Click and Release: A High-Content Bioorthogonal Prodrug with Multiple Outputs

Article abstract of DOI:10.1021/acs.orglett.9b01086

A high-content bioorthogonal prodrug with multiple outputs using the "click, cyclize, and release" concept is described. The proof of concept is established by the co-delivery of a gasotransmitter carbon monoxide, an anticancer drug floxuridine, and an in

Full text of DOI:10.1021/acs.orglett.9b01086

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Click and Release: A High-Content Bioorthogonal Prodrug with
Multiple Outputs
Xingyue Ji,†,‡ Robert E. Aghoghovbia,† Ladie Kimberly C. De La Cruz,† Zhixiang Pan,† Xiaoxiao Yang,†
Bingchen Yu,† and Binghe Wang*,†
†Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia 30303, United
States
‡Department of Medicinal Chemistry, College of Pharmaceutical Science, Soochow University, Suzhou, Jiangsu 215021, China
S
* Supporting Information
ABSTRACT: A high-content bioorthogonal prodrug with multiple
outputs using the “click, cyclize, and release” concept is described. The
proof of concept is established by the co-delivery of a gasotransmitter
carbon monoxide, an anticancer drug floxuridine, and an in situ
generated fluorescent reporter molecule for real-time monitoring of the
prodrug activation. Bioorthogonal prodrugs as such are invaluable tools
for the co-delivery of other drug payloads for multimodal therapy.
rodrug strategies have been successfully used in addressing
Pdrug developability issues by masking toxicity, improving
pharmacokinetic and physiochemical profiles, and affording
controlled delivery, among others.1 Traditional prodrug
strategies largely rely on chemical reactions mediated by
endogenous enzymes or small molecules to release the parent
drug.2 Recently, click chemistry has been creatively applied to
the design of bioorthogonal prodrugs, wherein the parent drug
is activated by a bioorthogonal reaction between the prodrug
and the another exogenously applied trigger/component.3
Such bioorthogonal prodrugs have been applied in the delivery
of anticancer agents4 and antibody drug conjugates (ADCs),5
yielding a controlled release of the payload at the tumor site.
Very recently, bioorthogonal prodrugs for gasotransmitters
including carbon monoxide (CO),6 carbonyl sulfide (COS),7
and sulfur dioxide (SO2)8 have also been devised to address
the unique challenging issues associated with the delivery of a
gaseous molecule. The need for a secondary trigger in using
bioorthogonal chemistry in drug delivery has also presented
unique challenges associated with the bimolecular nature of the
drug activation approach. Recently published pretargeting5 and
enrichment-triggered activation9 strategies have proven their
utility in animal models.
Figure 1. A schematic illustration of the “click, cyclize, and release”
concept. (A) Previous work with a single output, and (B) this work
with multiple outputs.
CO, an anticancer drug floxuridine (5-fluoro-2′-deoxyuridine),
and a fluorescent reporter (Figure 1B).
Previously, we have successfully developed a “click, cyclize,
and release” system for the delivery of doxorubicin to
mitochondria. However, such bioorthogonal prodrugs as well
as many others just yield a single output upon activation.
Herein, we are interested in going beyond the delivery of a
single payload (Figure 1A) by developing a general strategy of
prodrugs with multiple outputs, whether in payload, signal, or a
combination of both. We herein describe our efforts toward a
high-content bioorthogonal prodrug system using a similar
“click, cyclize, and release” concept with multiple outputs upon
activation, as exemplified by the co-delivery of gasotransmitter
Carbon monoxide has been well-established as a gasotrans-
mitter with pleiotropic pharmacologic effects.10 It has also
been shown to have synergistic effects with other drugs such as
anticancer,11 antibacterial,12 and anti-inflammatory agents.13 In
some cases, CO also alleviates some severe side-effects
observed with chemotherapies,14 making CO a very promising
adjuvant in disease treatment. Therefore, it would be highly
desirable to develop a drug delivery system for the co-delivery
Received: March 28, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b01086
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Letter
of CO and another payload for multimodal therapy. Along this
line, we describe our efforts toward a high-content
bioorthogonal prodrug system for the co-delivery of CO and
an anticancer drug payload.
The development of CO prodrugs represents a major
challenge due to its gaseous and chemically inert nature.
Generally speaking, CO delivery approaches reported so far are
classified into five categories: (1) gaseous CO for inhalation
administration, (2) CO in a drink (dissolved CO) for oral
delivery, (3) metal-immobilized carbonyls (commonly referred
to as CO-releasing molecules or CORMS) for oral or
parenteral administration,10a,15 (4) photosensitive organic
CO donors for topical application,16 and (5) organic CO
prodrugs for oral and parenteral administration.3b,17 In
developing organic CO prodrugs capable of CO release
under physiologic conditions, we have successfully employed
the click reaction between cyclopentadienones and (strained)
alkynes to trigger CO release without the need for photo-
irradiation.6 Herein, we utilize the same click reaction for CO
release. Meanwhile, a drug payload is appended to the dienone
moiety via an ester bond (1, Figure 1B), and a hydroxyl group
is installed at the propargyl position of the strained alkyne (2).
Ideally, the initial click reaction between 1 and 2 leads to CO
release and the formation of intermediate 3, which is expected
to readily undergo lactonization to liberate the drug payload
and compound 4. In addition, a naphthalene group is attached
