PH-Sensitive metal-free carbon monoxide prodrugs with tunable and predictable release rates

Article abstract of DOI:10.1039/c7cc04866a

Carbon monoxide prodrugs with triggered release profiles are highly desirable for targeted CO delivery to minimize their untoward side-effects. Herein, we describe a series of pH-sensitive metal-free CO prodrugs which are stable under acidic conditions and yet begin to release CO in response to increases in pH with tunable and predictable release rates.

Full text of DOI:10.1039/c7cc04866a

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pH-Sensitive metal-free carbon monoxide prodrugs
with tunable and predictable release rates†
Cite this:DOI: 10.1039/c7cc04866a
Xingyue Ji,
Binghe Wang
Ladie Kimberly C. De La Cruz, Zhixiang Pan, Vayou Chittavong and
*
Received 22nd June 2017,
Accepted 2nd August 2017
DOI: 10.1039/c7cc04866a
rsc.li/chemcomm
Carbon monoxide prodrugs with triggered release profiles are highly
desirable for targeted CO delivery to minimize their untoward side-
effects. Herein, we describe a series of pH-sensitive metal-free
CO prodrugs which are stable under acidic conditions and yet begin
to release CO in response to increases in pH with tunable and
predictable release rates.
Carbon monoxide, generated by heme oxygenase-mediated heme
degradation, is an endogenous signaling molecule with strong
cytoprotective and anti-inflammatory effects, among others.1 For
example, there have been remarkable efforts and success in
studying CO for the treatment of experimental ulcerative colitis
(UC).2 Because of the significant therapeutic potential of CO, much
Fig. 1 Schematic illustration of the beta-elimination triggered CO release
(EWG: electron withdrawing group; LG: leaving group).
effort has been devoted to the search for CO-releasing molecules Very interestingly, the cycloaddition product II between an alkene
(CO-RMs) with either spontaneous release upon dissolution3 or and cyclopentadienone is thermally stable (Fig. 1), and CO release
controllable release in response to various stimuli, including from II can only be initiated when the temperature is over 180 1C.10
photo-,4 enzyme-,5 and oxidation-sensitive6 and encapsulated Therefore, compound II could serve as a potential CO prodrug,
CO-RMs.7 Previously, we have successfully developed a series of provided that a double bond between C5 and C6 can be easily
metal-free CO prodrugs by using inter- or intra-molecular Diels– formed under physiological conditions to yield intermediate III for
Alder (DA) reactions to trigger CO release (Fig. 1).2b,8 Among all the spontaneous CO release. In doing so, the key is to regenerate a
successes, pH-sensitive CO release is considered to be a very double bond between the C5 and C6 positions under physiological
important method for achieving local delivery of CO. For example, conditions. In order to devise pH-sensitive CO prodrugs for delivery
CORM-A1 was reported to be pH-sensitive,9 which releases CO to the lower GI, we employed base catalyzed beta-elimination as a
under acidic pH and is thus ideal for gastric delivery. Herein, we strategy to form the double bond between C5 and C6 under
describe a series of pH-sensitive CO prodrugs, which are metal-free physiological conditions (Fig. 1, R5 is chosen for positioning an
and stable under acidic conditions. They begin to release CO at electron-withdrawing group, and R6 is a leaving group). CO prodrugs
pH above 5 with a tunable and predictable release rate for as such are expected to be very stable under acidic conditions,
future applications. Such prodrugs also complement CORM-A1 and would only undergo beta-elimination to release CO under
in allowing the prodrug to survive the stomach for CO delivery basic or possibly neutral conditions. In addition, by changing
to the lower gastric intestinal (GI) system.
