Norborn-2-en-7-ones as physiologically-triggered carbon monoxide-releasing prodrugs

Article abstract of DOI:10.1039/c7sc01647f

A prodrug strategy for the release of the gasotransmitter CO at physiological pH, based upon 3a-bromo-norborn-2-en-7-one Diels-Alder cycloadducts of 2-bromomaleimides and 2,5-dimethyl-3,4-diphenylcyclopentadienone has been developed. Examples possessing protonated amine and diamine groups showed good water solubility and thermal stability. Half-lives for CO-release in TRIS-sucrose buffer at pH 7.4 ranged from 19 to 75 min at 37 °C and 31 to 32 h at 4 °C. Bioavailability in rats was demonstrated by oral gavage and oCOm-21 showed a dose dependent vasorelaxant effect in pre-contracted rat aortic rings with an EC₅₀ of 1.6 ± 0.9 μM. Increased intracellular CO levels following oCOm-21 exposure were confirmed using a CO specific fluorescent probe.

Full text of DOI:10.1039/c7sc01647f

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Norborn-2-en-7-ones as Physiologically-Triggered Carbon
Monoxide-Releasing Prodrugs
Jui Thiang Brian Kueh,a Nathan J. Stanley,a Russell J. Hewitt,a Laura M.
Woods,a Lesley Larsen,a Joanne C. Harrison,b David Rennison,c Margaret A.
Brimblec, Ivan A. Sammut*b and David S. Larsena*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A prodrug strategy for the release of the gasotransmitter CO at physiological pH, based upon 3a-bromo-norborn-2-en-7-one
Diels-Alder cycloadducts of 2-bromomaleimides and 2,5-dimethyl-3,4-diphenylcyclopentadienone has been developed.
Examples possessing protonated amine and diamine groups showed good water solubility and thermal stability. Half-lives
for CO-release in TRIS-sucrose buffer at pH 7.4 ranged from 19 to 75 mins at 37 oC and 31 to 32 h at 4 oC. Bioavailability in
rats was demonstrated by oral gavage and oCOm-21 showed a dose dependent vasorelaxant effect in pre-contracted rat
aortic rings with an EC50 of 1.6 ± 0.9 µM. Increased intracellular CO levels following oCOm-21 exposure were confirmed
using a CO specific fluorescent probe.
heart valve replacement surgery13 and in renal transplant
procedure recipients.14 Inhalation of CO (100–125 ppm) in
chronic obstructive pulmonary disease (COPD) patients,
Introduction
Carbon monoxide (CO) is a key player in heme oxygenase
mediated cellular regulation and protection in health and
disease. Evidence, by our group and others,1, 2 has established
the endogenous existence of critical, CO-activated protective
signaling pathways (vasodilatory, anti-apoptotic, anti-
thrombotic and anti-inflammatory). For example, delivery of CO
gas at low concentrations protects organs from ischaemic injury
reduced sputum neutrophils and improved bronchial
responsiveness.15 In renal transplant procedures low dose CO
gas during surgery improves post-transplant kidney function.16
However, the difficulty of controlling gas delivery in a clinical
setting, combined with the hazardous consequences of any gas
leak, have been acknowledged as significant impediments in the
use of CO in gaseous form.6, 17 CO-releasing molecules (CORMs)
have been developed as an alternative CO delivery system
allowing a greater ease of administration and control, and
potentially enabling a safer, tightly regulated method of low
dose CO delivery. These molecules have been extensively
studied and their beneficial effects18 have been demonstrated
in transplant and disease models.19-22 A large proportion of
existing CORMs are based on transition-metal carbonyl
complexes [MxLy(CO)z, where M = Mn, Ru, Fe, W, Mo, Cr, Ir or
Re].23-25 Early metal carbonyl complexes suffered from low
water solubility and high toxicity. The attachment of organic
ligands to the metal centre resulted in a newer generation of
metal carbonyl complexes, such as CORM-3,26 with increased
water solubility. However, an issue with metal carbonyl
complexes is that the residual by-products (iCORMs) obtained
after CO release contain transition metals which is undesirable
and could contribute to toxicity.27-29
by decreasing cell death and most significantly inducing a
preconditioning response.3,
4
Carbon monoxide binds to
hemeproteins, to modulate the activity or level of expression of
key cellular targets such as soluble guanylate cyclase (sGC), and
inducible nitric oxide synthase to produce a plethora of
downstream signaling events.5, 6 The anti-inflammatory effects
of CO are mediated by p38 MAPK signaling and inhibition of the
translocation of the potent damage-associated molecular
pattern recognition molecule HMBG1. This result in a reduction
of pro-inflammatory cytokines, such as TNF-, and an increase
in the expression of the anti-inflammatory IL-10.7, 8
The protective and physiological effects of low levels of CO have
been studied in a number of both animal and clinical models
with the literature extensively reviewed.5, 8-12 The results of
these studies have been so compelling that the FDA has granted
approval for the application of low dose CO gas in a range of
clinical application trials such as CO gas delivery by inhalation in
Several CORMs that do not contain transition metals have been
reported.30 One example is sodium boranocarbonate
(CORM-
A1)31, which is water soluble, capable of liberating CO at pH 7.4
and was found to improve the renal function of rabbit kidney
transplants upon reperfusion.32 More recent organic examples
a.Department of Chemistry, University of Otago, Dunedin, New Zealand.
