Click, Release, and Fluoresce: A Chemical Strategy for a Cascade Prodrug System for Codelivery of Carbon Monoxide, a Drug Payload, and a Fluorescent Reporter

Article abstract of DOI:10.1021/acs.orglett.7b03348

A chemical strategy was developed wherein a single trigger sets in motion a three-reaction cascade leading to the release of more than one drug-component in sequence with the generation of a fluorescent side product for easy monitoring. As a proof of conc

Full text of DOI:10.1021/acs.orglett.7b03348

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Click, Release, and Fluoresce: A Chemical Strategy for a Cascade
Prodrug System for Codelivery of Carbon Monoxide, a Drug Payload,
and a Fluorescent Reporter
Ladie Kimberly C. De La Cruz,† Stephane L. Benoit,‡ Zhixiang Pan,† Bingchen Yu,† Robert J. Maier,‡
́
Xingyue Ji,*,† and Binghe Wang*,†
†Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia 30303, United
States
‡Department of Microbiology, University of Georgia, Athens, Georgia 30602, United States
S
* Supporting Information
ABSTRACT: A chemical strategy was developed wherein a single trigger sets in
motion a three-reaction cascade leading to the release of more than one drug-
component in sequence with the generation of a fluorescent side product for easy
monitoring. As a proof of concept, codelivery of CO with the antibiotic
metronidazole was demonstrated.
rug combination therapy often relies on coadministration
for treatment of idiopathic pulmonary fibrosis (ClinicalTrials.
Dof multiple drugs. In the area of antibacterials, the use of Gov identifier: NCT01214187) and for severe pulmonary
arterial hypertension (ClinicalTrials.Gov identifier:
NCT01523548).
Augmentin, which combines a β-lactam antibiotic with a β-
lactamase inhibitor,1 allows for the effective control of infections
by drug-resistant strains of bacteria. Another example is the
administration of a hybrid compound of sulfonamide and
dihydrofolate reductase inhibitor to achieve inhibition of two
enzymes on the same biosynthetic pathway leading to folic acid,
which is important for bacterial survival.2 Herein, we explore the
idea of using prodrug approaches to deliver multiple components
from a single chemical entity, with the aim of improved
pharmacokinetic control and ease of administration. In
appropriate situations, such an approach will be very useful. In
demonstrating the proof of concept, we select the delivery of
carbon monoxide (CO) and antibiotic metronidazole as a model
drug payload.
Despite being notorious for its toxicity at high concentration,
CO has recently been in the spotlight for its promising potential
as a medical gas.3 It is produced endogenously in the human body
from the degradation of heme by heme-oxygenase.4 The
discovery that CO, like nitric oxide (NO), activates soluble
guanylate cyclase5 was a milestone in understanding the
physiological relevance of CO and in triggering an ever-
increasing level of research interest in this area.6 Since then,
mounting evidence from a multitude of studies has demonstrated
that both endogenously produced and exogenously introduced
CO plays important roles in inflammation,7 cell proliferation,8
and cellular response to pathogens9 among other effects. Safety
concerns have been addressed by human safety trials wherein it
was shown that CO inhalation of 500 ppm for 1 h is well-
tolerated in healthy subjects−that is CO-bound hemoglobin did
not increase to more than 10% and there were no adverse events
observed in the course of the experiments.10 Due to convincing
preclinical studies, CO gas is now in several clinical trials such as
Aside from direct inhalation, other routes of CO admin-
istration involve the use of metal-based CO-releasing molecules
(CO-RMs)11 including photo-CORMs,12 boranocarbonate and
its derivatives,13 photosensitive organic CO-RMs,14 and
hemoglobin-based CO carriers.15 Adding to this list of CO gas
surrogates are a group of recently reported “spontaneously-
releasing”16 and triggered17 organic CO prodrugs. Generally
speaking, the design of such organic CO-prodrugs takes
advantage of an intramolecular cycloaddition reaction between
a dienone and an alkyne to form a bicyloheptenone intermediate
that spontaneously undergoes a cheletropic reaction to extrude
CO. The ability of this prodrug system to supply CO and to exert
its biological effects has been demonstrated through the
inhibition of LPS-induced TNF-α secretion in RAW 264.7
cells, and attenuation of 2,4,6-trinitrobenzensulfonic acid