different R6 groups with various leaving-group abilities, we are
The design takes advantage of the fact that intermediate I able to tune the CO release rate, which is a very important
under the right conditions can undergo a facile cheletropic reac- parameter in gasotransmitter prodrugs.2b,8b,11
tion to release CO as previously demonstrated (Fig. 1). Thus, we
To establish the proof of concept, we designed and synthesized
were interested in the design of precursors of this intermediate. several potential pH-sensitive CO prodrugs BW-CO-201–205
(Scheme 1). An aldehyde group is chosen as R5 due to its strong
electron withdrawing ability. Despite being very good leaving
Department of Chemistry and Center for Diagnostics and Therapeutics,
groups, we sought to avoid halides to minimize their alkylation
Georgia State University Atlanta, Georgia 30303, USA. E-mail: wang@gsu.edu
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7cc04866a capacity and issues related to the formation of hydrogen
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Table 1 The CO release kinetics of BW-CO-201–205
c
CO prodrugs
R
Hammett constanta
kb (hÀ1
)
t1/2 (h)
201
202
203
204
205
H
NO2
F
Cl
Me
0
0.9
0.77
0.33
0.65
0.58
1.2
0.78
0.06
0.23
À0.17
2.09d
1.07
1.2
0.58
a
The Hammett constant of the R group at the para-position was
extracted from a reported literature.14 b CO release rate constant was
determined in 30% of DMSO in PBS (pH = 7.4) at 37 1C by using HPLC.
Scheme 1 The chemical synthesis of the metal-free CO prodrugs. Reagents
and conditions: (a) DIBAL, CH2Cl2, À78 1C, 1–2 h, 75–80%; (b) xylene, 170 1C,
12 h; then HCl, MeOH, reflux, 1 h, 50–60%; (c) DIPEA, MOMCl, CH2Cl2, r.t.,
3 h, 85–90%; and (d) CH2Cl2, PCC, reflux, 30 min–1 h, 60–73%.
c
d
CO release half-life was calculated according to t1/2 = 0.693/k. The
CO release kinetics of BW-CO-202 was determined by monitoring the
formation of 4-nitrophenol using UV absorbance at 400 nm.
halides, which are strong acids.12 As a result, substituted
phenols were chosen to achieve a balance between the leaving
ability and reactivity. As shown in Scheme 1, CO prodrugs
BW-CO-201–205 were readily synthesized in 4 steps. Specifically,
compounds 1a–e, which were obtained following a literature
procedure,13 were reduced using DIBAL to yield the corresponding
alcohols 2a–e. The DA reaction between 2a (R = H) and 3 failed to
afford the desired product 6a, and compound 4 was obtained
instead. The formation of compound 4 is presumably due to the
generation of acrylaldehyde in situ from compound 2a via double
bond migration, tautomerization, and elimination of the phenol
Fig. 2 The correlation of CO release rate (k) to the Hammett constant (s).
group. Therefore, the hydroxyl group in compounds 2a–e was valuable for quantitatively predicting the half-lives/release rates
protected with MOMCl to form 5a–e before the DA reaction. As of newly synthesized CO prodrugs.
expected, compounds 6a–e were successfully obtained primarily
Having confirmed the CO release from BW-CO-201–205, we
as endo products (Fig. S1 and S2, ESI†) after the DA reaction next tested whether CO release is sensitive to the pH value of the
and deprotection of the MOM group. The final CO prodrugs buffer used. Toward this end, BW-CO-203 was employed to study
BW-CO-201–205 were obtained upon oxidation of the hydroxyl the CO release profiles in a buffer solution at different pH values
group in compounds 6a–e using PCC in CH2Cl2 at reflux by monitoring the consumption of the prodrug using HPLC. As
temperature. No beta-elimination was observed in the process, expected, the CO release from BW-CO-203 is dependent on the pH
indicating the desirable stability of CO prodrugs BW-CO-201–205 of the buffer solution, and is very sluggish in an acidic buffer
in an organic solvent. We also tested the stability of BW-CO-201 solution (pH = 3) with a half-life of over 9 h. However, when the pH
in organic solvents (e.g. CDCl3) at 37 1C, and no beta-elimination value reached 5, the CO release rate is significantly increased with
products were observed even after one week of incubation a half-life of 1.7 h. At pH 7.4 in PBS, the half-life decreased to
(Fig. S3, ESI†).
0.65 h. We also tested CO release from BW-CO-203 in a simulated
With these compounds in hand, we set out to test whether they gastric fluid (SGF) without pepsin (pH = B1), and, as expected,
would undergo a facile beta-elimination reaction to release CO around 80% of the prodrug remained intact after 8 h of incubation
under near physiological conditions. To our delight, all compounds at 37 1C (Fig. 3). Altogether, such CO prodrugs should be stable
readily underwent beta-elimination in a mixed aqueous solution in the stomach for delivery to the lower GI.
