Adapting decarbonylation chemistry for the development of prodrugs capable ofin vivodelivery of carbon monoxide utilizing sweeteners as carrier molecules

Article abstract of DOI:10.1039/d1sc02711e

Carbon monoxide as an endogenous signaling molecule exhibits pharmacological efficacy in various animal models of organ injury. To address the difficulty in using CO gas as a therapeutic agent for widespread applications, we are interested in developing CO prodrugs through bioreversible caging of CO in an organic compound. Specifically, we have explored the decarboxylation-decarbonylation chemistry of 1,2-dicarbonyl compounds. Examination and optimization of factors favorable for maximal CO release under physiological conditions led to organic CO prodrugs using non-calorific sweeteners as leaving groups attached to the 1,2-dicarbonyl core. Attaching a leaving group with appropriate properties promotes the desired hydrolysis-decarboxylation-decarbonylation sequence of reactions that leads to CO generation. One such CO prodrug was selected to recapitulate the anti-inflammatory effects of CO against LPS-induced TNF-α production in cell culture studies. Oral administration in mice elevated COHb levels to the safe and efficacious levels established in various preclinical and clinical studies. Furthermore, its pharmacological efficacy was demonstrated in mouse models of acute kidney injury. These studies demonstrate the potential of these prodrugs with benign carriers as orally active CO-based therapeutics. This represents the very first example of orally active organic CO prodrugs with a benign carrier that is an FDA-approved sweetener with demonstrated safety profilesin vivo.

Full text of DOI:10.1039/d1sc02711e

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Adapting decarbonylation chemistry for the
development of prodrugs capable of in vivo
delivery of carbon monoxide utilizing sweeteners
as carrier molecules†
Cite this: Chem. Sci., 2021, 12, 10649
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
a
a
b
b
Ladie Kimberly De La Cruz,‡ Xiaoxiao Yang,‡ Anna Menshikh, Maya Brewer,
a
c
a
a
a
d
Wen Lu, Minjia Wang, Siming Wang, Xingyue Ji, Alyssa Cachuela, Haichun Yang,
David Gallo, Chalet Tan, Leo Otterbein, Mark de Caestecker
e
c
e
b
a
and Binghe Wang
*
Carbon monoxide as an endogenous signaling molecule exhibits pharmacological efficacy in various animal
models of organ injury. To address the difficulty in using CO gas as a therapeutic agent for widespread
applications, we are interested in developing CO prodrugs through bioreversible caging of CO in an
organic compound. Specifically, we have explored the decarboxylation–decarbonylation chemistry of
1,2-dicarbonyl compounds. Examination and optimization of factors favorable for maximal CO release
under physiological conditions led to organic CO prodrugs using non-calorific sweeteners as leaving
groups attached to the 1,2-dicarbonyl core. Attaching a leaving group with appropriate properties
promotes the desired hydrolysis–decarboxylation–decarbonylation sequence of reactions that leads to
CO generation. One such CO prodrug was selected to recapitulate the anti-inflammatory effects of CO
against LPS-induced TNF-a production in cell culture studies. Oral administration in mice elevated
COHb levels to the safe and efficacious levels established in various preclinical and clinical studies.
Furthermore, its pharmacological efficacy was demonstrated in mouse models of acute kidney injury.
These studies demonstrate the potential of these prodrugs with benign carriers as orally active CO-
based therapeutics. This represents the very first example of orally active organic CO prodrugs with
a benign carrier that is an FDA-approved sweetener with demonstrated safety profiles in vivo.
Received 17th May 2021
Accepted 22nd June 2021
DOI: 10.1039/d1sc02711e
rsc.li/chemical-science
7,8
9
the gastrointestinal tract, and the liver, among others.
Introduction
Because of the gaseous nature of CO, the difficulty in delivering
With the rm establishment of carbon monoxide (CO) as an a gas as a therapeutic to patients, and the potential risks it poses
endogenous signaling molecule, there has been an increasing to healthcare workers as well as patients in terms of accidental
level of interest in developing CO-based therapeutics by taking poisoning, there have been intense efforts in developing alter-
1
,10,11
advantage of its demonstrated cyto- and organ-protective native delivery forms.