at the C3, 4 positions to enable fluorescence turn-on upon the
release of CO and the other payload, which can be leveraged
for real-time monitoring of payloads release.
1
Figure 2. CO release reaction monitored by H NMR. A solution of
1a and 2 in DMSO-d6 was incubated at 37 °C.
fold higher than that of the reaction between 2 and 1a. The
release of floxuridine from the reaction between 2 and 1b was
monitored using HPLC, and the results showed that the
cycloaddition reaction between 2 (5 mM) and 1b (100 μM) in
30% of DMSO/PBS was finished within about 4 h with the
floxuridine recovery yield being about 50% (Figure 3A). The
remaining 50% was trapped as compound 4b′. The chemical
structures of 4b and 4b′ have been thoroughly elucidated by
NMR and HRMS (SI), confirming the proposed payload
release mechanism (Figure 3A). However, incubation of 1b
only under the same conditions led to <3% of floxuridine
In order to examine the feasibility of such a design, a model
compound 1a with benzyl alcohol as the model payload was
synthesized (SI). Indeed, the click reaction between 1a and 2
resulted in a blue fluorescence turn-on. The second-order
reaction rate constant was determined to be 0.018 M−1 s−1 in
30% of DMSO/PBS at 37 °C by monitoring the fluorescence
intensity increase, and CO release was validated by a CO-
myoglobin assay (Figure S5). In order to further confirm the
release of benzyl alcohol, the reaction between 1a and 2 was
1
studied with H NMR in DMSO-d6 at 37 °C by monitoring
the featured protons Ha in 1a, Hb, c in 3a, Hb′, c′ in 3a′, and
He, f in 4a. Noteworthy, protons Hc, Hc′, and He are shifted
significantly to high field (6.0−6.3 ppm) because they are in
the shielding cone of phenyl ring A. As shown in Figure 2, the
click reaction between 1a and 2 completed within about 4.5 h
with the formation of 3a (Hb, c), 3a′ (Hb′, c′), benzyl alcohol
(Hd), and the lactone 4a (He, f). The peaks of intermediate 3a
(Hb, c) decreased gradually with the increase in intensity for
the peaks of benzyl alcohol (Hd) and 4a (He, f). After 21 h, all
the intermediate 3a was totally consumed with 3a′, 4a, benzyl
alcohol, and excessive 2 as the only compounds in the solution.
Due to a regioselectivity issue, the release yield of the benzyl
alcohol was around 58% based on the integration ratio
between Hd and Hb′. Altogether, these results unambiguously
validated the release of benzyl alcohol via a cascade of click,
cyclization, and release process.
Having confirmed the feasibility of the “click and release”
strategy, we next appended a real drug floxuridine to the
dienone moiety. In addition, in order to enhance the reaction
rate, an electron-withdrawing group morpholine-4-carbonyl
was attached to the C2 position of the dienone ring to further
lower its LUMO energy level and thereby to facilitate the
cycloaddition reaction.17 Indeed, the reaction rate constant
between 2 and 1b increased to 0.17 M−1 s−1, which is about 9-
Figure 3. Release profiles of bioorthogonal prodrug 1b upon clicking
with strained alkyne 2. (A) The drug release yield upon the click
reaction between 1b (100 μM) and 2 (5 mM) in 30% of DMSO/PBS
at 37 °C. (B) The HPLC chromatogram of the reaction between 1b
and 2 at different time points.
B
DOI: 10.1021/acs.orglett.9b01086
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Letter
release. It is worth noting that lactonization also occurred for
regioisomer 3b′ with the release of morpholine, suggesting the
utility of this approach for the delivery of either alcohol- or
amine-containing drugs. In both cases (3b and 3b′), the build-
up for the initial cycloaddition intermediates 3b and 3b′ was
not observed under the experimental conditions, indicating the
rate-limiting nature of the cycloaddition reaction in this
cascade reaction sequence. It is worth pointing out that for the
click reaction between 2 and 1b, four different outputs were
generated, namely CO, floxuridine, morpholine, and blue
fluorescence, albeit release yields for floxuridine and morpho-
line were moderate (ca. 50% each).
anticancer drug floxuridine. The fluorescent nature of the
byproduct after prodrug activation can also be used as a
reporter molecule for real-time monitoring of payload release.
Such a multimodality of this click and release system would be
invaluable for drug delivery, especially for combination
therapy.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.9b01086.
Having confirmed the drug release from the reaction
between 2 and 1b, we moved to test if that is the case in a
real biological milieu. The fluorogenic nature of the click
reaction greatly facilitated the real-time monitoring of the
intracellular payload release. For this purpose, MB-231 cells
were co-treated with compounds 2 and 1b for 24 h. Then cells
were fixed for imaging studies under the DAPI channel. As
expected, cells co-treated with 2 and 1b (SI, Figure S6e,g)
exhibited much enhanced blue fluorescence as compared to
the control cells treated with vehicle (Figure S6a) or 1b
(Figure S6c) only. It is worth pointing out that the turned-on
fluorescence can only indicate the release of CO but not
floxuridine in that both 4b and 4b′ are fluorescent.
Synthetic procedure and copies of spectra, CO-
myoglobin assay, and biology experimental details
(PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: wang@gsu.edu.
ORCID
Binghe Wang: 0000-0002-2200-5270
Notes
The authors declare no competing financial interest.
Having confirmed the intracellular payloads release, we next
assayed the antiproliferative activity of the prodrug against
MB-231 cells. As shown in Figure 4, co-treatment with 1b and
ACKNOWLEDGMENTS
■
The authors acknowledge GSU Molecular Basis of Disease
(Z.P.), Brains and Behaviors (B.Y.), and University 2CI
(L.K.C.d.l.C.) fellowships for their support.
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