(TNBS)-induced experimental colitis in mice.16b,17a,b
Pushing forward and building on the flexibility of the design of
this click-and-release organic scaffold, we were interested in
exploring the possibility of coupling the release of CO to another
drug payload for potential multidrug therapy applications
(Figure 1). The other two gasotransmitters, nitric oxide and
hydrogen sulfide, have been conjugated to compatible
therapeutic agents such as anticancer, cytototoxic, and anti-
inflammatory drugs to expand the therapeutic scope of these
gases.18 Based on previous reports of CO’s ability to work
additively and/or synergistically with other entities such as
cytotoxic agents (doxorubicin),19 antibiotics (metronidazole),9a
Received: October 26, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b03348
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Letter
by 1H NMR experiments. A 10 mM solution of model prodrug
10a in DMSO-d6: PBS in D2O was incubated at 37 °C while
taking NMR spectra at indicated time points. The simplicity of
the structure of prodrug 10a allowed for easy monitoring of
reaction progress. Scrutinizing the spectroscopic changes of four
sets of protons from prodrug 10a showed that three CH2 peaks
labeled b−d and the most deshielded aromatic proton labeled as
a proved to be informative (Figure 2). As expected, the peaks
Figure 1. Cascade prodrug system for delivery of CO, another drug
payload, and a fluorescent byproduct.
and anti-inflammatory agents,20 we sought to develop a platform
where compatible drugs can be easily conjugated to our
previously developed CO releasing scaffold.16b By combining a
cascade of three bioorthogonal reactions−an inverse electron-
demand Diels−Alder reaction, a retro-Diels−Alder reaction/
cheletropic extrusion, and lactonization, three key components
are released (Figure 1). This design features a prodrug that is
activated under near physiological conditions (PBS buffer, pH 7,
37 °C) wherein the rate of the inverse electron-demand Diels−
Alder reaction is expected to be greatly enhanced. Then the
formed norbornenone intermediate readily undergoes a
cheletropic reaction to extrude CO while forming an aromatic
ring. Postaromatization, the hydroxyl group is now poised to
undergo an anticipated facile lactonization21 to release the free
drug. Release of both CO and free drug leaves the original
scaffold as a blue fluorescent compound that can be used to track
release progress (Figure 1).
To probe the potential of this design, model prodrug 10a with
benzyl alcohol as the model payload was prepared first according
to the procedure detailed in the Supporting Information. The
synthetic scheme was designed such that any compatible drug or
agent bearing a free hydroxyl group or amino group can be easily
conjugated. The latent CO is caged in the scaffold containing the
carboxylic acid portion from readily available starting material, 2-
hydroxyphenylacetic acid. With straightforward chemistry,
compound 7 was readily obtained. A compatible payload such
as the antibiotic metronidazole can be easily conjugated via
amidation or esterification. Once the payload is appended, the
alkyne component will then be introduced in the ortho-phenolic
group to complete the prodrug. The modularity of this synthetic
design offers possibility to synthesize prodrugs of CO that can
release various drugs.
Initially, the feasibility of this design was investigated by
studying CO and payload releasing capacity of a simple model
compound 10a through fluorescence spectroscopy, CO-
myoglobin assay, and NMR studies. Because a fluorescent
cyclized product is also produced as CO and the payload are
released, the progress of cascade reaction of the prodrug can be
monitored by fluorometric studies. The fluorescent cyclized
product is characterized by excitation wavelength of 355 nm and
emission wavelength of 460 nm (Figure S1a). Time-dependent
increase in fluorescence intensity was observed, indicating that
CO and the payload are released as the prodrug is incubated in
aqueous solution at physiological pH, providing indirect
confirmation of CO release (Figure S1b). Correlation of the
data using Sigmaplot revealed that the half-life of the model
prodrug is 0.66 0.05 h. To supply direct proof that indeed CO
is being released, a modified CO-myoglobin assay16b was
conducted. Indeed, it was shown that incubation of deoxy-
myoglobin with the prodrug produces CO-myoglobin as
demonstrated by the change in the absorbance spectrum that
is characteristic of CO-bound myoglobin (Figure S2).