Earlier efforts focused on metal-
1–3
1
effects. Along this line, the organ protective effects of CO have based CO releasing molecules (CORMs), photo-sensitive-
4,5
6
11–13
14,15
been demonstrated in injury models of the kidneys, lungs,
organic CO donors,
CO in solution,
and CO immobilized
16
on modied hemoglobin. There are several reviews discussing
in detail the various existing delivery forms of CO.
1,10,11
We are
a
interested in developing organic CO prodrugs as donors for
Department of Chemistry, Georgia State University, Atlanta, GA, 30303, USA. E-mail:
wang@gsu.edu
developing CO-based therapeutics as “CO in a pill” for eventual
b
17
18
19
Department of Medicine, Division of Nephrology, Vanderbilt University Medical clinical applications. Along this line, we and Larsen's lab
Center, Nashville, TN, 37232, USA
developed cheletropic extrusion-based CO prodrugs with
tunable release rates, triggered release, and the ability to target
c
Department of Pharmaceutics and Drug Delivery, University of Mississippi, MS,
3
8677, USA
20
the mitochondria. Fig. 1 shows some representative CO
donors. In terms of drug-like features, each class has its own
features that need improvement including metal reactivity for
d
Department of Pathology, Microbiology, and Immunology, Vanderbilt University
Medical Center, Nashville, TN, 37232, USA
e
Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical
21,22
School, Boston, MA 02115, USA
some metal-based CO-RMs (Fig. 1A),
high hydrophobicity for
†
1
‡
Electronic supplementary information (ESI) available. See DOI: some of the organic prodrugs due to the presence of multiple
0.1039/d1sc02711e
18
aryl groups (Fig. 1B), and the known and unknown toxicity of
These authors contributed equally to this work.
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2021 The Author(s). Published by the Royal Society of Chemistry
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chemistry, oxalyl chloride has been used as a convenient CO gas
35,36
donor in carbonylation reactions.
However, its use for bio-
logical and therapeutic applications presents signicant chal-
lenges because of the release of HCl, and its corrosive and
37
acutely toxic nature. With CO xed in the 1,2-dicarbonyl
moieties, our goal was to design a new class of CO donors
involving benign carriers (Fig. 2B).
Results and discussion
Fig. 1 Examples of CO donors that are activated by physiological Because of enhanced electrophilicity arising from vicinal keto
stimuli – (A) ligand exchange from endogenous nucleophiles, and (B)
exposure to an aqueous environment and/or at pH 7.4.
groups, 1,2-dicarbonyl compounds with appropriate leaving
groups are susceptible to hydrolysis, expelling one leaving
group and generating a ketoacid intermediate (Fig. 2C). CO is
generated once this ketoacid intermediate undergoes a decar-
2
3,24
transition metals
and the organic “carrier” portions of the
boxylation–decarbonylation sequence of reactions. For oxalyl
chloride, this path predominates and thus forms CO, CO , and
organic CO prodrugs.
2
In search of CO prodrugs with improved pharmaceutical
properties, we are especially interested in those that use
a “carrier” moiety with known safety proles. Along this line, we
look to known decarbonylation chemistry for our design.
Several types of decarbonylation chemistry are known in
synthetic chemistry in organic solvents, including decarbon-
ylation of formic acid and its derivatives, a-hydroxyacid, 5-
HCl as end-products when placed in water. However, when the
chloride leaving group is replaced by other leaving groups,
another decomposition path may compete with the CO gener-
38,39
ating path.
The ketoacid intermediate can undergo hydro-
lysis to expel the second leaving group and form oxalic acid
without CO generation. Therefore, the identity of the leaving
group must be carefully selected to tune the reaction in favor of
the CO generating path.
For understanding the basic chemistry and parameters that
control the partitioning between the two pathways, chloride was
25,26
(
hydroxymethyl)furfural
and other aldehydes and carbonyl-
However, most of these reactions
27–33
containing compounds.
happen under non-physiological conditions or require harsh
acids, heating, or metal catalysis for CO release. In most cases,
especially those that require metal catalysis, application under
near physiological conditions is hard to achieve.