b.Department of Pharmacology, University of Otago, Dunedin, New Zealand.
c. School of Chemical Sciences, University of Auckland, Auckland, New Zealand.
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [experimental details for the
synthesis of compounds, supporting figures and schemes, and NMR spectra]. See
DOI: 10.1039/x0xx00000x
(Figure 1) include the silacarboxylic acids
analogue 434 and the cyclic α-diketones 5-7 35
1-
333 the fluorescein
.
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generate unstable norbornadienone intermediates with
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concomitant loss of CO.40 An intramolecuDlaOrIv: 1a0r.i1a0n3t9,/Cw7hSCer0e16t4h7eF
alkyne was tethered to the diene (Figure 2B, 9) provided a series
of CORMs with CO release half-lives, controlled by the rate of
the Diels-Alder reaction, ranging from 2 minutes to one week.38
These compounds demonstrated possible therapeutic
applications in an anti-inflammatory assay and a colitis animal
model, however DMSO was required to solubilize them.
We sought an alternative approach where physiological pH
controls the release of CO using bridged carbonyl compounds
based upon a norbornenone substructure. These well-known
compounds are generally prepared using Diels-Alder reactions
of substituted cyclopentadienones and activated dienophiles,
such as maleic anhydride, giving stable cycloadducts. Early work
on these compounds carried out by Allen et al.41 and Fuch et
al.42,43 showed that compounds such as 10 could decompose
under thermal and/or photochemical conditions to release
carbon monoxide (Scheme 1).
Figure 1: Selected examples of organic CORMs
Silacarboxylic acids 1-3 have only been employed as a source of
CO gas for carbonylation reactions and require the addition of
fluoride-based desilylating reagents to induce CO release.33, 36
The fluorescein analogue
4
requires exposure to visible light
need
(500 nm) for CO release,34 while the cyclic α-diketones
5-7
to be encapsulated within pluronic micelles so that
photoactivation (400-550 nm) can result in effective CO release.
However, these examples have yet to show clinical relevance
because of issues associated with the mechanism of CO release,
solubility and cellular toxicity.
The focus of the current work is the development of organic
CORMs that would release CO on physiological stimulus.
Furthermore, the formation of an easily characterized organic
by-product after CO release would allow determination of the
CO-release kinetics whilst potentially reducing residual
compound toxicity. Our approach is based upon the bridged
carbonyl compound norbornadien-7-one (8)
37 which undergoes
cheletropic loss of carbon monoxide (CO) giving benzene as a
by-product (Figure 2A).
Scheme 1: Formation and aromatisation of cycloadduct 10.41,42
However, the conditions for CO release by heating (200 oC)40 or
photochemical activation42, 43 as reported for 10 preclude the
use of such compounds as CORMs in a biological setting. Given
this we have developed a strategy that utilises the properties of
both the norbornenone (stability) and norbornadienone (rapid
decomposition releasing CO) class of compound.
This approach provides a prodrug for an organic CO donor
molecule, the general strategy of which utilizes compounds
with the functionalised norbornenone structure represented by
12 (Figure 3). Elimination of HX would produce the transient
norbornadienone which would undergo rapid/spontaneous
cheletropic loss of CO and concomitant aromatisation to by-
product 13. X would be a halogen (Cl, Br or I) and R4 an electron
withdrawing group. The rate determining step, an E1CB process,
would be affected by the substituents R3 and R4 and thus would
control the rate of CO release. This could be achieved when
R3/R4 was a cyclic imide, which would be less susceptible to
hydrolysis than an anhydride and also provide a handle for
further functionalization. This structural motif would allow the
process to be triggered by raising the pH to give an intermediary
Figure 2
: A; Cheletropic loss of carbon monoxide from norbornadien-7-one (8).