Figure 2. (A) 10a in pure DMSO-d6. (B) Incubation in DMSO-d6/PBS
in D2O after 30 min, (C) 75 min, (D) 120 min, (E) 165 min. (F)
Proposed mechanism of release.
corresponding to the norbornenone intermediate were not
observed, confirming that the CO extrusion step is very fast22 and
the Diels−Alder reaction step is rate-limiting for CO release
(Figure 1). Aside from the peaks corresponding to the intact
prodrug 10a, cyclized product 11 and benzyl alcohol, another set
of peaks possibly from the prelactonization intermediate Y was
also present. As the reaction progressed, peaks from the intact
prodrug and intermediate Y decreased while peaks correspond-
ing to the cyclized product 11 and benzyl alcohol steadily
increased with a ratio of 1:1. After 24 h, both intact prodrug 10a
and intermediate Y were no longer observable and only peaks
corresponding to the cyclized product 11 and benzyl alcohol
were present (Figure S3).
Having confirmed the release of the two drug components
from model compound 10a, we sought to use this design to
deliver CO together with a real drug payload. CO has been well-
documented to exert antimicrobial effects either through directly
targeting bacterial survival machinery23 or through activating the
killing potential of the host’s phagosome in response to microbial
invasion.9b,24 Co-administration of CO with known antibiotics
such as metronidazole, amoxicillin, and clarithromycin has been
shown to enhance the efficacy of these antibiotics against
Helicobacter pylori,9a the etiological agent of peptic ulcer disease
or chronic atrophic gastritis.25 Hence, we prepared prodrug 10b
such that it would release three componentsCO, an antibiotic
payload (metronidazole), and a fluorescent reporter molecule.
The formation of these three components following
incubation of prodrug 10b in PBS buffer was examined using
RP-HPLC (Figure 3A). For reference, a mixture containing 25
To gain preliminary insights into the possible mechanism of
CO and payload release, we decided to follow reaction progress
B
DOI: 10.1021/acs.orglett.7b03348
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Letter
dose-dependent growth inhibitory effect against H. pylori strain
26695 with MIC90 of 2.5 μg/mL. In contrast, it took only 0.31
μg/mL of prodrug 10b to achieve greater than 90% growth
inhibition against H. pylori (Figure 5). This is an enhancement of
Figure 3. (A) Release profile of 10b in PBS buffer, pH 7, 37 °C. (B)
Quantification of accumulated free metronidazole released over time.
μM each of the prodrug 10b, free drug metronidazole, and
cyclized product 11 were prepared (Figure S4A). Then, a 25 μM
solution of prodrug 10b was incubated at 37 °C, pH 7. At time 0
(Figure S4B), only the peak for the prodrug was observable. After
30 min (Figure S4C), two new peaks corresponding to the
cyclized product and free metronidazole emerged while the
intensity of the peak for the intact prodrug decreased. As 10b was
being consumed, the free drug and 11 were in turn produced.
After 6 h (Figure S4F), the peak for the intact prodrug was almost
undetectable while the peaks corresponding to the free drug and
cyclized byproduct appeared to level off to the same peak area as
that of the 25 μM mixture shown in Figure S4A. Quantification of
the release of the free drug showed that at the 6-h time point,
around 85% of the free drug had already been released (Figure
3B). It is also worth noting that the prodrug fell apart in PBS
buffer, pH 7 at 37 °C in a clean manner without any other
discernible artifacts aside from the three components (Figure
S4).