The hydrolysis, decarboxylation, decarbonylation reaction
sequence characteristic of 1,2-dicarbonyl compounds with
appropriate leaving groups, presented an opportunity to install
benign carriers as leaving groups. As a literature precedent,
oxalyl chloride, one of the simplest 1,2-dicarbonyl compounds
in the form of oxalates, is known to decompose in water to
replaced by various leaving groups having different pK
a
values
to survey the effect of pK on CO release proles (Fig. 3 and
a
Table S1†). In doing so, we utilized a selective uorescent CO
probe for monitoring CO production in situ. Specically, COP-1
(ref. 40) is a widely tested uorescent CO probe based on the Pd-
mediated carbonylation reaction. We selected a number of
leaving groups with different pK values including phenol, 2,4,6-
a
trichlorophenol, 2,4-dinitrophenol, imidazole, and an imide
compound, phthalimide.
A 6-fold increase in uorescence turn-on intensity was
observed when the leaving group was 2,4-dinitrophenol. In
2
produce CO, HCl, and CO through rst hydrolysis of one acyl
chloride, then decarboxylation, and nally decarbonylation to
34
release CO (Fig. 2A). Because of the reliability of this
a
general, compounds with leaving groups having pK greater
than 4 were incapable of CO release, except for a marginal 0.1-
fold increase with imidazole. According to previous reports,
Fig. 2 (A) Decomposition of oxalyl chloride to produce CO. (B) Design
of CO donors based on the hydrolysis, decarboxylation, decarbon-
ylation chemistry of oxalyl derivatives. (C) Competing paths by which
1
,2-dicarbonyl CO prodrugs can breakdown in aqueous solution – (i) Fig. 3 Examination of the effect of pK
a CO-generating pathway; (ii) non-CO producing pathway that releasing capacity of 1,2-dicarbonyl compounds using a CO probe,
generates oxalic acid as the product. COP-1.
a
of the leaving group on the CO
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hydrolysis of symmetrical oxalates occurs in two steps with the of only around 40% (Fig. 4B) indicates that installation of two
rst step being at least two orders of magnitude faster than the leaving groups with pK less than 4 into the 1,2-dicarbonyl core

a
38
second hydrolysis. In certain cases and as is shown in the later favors the CO generating reaction pathway. An LC-MS method
section, the ketoacid with a good leaving group is stable enough was used to analyze the proportion of prodrug BW-CO-306 that
to be isolated. The nature of the attached leaving group to the is converted to oxalic acid through the non-CO generating
keto acid inuences the decomposition pathway it goes through pathway. Oxalic acid (47 mM) was produced from 200 mM of
(
Fig. 2C). Leaving groups with pK
releasing pathway. Based on these results, additional leaving be about 75%, which agrees well with the results generated
groups with pK less than four were considered. Two “carrier” from the GC-TCD experiments.
molecules seemed especially interesting to us for CO delivery Since CO release from the prodrug involves hydrolysis, low
a
less than 4 favor the CO- prodrug BW-CO-306. Therefore, CO production is estimated to
a
under physiological conditions: saccharin and acesulfame. pH might enhance the hydrolysis rates and therefore decrease
Both are FDA-approved, non-caloric sweeteners, having pKa CO yield. This is indeed what was observed with prodrug BW-
values that fall within the range of 1.6 and 3.0, respectively CO-306 wherein CO yield decreased from 60% to 30% with
(Fig. 4A). The oxalyl derivative with saccharin as a leaving group decreasing pH (Fig. 4C). In contrast, the CO release yield for
was previously reported as a coupling reagent for the synthesis prodrug BW-CO-307 is pH-independent with CO yields constant
41
of carboxylic acid derivatives. In this report it was noted that at around 90% across a broad pH range. Furthermore, RP-HPLC
CO and CO
conditions.
2
are released but under non-physiological studies revealed that prodrugs BW-CO-306 and BW-CO-307 are
stable in the solid state for at least 24 days exposed to ambient
Therefore, CO prodrugs BW-CO-306 and BW-CO-307 were light and temperature. These prodrugs are also stable in solu-
made in one step from the reaction between commercially tions of acetonitrile stored at room temperature for at least
available sweeteners saccharin and acesulfame, respectively, seven days. Kinetic experiments using HPLC revealed that the
with oxalyl chloride in the presence of triethylamine (Scheme half-lives of compound BW-CO-306 and BW-CO-307 in 60%
S1†). Qualitative determinations using
a
household CO phosphate buffered saline are 1.28 ꢀ 0.03 min and 9.5 ꢀ 0.14
detector, COP-1 probe, and CO-myoglobin assay indicated CO min, respectively (Fig. 4D and E). HPLC and NMR analyses both
production with BW-CO-306 (Fig. S1–S3†). The CO release yields provide strong evidence that products aer CO release are
were then quantied using an Agilent 7820A GC with a thermal mainly the carriers saccharin and acesulfame (Fig. S8–S11†).