B
; Strategy reported by Wang et al.38
It is well known that compound
8 is remarkably prone to
fragmentation with loss of CO and several computation
studies37, 39 have been carried out on this reaction where the
activation energy for this process was calculated to be between
11 to 12 kcal mol-1 with the corresponding rate constant at 300
K (27 oC) of 4.2 x 105 s-1 (t1/2 = 1.6 x 10-5 s).
With this in mind, Wang et al. have recently developed an
elegant “click and release” CO prodrug utilising intermolecular
Diels-Alder
reactions
of
functionalised
tetraphenylcyclopentadienones with strained alkynes to
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anion, stabilized by the imide, followed by loss of halide to give
a norbornadienone intermediate.
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DOI: 10.1039/C7SC01647F
Scheme 2: Synthesis and decarbonylation of CORM 16
Figure 3: Strategy for CO-release from a functionalized norbornen-7-one.
Discussion
Einhorn et al.44 reported the synthesis of polyarylphthalimides
from the Diels-Alder reaction of tetraarylcyclopentadienones
and 2-bromomaleimide in refluxing bromobenzene which
occurred with concomitant decarbonylation. Our attempts to
carry out the reaction of tetraphenylcyclopentadienone and 2-
bromo-N-phenylmaleimide (14) at lower temperatures in order
to isolate the Diels-Alder cycloadduct were unsuccessful due to
the slow rate of reaction in refluxing toluene. We surmised that
this was due to steric interactions between the 2- and 5- phenyl
groups and the bromine substituent of the maleimide.
Pleasingly, the less sterically-encumbered 2,5-dimethyl-3,4-
diphenylcyclopentadienone (15), which exists as a reversible
dimer, reacted smoothly with 14 to give endo-16 in 71% yield
after crystallisation from diethyl ether (Scheme 2). To test
whether this compound could release CO it was treated with
triethylamine in THF. Effervescence occurred and phthalimide
17 was isolated in 93% yield. GC/MS headspace analysis of a
solution of 16 in DMSO in a sealed tube after treatment with
1
DBU confirmed that the gas released was CO. H NMR analysis
of the solution indicated the complete conversion of 16 into 17
.
Scheme 3: Synthesis of oCOm-19 Reagents and conditions: (a) BocNH2CH2CH2NH2,
CH2Cl2, 0 °C to rt (69%). (b) Br2, CCl4, reflux. (c) NEt3, THF, 0 °C (57% over two steps).
(d) PhCH3, 15, reflux, (93%). (e) DBU, THF, rt, (88%). (f) 6 M HCl, 1,4-dioxane, 0 °C
to rt, A- = Cl- (99%), A- = TFA-, (34% + inseparable mixture of oCOM-19 and BP-CO-
19). (g) 6 M HCl, 1,4-dioxane, 0 °C to rt, (95%).
With the proof of principle that cycloadducts like 16 can release
CO on treatment with base, attention turned towards making
water soluble analogues. To this end a series of CORMs
functionalized with amine salts were prepared. Maleimide 18 45
,
sensitive to base and on treatment with DBU in THF gave
phthalimide 22 (88%). Removal of the Boc-group of adduct 21
was achieved on reaction with 6 M HCl in dioxane to give oCOm-
19 as its HCl salt (99%, 3:1 mixture of endo and exo isomers). In
a separate reaction the reaction product was subjected to
reverse phase chromatography on a C-18 solid phase extraction
cartridge using 1% aqueous TFA and acetonitrile which resulted
with a pendant activated ester was reacted with mono-Boc
protected ethylene diamine giving 19 which was subjected to a
bromination-dehydrobromination
sequence
to
give
bromomaleimide 20 in 57% yield over the two steps (Scheme
3). Cycloaddition of diene 15 and 20 gave adduct 21 as a 3:1
mixture of endo and exo-isomers in 93% yield. Adduct 21 was
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in partial separation of the endo- and exo-isomers (10:1, 34%) reactions of diene 15 and maleimides 19
Journal Name
,
24, VaienwdArti2cl5e Oanlninde
DOI: 10.1039/C7SC01647F
of oCOm-19 as the TFA salt, with the remainder of the material subsequent deprotection of the Boc groups for use as control
as a mixture of oCOm-19 and aromatised product, iCORM BP- compounds (Scheme S1, ESI).
