 1,4-inv-DA adducts 10a/13a (R ,R =CH3;
octene-derived 3,6-diphenyl- R3 =Bn); : benzylamine (14-Bn).
*
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that 4,5- to 1,4-tautomerization may occur readily under
water-free conditions by virtue of a 1,3-hydride shift,[11,12] and
therefore it is possible that 9 leads to a short-lived inter-
mediate 10, which liberates 14, whereas formed 13 does not
release this moiety. However, the results so far also support
direct elimination from 9 to 11 (or similarly from 13), whereby
10 cannot liberate 14. Importantly, from the product-forma-
tion plot it follows that aromatization to 12 cannot be the
driver of the elimination, as it occurs on a longer time scale
than the formation of 14 (see Figure S4 in the Supporting
Information).
We subsequently employed HPLC–MS/PDA to study the
release of doxorubicin (14-Dox) from the TCO conjugate 8a-
Dox (25 mm) as induced by 2–4 (10 equiv) in 25% MeCN in
PBS at 378C (Table 2 and Figure 3b; see also Figure S6). The
Table 2: Doxorubicin (14-Dox) release from 8a-Dox following the
addition of tetrazine 2–4 (10 equiv) in 25% MeCN in PBS or 50% serum
at 378C, as measured by HPLC–MS/PDA at 4 h (n=3).
Probe
Doxorubicin release [%]
in PBS/MeCN (3:1)
in serum
2
3
4
7Æ3
55Æ4
79Æ3
0
12Æ1
46Æ3
75Æ4
0
[a]
–
[a] No release of 14-Dox from 8a-Dox was observed at 378C in PBS
(72 h) or serum (24 h).
reactions were complete within minutes and, in analogy to the
NMR spectroscopic experiments, treatment with 2 gave
almost no 14-Dox (7%), whereas 3 and 4 afforded free 14-
Dox in 55 and 79% yield, respectively, after 4 h (Table 2). In
fact, the elimination reached its maximum within 4 min for 3
and 16 min for 4 (limited by its lower reactivity; Figure 3b).
Analogous to 8a-Bn, the reaction between 8a-Dox and
tetrazines 2–4 afforded dihydropyridazine products that
either led to rapid doxorubicin release or remained stable.
No evidence was found by MS for the deactivation of 9, 10, or
13 by oxidative aromatization. To determine whether the
elimination is dependent on the TCO isomer, we monitored
the reaction between 8e-Dox and 3 in PBS at 378C and found
a similar value for liberated 14-Dox (51 Æ 1.5%; n = 3) after
4 h. Next, we tested the release from 8a-Dox in serum with
tetrazines 2–4 (largely stable in serum for 4 h; Table 1) and
found similar values to those obtained in PBS (Table 2). This
finding and the fact that 8a-Dox by itself did not liberate 14-
Dox for at least 24 h in PBS or serum at 378C support the
bioorthogonality of the system. The faster elimination
observed in the HPLC experiments in 25% MeCN in PBS
as compared to the NMR experiments in [D3]MeCN indicates
an important role of water in the release and possibly
supports the mechanism involving the conversion of 10 into
11. The slightly higher yields relative to those observed in the
NMR experiments in 33% [D3]MeCN in PBS are possibly
due to the increased percentage of water and/or the increased
temperature. The formic acid used in HPLC–MS/PDA was
shown by NMR spectroscopy to facilitate the aromatization
Figure 3. a) HPLC–MS/PDA analysis of the reaction of tetrazine 4 with
TCO 8a-Bn in [D3]MeCN at 208C after 18 h. The upper graph is the
MS trace in the positive mode; the lower graph is the PDA trace.
^
*
Legend: : elimination product 12; : inv-DA adduct 10a/13a
1
2
3
~
*
(R ,R =CH3; R =Bn); : excess TCO 8a-Bn; : oxidized inv-DA
adduct (possibly formed during analysis); 14-Bn is not visible.
b) Doxorubicin (14-Dox) release (%) from 8a-Dox following the
addition of tetrazine 2–4 (10 equiv) in 25% MeCN in PBS as measured
by HPLC–MS/PDA. Legend: dotted line: 2; dashed line: 3; solid line:
4.
1
DA product, key H NMR signals in dry [D3]MeCN, such as
the triplet for the alkylidene hydrogen atom of 11 at d =
6.75 ppm, were similar to those observed for the reaction of
4 (see Figure S3 and Section S6 in the Supporting Informa-
tion).