(Table 1) with concomitant release of CO. The CO release was
Having confirmed CO release from BW-CO-201–205, we next
confirmed by a thorough elucidation of the structure of by-product tested whether those CO prodrugs could be used to deliver
7 and a widely accepted CO-myoglobin assay (Fig. S4, ESI†). As enough CO intracellularly to recapitulate CO-associated anti-
shown in Table 1, by varying the leaving groups, the CO release inflammatory effects. All CO prodrugs were initially screened
half-life can be readily tuned from 20 min (BW-CO-202) to 1.2 h for their cytotoxicity against Raw 264.7 cells, and the results
(BW-CO-205). Generally, the more electron withdrawing the showed that no cytotoxicity was observed for these CO prodrugs
R group is, the faster the CO release is. The observed CO release along with their respective inactive products (7 and substituted
rate constant (Table 1) also showed a very good correlation with phenols) at concentrations up to 50 mM after 24 h of incuba-
the Hammett constant of the R groups (Fig. 2, R2 = 0.99), which is tion, but all CO prodrugs showed obvious cytotoxicity when the
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Fig. 3 CO release profiles of BW-CO-203 in a buffer containing 30% of
DMSO at 37 1C and different pH values. SGF: simulated gastric fluid without
pepsin containing 0.2% of NaCl (w/v) and 0.7% of HCl (v/v).
Fig. 5 The intracellular CO release from BW-CO-203/205. (a and b) The
FITC channel and bright field of cell only; (c and d) the FITC channel and
bright field of COP-1 (1 mM); (e and f) the FITC channel and bright field of
COP-1 (1 mM) + BW-CO-203 (50 mM); (g and h) the FITC channel and bright
field of COP-1 (1 mM) + BW-CO-205 (50 mM); (i) the fluorescence
quantification (lex: 485 nm, lem: 525 nm) of cells treated with COP-1 (1),
BW-CO-203 + COP-1 (2) or BW-CO-205 + COP-1 (3). The experiment
was repeated six times. ****: p o 0.0001. Scale bar: 20 mm. The images
were taken using a 40Â objective.
concentration reached 100 mM (Fig. S15 and S16, ESI†). BW-CO-203
and 205 were then chosen for determining CO’s anti-inflammatory
effects using an Elisa assay for TNF-a. Specifically, Raw 264.7 cells
were pretreated with BW-CO-203 and 205 and their respective inactive
products for 4 h, followed by the treatment of LPS (1 mg mLÀ1) for
another 1 h. Then the cell supernatants were collected to determine
the TNF-a level by using a commercially available Elisa kit. As shown
in Fig. 4, both BW-CO-203 and 205 dose-dependently inhibited
LPS-induced secretion of TNF-a, and no similar effects were
observed for their respective inactive products, suggesting that
the observed TNF-a suppression effects were attributed to the
CO released from BW-CO-203 and 205.
As shown in Fig. 5, the cells co-treated with COP-1 and BW-CO-203
or 205 showed much enhanced green fluorescence compared to
the ones treated with COP-1 only, indicating ready intracellular
CO release from BW-CO-203 and 205.
In conclusion, CO prodrugs with a triggered release mechanism
are highly desirable for local delivery of CO to minimize
unintended side effects. We have successfully developed a
series of pH-sensitive metal-free CO prodrugs with a tunable
and predictable release rate, which are very stable under acidic
conditions (e.g. simulated gastric fluid), and yet elicit CO
release in a buffer in a pH-sensitive fashion. Two CO prodrugs
(BW-CO-203/205) were studied as examples for their ability to
deliver enough CO intracellularly to recapitulate CO-associated
anti-inflammatory effects. Beyond the chemistry work described,
we are in the process of examining these CO prodrugs in animal
model studies.
In order to further confirm intracellular CO release from
these CO prodrugs, BW-CO-203 and 205 were chosen for cell
imaging studies using a reported CO fluorescent probe, COP-1.15
We gratefully acknowledge the partial financial support
by the Molecular Basis of Disease fellowship (Z. Pan) and the
University Center for Diagnostics and Therapeutics fellowship
(L. De La Cruz) programs.
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