conductivity detector (TCD). CO prodrugs BW-CO-306 and BW-
To demonstrate that these compounds are compatible CO
CO-307 released 76% ꢀ 6 and 92% ꢀ 3 CO, respectively when donors for biological applications and that these donors can
ꢁ
dissolved in 4 : 1 ACN : H
2
O and incubated at 37 C for 1 h recapitulate CO-associated anti-inammation activity, an ELISA
(Fig. 4B). The ketoacid form of BW-CO-306 was also synthesized assay for the pro-inammatory cytokine TNF-a was conducted.
and isolated as a white solid (Scheme S2†). The lower CO yield Because the half-life of BW-CO-306 is very short, the produced
CO may escape readily in a cell culture experimental setting.
Thus, we repeatedly exposed the cells to the prodrug ve times.
BW-CO-307, with a slightly longer half-life, was added only
once. Dose-dependent inhibition of LPS-induced secretion of
TNF-a was observed for both prodrugs (Fig. 5A and B). For in
vivo applications, the utility of orally administered BW-CO-306
was demonstrated in a pharmacokinetic study wherein COHb
levels in the blood were monitored by a CO-oximeter. Elevation
of COHb was observed up to around 4–5.5% and remained
above the baseline for about an hour (Fig. 5C). In comparison,
this COHb elevation is within the limits established by human
4
2,43
clinical trials to be safe
established in preclinical animal studies.
the in vitro kinetic properties of BW-CO-306 and BW-CO-103
another CO prodrug which has been shown to be efficacious in
and comparable to the efficacy levels
44,45
Consistent with
(
46
various animal models such as colitis), with half-lives of 1.3
min and 1.2 h, respectively, BW-CO-103 gave a more sustained
17
elevation of COHb with a tmax of 60 min while BW-CO-306 gave
a burst of CO resulting in a spike of COHb elevation with a tmax
of 10 min that only lasted for around 30 min (Fig. 5D). The
dose–response relationship and the increase of COHb above the
blank vehicle control aer oral administration of BW-CO-306
indicated a facile CO release in vivo and demonstrated the
feasibility of systemic CO delivery.
Fig. 4 (A) Structures of CO prodrugs. (B) GC-TCD analysis of CO and
ꢁ
CO
time. (C) Effect of pH on CO and CO
glycine/NaCl/HCl, pH 3 – citric acid/NaOH/HCl, pH 7.4 – phosphate
2
yields of CO prodrugs in 4 : 1 ACN : H
2
O at 37 C, 1 h incubation
2
yields in 4 : 1 ACN : buffer (pH 1
–
ꢁ
The rst in vivo validation of heme oxygenase (HO-1, the
enzyme responsible for endogenous CO production through
buffer) at 37 C, 1 h incubation time. (D) and (E) Decomposition profile
of CO prodrugs in 60% PBS analyzed by RP-HPLC.
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Fig. 5 (A) and (B) The anti-inflammatory effects of BW-CO-306 and
BW-CO-307 in RAW 264.7 cells. Mean of each concentration vs. LPS 1
ꢂ1
mg mL : *p < 0.05, **p < 0.01 and ***p < 0.001; mean of each
#
concentration vs. the corresponding saccharin only group: p < 0.05
###
and
p < 0.001 (C) COHb elevation of BW-CO-306 in mice (D)
COHb level elevation of BW-CO-306 compared to another CO pro-
drug, BW-CO-103.
heme catabolism) induced protection in response to tissue
injury was conducted using a rhabdomyolysis induced acute
47
kidney injury (AKI) model in rats. The model recapitulates the
mechanisms of heme-induced kidney injury that occurs in
patients with extensive muscle damage, and intravascular
hemolysis associated with sickle cell disease, falciparum
47–50
malaria, cardiopulmonary bypass surgery and severe sepsis.