The inv-DA reaction and the subsequent cleavage of the
carbamate can give rise to multiple interconverting tautomers
and stereoisomers, the relative abundances of which are
probably dependent on the tetrazine substituents R1 and R2
and the reaction medium, among other factors. Therefore,
further work is required to fully elucidate the elimination
pathway. Nevertheless, the elimination has been shown to
occur via 9, to depend on the nature (e.g. electron density) of
the tetrazine, and to lead to the key intermediate 11, thus
supporting the hypothesis that it is driven by the inv-DA
reaction and dihydropyridazine rearrangement. It is known
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of 11 to give 12, but not the formation of benzylamine (14-Bn)
from 8a-Bn (data not shown).
cleavable linkers or masking moieties in bi- and trispecific
biologics, chemical biology, life-science assays, and materials
chemistry to enable the controlled (dis)assembly of mole-
cules, proteins, cells, or biomaterials. The availability of an
elimination reaction with the bioorthogonality and speed of
the inv-DA will enable enhanced control over chemical and
biological manipulations in vitro and in living systems.
Finally, having established the proof of principle of the
inv-DA pyridazine elimination, we were interested in testing
the system in a cellular environment. Although future efforts
will be focused on applying the system in ADCs, we expected
that unconjugated 8a-Dox would still have a lower cytotox-
icity than that of the parent 14-Dox, possibly also owing to
differences in cellular uptake, thus allowing the monitoring of
its activation in an in vitro cytotoxicity assay.[5] Indeed, when
tetrazines 2–4 (10 mm) were combined with the TCO con-
jugate 8a-Dox in an A431 cell culture, increased cytotoxicity
Received: July 9, 2013
Revised: October 15, 2013
Published online: November 26, 2013
Keywords: cycloaddition · trans-cyclooctene · elimination ·
by a factor of 14, 53, and 78 (EC : 0.280, 0.072 and 0.049 mm),
.
50
pyridazines · tetrazines
respectively, was observed relative to that of 8a-Dox alone
(EC50: 3.834 mm). These values reflect the different maximal
release percentages and the EC50 value of 0.037 mm of 14-Dox
alone (Scheme 1d and Table 3; see also Figure S7). This
effective recovery of the cytotoxicity of the parent drug,
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Table 3: EC50 (half-maximal effective concentration) values for doxoru-
bicin (14-Dox), tetrazines 2–4, and the TCO conjugate 8a-Dox alone and
in the presence of tetrazines 2–4, as obtained from proliferation assays
on A431 tumor cells.
[5] R. van Brakel, R. C. Vulders, R. J. Bokdam, H. Grꢁll, M. S.
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Compound
EC50 [mm][a]
14-Dox
8a-Dox
8a-Dox+2 (10 mm)
8a-Dox+3 (10 mm)
0.037 (0.026–0.052)
3.834 (2.051–7.166)
0.280 (0.208–0.375)
0.072 (0.053–0.097)
0.049 (0.036–0.066)
18.98 (11.99–30.04)
21.05 (12.22–36.24)
48.23 (27.28–85.24)
8a-Dox+4 (10 mm)
2
3
4
[a] The EC50 95% confidence interval (n=3) is given in parentheses.
combined with the higher EC50 values found for tetrazines 2–4
alone, indicates that 8a-Dox was efficiently activated.
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In summary, we have developed a new bioorthogonal
elimination reaction that enables instantaneous, self-immola-
tive, and traceless release of a substance from trans-cyclo-
octene following tetrazine ligation. In this proof-of-principle
study, the inv-DA pyridazine elimination reached 79% yield
within minutes under ambient conditions at micromolar
concentrations. The components are essentially the same as
those used successfully in living systems, and we expect that
further optimization of the reactivity and release will lead to
application as a novel chemically cleavable ADC linker.[20]
Identification of the drug-releasing tautomer is key, as this
may provide inroads to new inv-DA components that will
favor its formation. The trans-cyclooct-2-enol building block
is readily accessible, and ample options exist for the prepa-
ration of bifunctional derivatives for conjugation. The use of
a radiolabeled tetrazine as the drug-releasing probe would
possibly allow a combination of the ADC approach with
pretargeted radioimaging or radiotherapy to monitor drug
release or for combination therapy, respectively. Besides
ADC applications, we also envision the use of inv-DA-
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[20] The reactivity between 3 and 8a-Bn is only ca. 20-fold lower than
that exploited successfully for pretargeting in mice in Ref. [7]. In
an ADC application, the administration of 3 in excess, which is
not possible in pretargeting, can compensate this lower reac-
tivity.
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