CO-19. The latter compound was also prepared in 95% yield by
treatment of 22 with 6 M HCl in dioxane.
Using a similar process ethylenediamine and diethyltriamine
derived CORMs were prepared from carboethoxymaleimide 23
(Scheme 4).
To assess whether CORMs oCOm-19, -21 and -23 have
therapeutic potential their water solubility and CO release
profiles were measured at pH 7.4. Pleasingly all three were
soluble in tissue culture water at 20 oC at 3.2, 10.8 and 18.2 mg
mL-1 respectively. Transplant organ preservation solutions are
normally maintained at 8 oC. Unfortunately, the solubility of
oCOm-19 at 8 oC was low and could not be determined, whereas
those lacking the amide linking group, oCOm-21 and -23, were
measured as 3.9 and 17.6 mg mL-1 respectively. The CO release
profiles for the more soluble CORMs oCOm-21 and -23 were
assessed by monitoring the formation of the corresponding
iCORMs, BP-CO-21 and BP-CO-23 in TRIS-sucrose buffer at pH
7.4 using an HPLC method. oCOm-21 showed half-lives for CO
release of 19 min at 37 oC (Figure S1, ESI) and 31 h at 4 to 5 oC.
oCOm-23 half-lives at the same temperatures were 75 min and
Scheme
4
:
Synthesis of oCOm-21 and -23. Reagents and conditions: (a) 32 h, respectively. The CO release was also supported by
BocNH(CH2)2NH2, THF, H2O, 0 °C to rt, 24 (58%); BocNH(CH2)2NHBoc(CH2)2NH2 THF,
H2O, 0 °C to rt, 25 (mixture with ethyl carbamate). (b) Br2, CCl4, reflux. (c) NEt3, monitoring via a CO-selective electrode over a range of pH and
THF, 0 °C, 26 (73% over two steps), 27 (25% over two steps + 1:1 inseparable
mixture of 27 and ethyl carbamate). (d) PhCH3, 15, reflux, 28 (84% 2.5:1 endo:exo), myoglobin assays (Figure S2a to S2c, ESI). There was no sign of
29 (60% endo) (e) DBU, THF, rt, 30 (61%), 31 (76)%. (f) and (g) 6 M HCl, 1,4-dioxane,
0 °C to rt, oCOM-21 (63%), oCOM-23 (63%). (h) 6 M HCl, 1,4-dioxane, 0 °C to rt the formation of the corresponding iCORMs from solutions of
(78%). (i) 6 M HCl, 1,4-dioxane, rt to 50 °C (82%). †Purified by reverse phase C-18
solid phase extraction cartridge using 1% aqueous TFA and acetonitrile.
oCOm-19, -21 and -23 in aqueous media at pH < 7 after
prolonged periods of time (> 1 week). Samples showed no
deterioration after being left at room temperature exposed to
ambient light for over one month. The thermal stability of
oCOM-21 was investigated by thermogravimetric analysis and
Reaction of bromomaleimide 26 with diene dimer 15 gave
cycloadduct 28 (endo:exo = 2.5:1) in 84% yield after purification
by column chromatography. Partial separation was achieved
with a sample of endo-28 isolated in 32% yield with the
remaining 52% a 1:1 mixture of endo- and exo-isomers.
Similarly, reaction of bromomaleimide 27 with 15 gave a 3:1
mixture of endo- and exo-adducts 29 which after column
chromatography afforded the endo-isomer (60%) along with a
mixture of the isomers in 27% yield. Removal of the Boc-
protecting groups of endo-28 and -29 was achieved on
treatment with 6 M HCl in dioxane and gave, after purification
on reverse-phase solid-phase cartridges and lyophilization,
oCOm-21 and -23 both in 63% yield as their mono- and bis-
trifluoroacetate salts as white powders. Aromatisation of 28
and 29 on treatment with DBU gave phthalimides 30 and 31 and
subsequent removal of the Boc-group provided the respective
iCORMs BP-CO-21 and BP-CO-23 in 48 and 62% yield
respectively over the two steps. A series of desbromo-
o
was shown to undergo decomposition at 125 C (Figure S3a,
ESI). Further analysis showed that heating a sample of oCOM-
21 to 90 oC and holding it at that temperature for 30 mins
resulted in a 4% loss of mass (Figure S3b, ESI). HPLC analysis of
this sample showed that it was a 96:4 mixture of oCOm-21 and
its corresponding iCORM (Figure S3c, ESI).
