Mitochondrial biotransformation of ω-(phenoxy)alkanoic acids, 3-(phenoxy)acrylic acids, and ω-(1-methyl-1H-imidazol-2-ylthio)alkanoic acids: A prodrug strategy for targeting cytoprotective antioxidants to mitochondria

Article abstract of DOI:10.1016/j.bmc.2010.01.019

Mitochondrial reactive oxygen species (ROS) generation and the attendant mitochondrial dysfunction are implicated in a range of disease states. The objective of the present studies was to test the hypothesis that the mitochondrial β-oxidation pathway could be exploited to deliver and biotransform the prodrugs ω-(phenoxy)alkanoic acids, 3-(phenoxy)acrylic acids, and ω-(1-methyl-1H-imidazol-2-ylthio)alkanoic acids to the corresponding phenolic antioxidants or methimazole. 3- and 5-(Phenoxy)alkanoic acids and methyl-substituted analogs were biotransformed to phenols; rates of biotransformation decreased markedly with methyl-group substitution on the phenoxy moiety. 2,6-Dimethylphenol formation from the analogs 3-([2,6-dimethylphenoxy]methylthio)propanoic acid and 3-(2,6-dimethylphenoxy)acrylic acid was greater than that observed with ω-(2,6-dimethylphenoxy)alkanoic acids. 3- and 5-(1-Methyl-1H-imidazol-2-ylthio)alkanoic acids were rapidly biotransformed to the antioxidant methimazole and conferred significant cytoprotection against hypoxia-reoxygenation injury in isolated cardiomyocytes. Both 3-(2,6-dimethylphenoxy)propanoic acid and 3-(2,6-dimethylphenoxy)acrylic acid also afforded cytoprotection against hypoxia-reoxygenation injury in isolated cardiomyocytes. These results demonstrate that mitochondrial β-oxidation is a potentially useful delivery system for targeting antioxidants to mitochondria.

Full text of DOI:10.1016/j.bmc.2010.01.019

Bioorganic & Medicinal Chemistry 18 (2010) 1441–1448
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Mitochondrial biotransformation of
x
-(phenoxy)alkanoic acids,
3-(phenoxy)acrylic acids, and
x
-(1-methyl-1H-imidazol-2-ylthio)alkanoic acids:
A prodrug strategy for targeting cytoprotective antioxidants to mitochondria
Kurt S. Roser a, Paul S. Brookes a, Andrew P. Wojtovich b, Leif P. Olson a, Jalil Shojaie a,
Richard L. Parton a, M. W. Anders a,b,
*
a Mitochondrial Research and Innovation Group, Department of Anesthesiology, Rochester, NY 14642, United States
b Department of Pharmacology and Physiology, University of Rochester Medical Center, 601 Elmwood Avenue, Box 711, Rochester, NY 14642, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Mitochondrial reactive oxygen species (ROS) generation and the attendant mitochondrial dysfunction are
implicated in a range of disease states. The objective of the present studies was to test the hypothesis that
Received 29 October 2009
Revised 6 January 2010
Accepted 7 January 2010
Available online 15 January 2010
the mitochondrial b-oxidation pathway could be exploited to deliver and biotransform the prodrugs
x-
(phenoxy)alkanoic acids, 3-(phenoxy)acrylic acids, and -(1-methyl-1H-imidazol-2-ylthio)alkanoic acids
x
to the corresponding phenolic antioxidants or methimazole. 3- and 5-(Phenoxy)alkanoic acids and
methyl-substituted analogs were biotransformed to phenols; rates of biotransformation decreased mark-
edly with methyl-group substitution on the phenoxy moiety. 2,6-Dimethylphenol formation from the
analogs 3-([2,6-dimethylphenoxy]methylthio)propanoic acid and 3-(2,6-dimethylphenoxy)acrylic acid
Keywords:
Mitochondrial b-oxidation
Antioxidants
Mitochondrial prodrug targeting
Hypoxia-reoxygenation injury
was greater than that observed with
x-(2,6-dimethylphenoxy)alkanoic acids. 3- and 5-(1-Methyl-1H-
imidazol-2-ylthio)alkanoic acids were rapidly biotransformed to the antioxidant methimazole and con-
ferred significant cytoprotection against hypoxia-reoxygenation injury in isolated cardiomyocytes. Both
3-(2,6-dimethylphenoxy)propanoic acid and 3-(2,6-dimethylphenoxy)acrylic acid also afforded cytopro-
tection against hypoxia-reoxygenation injury in isolated cardiomyocytes. These results demonstrate that
mitochondrial b-oxidation is
a
potentially useful delivery system for targeting antioxidants to
mitochondria.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
the mitochondrial b-oxidation of a series of
acids and 3-(phenoxy)acrylic acids to the corresponding phenols
and of -(1-methyl-1H-imidazol-2-ylthio)alkanoic acids to meth-
x-(phenoxy)alkanoic
There is considerable contemporary interest in targeting antiox-
idants to mitochondria. Mitochondria account for >90% of cellular
oxygen consumption and are a major source of reactive oxygen
species (ROS); indeed, 1–2% of the oxygen consumed by mitochon-
dria is converted to ROS.1 Moreover, a role for mitochondrial ROS
in a range of disease states has been established.2
A range of strategies for targeting of antioxidants to mitochon-
dria has been reported, including targeting based on the mitochon-
drial electrochemical potential, on binding to mitochondrial
membrane components, on the unique mitochondrial expression
of enzymes that catalyze the release of drugs from prodrugs, and
on transporter-dependent delivery of prodrugs or drugs.3–8
The objective of the experiments reported herein was to test the
hypothesis that the mitochondrial location of the enzymes of b-
oxidation could be exploited to deliver phenolic chain-breaking
and thiol-based antioxidants to mitochondria (Fig. 1). Accordingly,
x
imazole was undertaken (Fig. 2).
2. Targeting strategy
The enzymes of mitochondrial b-oxidation catalyze the biotrans-
formation of dietary and endobiotic fatty acids along with a range of
xenobiotic alkanoic acids, including tianeptine, chlorophenoxybu-
tyric acids, and 5-hydroxydecanoic acid.9–11 Moreover, the toxicity
of several xenobiotic alkanoic acids, including haloalkene-derived
4-thiaalkanoates,12–14 4-bromo-2-butenoic acid,15 valproic acid,16
and hypoglycin,17 is associated with their bioactivation by the mito-
chondrial b-oxidation pathway.
3. Results and discussion
Studies on the metabolism of
x-phenylalkanoic acids by Knoop
and others established the concept of fatty acid b-oxidation.18–22
* Corresponding author. Tel.: +1 585 230 5163; fax: +1 585 273 2652.
E-mail address: mw_anders@urmc.rochester.edu (M.W. Anders).
These studies were extended by Levey and Lewis,23 who showed
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.019