To verify CO release in biological systems, we monitored the
formation of blue fluorescence of the cyclized product 11 in cell
imaging studies using RAW 264.7 cells. After incubation of 10b
for 6 h, we found that the cells exhibited a dose-dependent
increase (qualitative) in blue fluorescence formation (Figure S5)
indicating that indeed CO was released from prodrug 10b. To
directly assay CO release in cell cultures, a CO-selective turn-on
fluorescent probe COP-126 was used. An increase in green
fluorescence (qualitative) relative to the negative control (Figure
4) was observed verifying that indeed CO was present inside the
Figure 5. Inhibition of H. pylori growth in BHI-β-cyclodextrin medium
supplemented with metronidazole, 10b, 10a, and 11 in 1% DMSO.
Results are expressed as (mean and standard deviation) percentage of
OD600 at 24 h for each compound compared to OD600 of control bottles
(1% DMSO only). Growth experiments were done in triplicate. *
indicates no growth.
over 8-fold in potency. Further, with metronidazole’s molecular
weight (171) being only less than 1/3 of that of 10b (MW =
562), the true enhancement in potency for 10b was over 26-fold
in terms of molar concentrations. As controls, prodrug 10a,
which releases CO only without metronidazole, and cyclized
product 11 exhibited no or minimal inhibition at the same
concentration (0.31 μg/mL). Such results are consistent with the
observed bacterial sensitization effect of CO toward existing
antibiotics.9a Collectively, these results demonstrated the
feasibility of using prodrug 10b to codeliver CO and
metronidazole in a biological milieu. These results also herald
the potential of combinational therapy of CO and other drugs
such as metronidazole using a single prodrug entity.
The viability of this prodrug to act as an antimicrobial agent
depends on its opposite toxicity profiles in microbes and in host
cells. Therefore, the toxicity profiles of the prodrug (10) and the
cyclized product (11) were tested in a crystal violet assay of
RAW264.7 macrophage cells. No toxicity was observed at up to
100 μM of both prodrugs 10a (50 μg/mL) and 10b (56 μg/mL)
and fluorescent byproduct 11 (36 μg/mL) after 24 h of
incubation (Figure S6).
In summary, we have developed a modular platform that
features a cascade reaction sequence that is set in motion once
the prodrug is placed in an aqueous environment leading to the
release of CO, a compatible drug payload, and a fluorescent
reporter molecule. The release of both CO and drug payload was
monitored by the increase in characteristic blue fluorescence of
the reporter molecule byproduct. Further verification through
NMR studies, RP-HPLC kinetic studies, cell imaging, and
antibacterial assay provide unambiguous evidence in support of
the feasibility of the cascade prodrug design.
Figure 4. Concentration-dependent increase in fluorescence intensity of
11 (blue channel) and COP-1 probe (green channel) after 6 h
incubation of prodrug 10b.
cell. Certainly, these results indicate that cyclized product 11 can
be used as a fluorescent reporter molecule to monitor CO and
drug release from the prodrug. To further demonstrate that this
cascade prodrug concept can work in living systems, we probed
the ability of prodrug 10b to deliver metronidazole and CO in H.
pylori, and juxtaposed its growth inhibitory activity against the
antibiotic metronidazole. As expected, metronidazole exhibited a
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.7b03348.
■
S
Full experimental procedure and characterization data
(PDF)
C
DOI: 10.1021/acs.orglett.7b03348
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Letter
2014, 2, 1454. (b) Carrington, S. J.; Chakraborty, I.; Bernard, J. M. L.;
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AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: xji@gsu.edu.
*E-mail: wang@gsu.edu.
ORCID
55, 1056. (f) G, U. R.; Axthelm, J.; Hoffmann, P.; Taye, N.; Glaser, S.;
̈
Binghe Wang: 0000-0002-2200-5270
Notes
Gorls, H.; Hopkins, S. L.; Plass, W.; Neugebauer, U.; Bonnet, S.; Schiller,
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A. J. J. Am. Chem. Soc. 2017, 139, 4991. (g) Motterlini, R.; Clark, J. E.;
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We gratefully acknowledge the Center for Diagnostics and
Therapeutics Fellowship for L.K.C.D.L.C.
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