This pivotal paper demonstrated that the kidneys employ HO-1
coupled with ferritin synthesis (to sequester free iron) to launch
a rapid, protective response against acute kidney injury (AKI).
Since that time, a number of pharmacological and genetic loss
Fig. 6 BW-CO-306 protects against rhabdomyolysis-induced acute
kidney injury (AKI). Male BALB/c mice were treated with 50 mg kg
51
ꢂ1
BW-CO-306 (or release product saccharin) by oral gavage starting 24
ꢂ1
and gain of function studies have shown that HO-1 is protective hours before and then daily after intramuscular injection of 5.4 mL kg
5
2
62
in a number of different models of AKI, including sepsis,
50% glycerol in water to induce rhabdomyolysis, as described. Blood
53–56
57–59
was collected for BUN measurements 24 hours after glycerol injection,
and tissues were collected after euthanasia at day 3. (A) Blood urea
nitrogen (BUN) 24 hours after glycerol injection; (B) survival curves
with numbers of mice at each time point indicated; (C) semi-
cisplatin,
and ischemia reperfusion induced (IRI)-AKI.
These data indicate that induction of HO-1 may be a viable
60
therapeutic strategy against AKI. However, heme catabolism
also produces free iron which if not sequestered by ferritin can quantitative analysis of renal tubular injury (tubular injury score 0–4).
60
also exacerbate AKI. A different pharmacological strategy is to Representative images of PAS-stained sections shown in Fig. S14;† (D)
QRT-PCR for Kim1 mRNA in the kidneys. (E) and (F) Quantification of
the % surface area immunofluorescence staining for Kim1 with
representative images of Kim1 (magenta), and LTL (green), staining in
the OSOM of CP-306 and CO-306 treated mouse kidneys 3 days after
employ CO, one of the end-products of heme catabolism as the
therapeutic agent. For this reason, we were interested in testing
our CO prodrug in this rhabdomyolysis model of kidney injury.
BW-CO-306 was prepared as an oral formulation in activated rhabdomyolysis-induced AKI, as indicated. Scale bars, 100 mM. Mouse
ꢂ1
charcoal and 50 mg kg was administered to groups of 13 week numbers as indicated, with individual data points, mean ꢀ SEM indi-
ꢂ1
cated. One-way ANOVA was used to determine statistical significance
of differences between multiple groups, and a post-hoc Dunnett's test
used to compare pairwise significance, as shown (A) and (C). A two-
sided unpaired T-test was used to compare two groups (C). *p < 0.05;
male BALB-c mice injected with 5.4 mL kg of 50% glycerol to
the muscle of hindlegs. We utilized BW-CP-306 (i.e. saccharin),
the “carrier” portion of BW-CO-306 aer CO release, as an
inactive control. Intramuscular injection of glycerol-induced **p < 0.01; ****p < 0.0001.
AKI was associated with a rapid increase in blood urea nitrogen
(
BUN) levels 24 hours aer injection while CO treatment using
BW-CO-306 resulted in signicant lowering of BUN levels
Fig. 6A). BW-CO-306 also increased survival aer glycerol-
not statistically signicant (log-rank test, p ¼ 0.3). Seventy-two
hours aer glycerol injection, histological studies revealed
a marked reduction in features of acute tubular injury including
(
induced rhabdomyolysis from 3/7 mice (42%) in BW-CP-306
treated mice to 6/8 (75%) (Fig. 6B), although these changes were
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
attened, dedifferentiated tubular epithelial cells, and tubular Mississippi, Vanderbilt University, and BIDMC/Harvard
casts (Fig. 6C and S14†) in mice treated with BW-CO-306. In Medical School). The authors are grateful for nancial support
support of the reduced histological evidence of renal injury, from the National Institutes of Health (R01DK119202,
both QRT-PCR (Fig. 6D) for kidney injury molecule 1 (Kim1) R01DK112688), the Georgia Research Alliance, Eminent Scholar
mRNA, a marker of renal tubular injury, and immunouores- fund, and internal resources at Georgia State University.
cence staining (Fig. 6E) for Kim1 in the outer stripe of the outer
medulla (OSOM) indicated reduced expression in comparison
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1
Conflicts of interest
There are no conicts to declare.
2
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10654 | Chem. Sci., 2021, 12, 10649–10654
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