In context to applications such as organ transplantation, the
toxicity of oCOm-19
MDCK cells at 37 oC and EC50 values were measured as 86, 11.2
and 10.1 µM, respectively (Figure S4, ESI). iCORMs BP-CO-2
, -21 and -23 was assessed using sensitive
1
and -23 gave EC50 values of 21 and 100 µM, respectively. The
toxicity of oCOm-21 was also measured against this cell line at
8 oC and gave an EC50 > 100 µM.
o
CO delivery to MDCK cells by oCOm-21 and -23 at 8 and 37 C
was confirmed using the CO-selective fluorescent probe COP-
developed by Chang et al.46 (Figure 4).
1
analogues of oCOm-19, -21 and -23, DB-CO-19, -21 and -23
respectively, which lack the ability to release CO under
physiological conditions, were also synthesised by Diels-Alder
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Oral bioavailability in rats was demonstrated by
carboxyhaemoglobin analysis following administration of
oCOm-19 and -21 by oral gavage. Moderate but significant
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DOI: 10.1039/C7SC01647F
Figure 4: CO specific fluorescent probe (COP-1) confirms increased intracellular
CO levels following exposure to oCOm-21 (10 µM) or oCOm-23 (10 µM) against
Figure 6: Protective effect of oCOm-21 on heart tissue (rat myocardium):
(
a) fresh
stain;
heart tissue samples (i) untreated and not stained (ii) untreated and stained with
the respective iCORM (50 µM) at both 37 C and 8 C.
TTC
(b) heart tissue samples after 48 hours incubation (i) control heart tissue samples
stained with TTC stain (ii) heart tissue samples incubated with oCOm-21 (3 μM)
after 48 hours and stained with TTC stain (iii) heart tissue samples incubated with
DB-CO-21 (3 μM) after 48 hours and stained with TTC stain.
increases in carboxyhaemoglobin levels in the plasma were
obtained after 3 h of administration for the less soluble oCOm-
19 and within 1 h for oCOm-21 (Table S1, ESI). BP-CO-19, which
cannot release CO, showed no increase in carboxyhaemoglobin
at the same time points.
Conclusions
CO relaxes vascular tissues by acting on the cGMP pathway and
high conductance calcium-activated K+(KCa) channels. To assess
the vasorelaxant effect of our compounds, pre-contracted rat
aortic rings were treated with oCOm-19 and -21. A dose-
dependent relationship was observed for both compounds with
EC50 values of 18.0 ± 0.8 µM and 1.6 ± 0.9 µM respectively
(Figure 5). This vasodilatory effect was reversed by the addition
of 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an
inhibitor of soluble guanylate cyclase (sGC).
In conclusion, we have developed CO prodrugs based upon
norbornenones which function via E1CB elimination of HBr
under physiological conditions, producing
norbornadienone intermediate that rapidly undergoes
chelotropic loss of CO. The three prodrugs, oCOm-19 -21, and
a
reactive
,
-
23 are thermally stable solids that show good water solubilities
and CO release profiles. Under physiological conditions they
have been shown to deliver CO to cells, have a vasorelaxant
effect and have the potential to preserve tissue sample viability.
We are developing new generations of these compounds and
investigating their applications as additives for organ
transplantation preservation solutions.
Acknowledgements
This work is supported by a University of Otago Research Grant
and by the Ministry of Business, Innovation and Employment
grant (CONT-29537-HVMSSI-UOO).
Figure 5: Vasorelaxant effect of oCOm-19 and -21 in isolated rat aortas at 37 °C.
A. Relaxation responses in precontracted aortic rings expressed as a percent
change of the contractile response to phenylephrine (100 nM). B. Responses to
EC50 concentrations of oCOm-19 repeated in the presence of the guanylate
cyclase inhibitor ODQ. Data was analysed using a One-way ANOVA with Bonferroni
post-hoc test and expressed as mean ± SEM. ** denotes P<0.01= vs the non (-)-
ODQ inhibited group.
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