1442
K. S. Roser et al. / Bioorg. Med. Chem. 18 (2010) 1441–1448
Figure 1. Mitochondrial biotransformation of
x
-(phenoxy)alkanoic acids. FACL, fatty-acid-CoA ligase; CPT-I, carnitine palmitoyltransferase-I; CACT, carnitine-acyl carnitine
translocase; CPT-II, carnitine palmitoyltransferase-II; MCAD, medium-chain acyl-CoA dehydrogenase; SCAD, short-chain acyl-CoA dehydrogenase.
activity and because the compounds afforded insight into the sub-
strate selectivity of the b-oxidation pathway.24–27 Finally, methim-
azole is an antioxidant and cytoprotective agent,28,29 and the
methimazole prodrugs
x-(1-methyl-1H-imidazol-2-ylthio)alka-
noic acids 4a and 4b were investigated as substrates and as cardio-
protective agents.
The data presented herein show that
x-(phenoxy)alkanoic acids
are substrates for mitochondrial b-oxidation, but that the rates of
biotransformation differ markedly depending on the structure.
All 3-(phenoxy)propanoic acids and 5-(phenoxy)pentanoic acids
studied were biotransformed to phenolic products in isolated rat
liver mitochondria (Table 1), but the rates of biotransformation dif-
fered markedly: with 3-(phenoxy)propanoic acids (1a–1d), the
rates of biotransformation decreased with number and position
of the methyl groups on the phenoxy moiety in the order:
1a ꢀ 1b > 1d > 1c. A similar pattern was observed with 5-(phen-
oxy)pentanoic acids where the relative rates of biotransformation
were 2a ꢀ 2b > 2c > 2d. In general, the rates of biotransformation
Table 1
Rates of mitochondrial biotransformation of
x
-(phenoxy)alkanoic acids, 3-(phen-
oxy)acrylic acids, and
x
-(1-methyl-1H-imidazol-2-ylthio)alkanoic acidsa
Compound
Rate
1a
1b
1c
1d
2a
2b
2c
2d
3
4a
4b
5
6a
6b
2.06 0.50
0.39 0.12
0.07 0.02
0.10 0.06
1.27 0.37
0.39 0.12
0.06 0.01
0.01 0.06
1.54 0.17
2.50 0.31
0.97 0.16
0.54 0.06
1.55 0.14
N.D.
Figure 2. Structures of
x-(phenoxy)alkanoic acids, 3-(phenoxy)acrylic acids, and
x
-(1-methyl-1H-imidazol-2-ylthio)alkanoic acids.
that 4-(phenoxy)butanoic acid and 6-(phenoxy)hexanoic acid are
metabolized to phenoxyacetic acid. The present study exploits
these seminal investigations to determine whether alkanoic acid-
based prodrugs could be used to target antioxidants to
mitochondria.
Octanoic acid
40.3 6.7
a
The alkanoic acids (Fig. 2) were incubated with rat liver mitochondria, and
product formation was quantified, as described in Section 5. Rates are expressed as
nmol minÀ1 mg proteinÀ1. Data are shown as mean SD, n = ꢁ 3. Statistical analysis
(unpaired t-test): 1a versus 2a, p <.05; 1b versus 2b, NS; 1c versus 2c, NS; 1d versus
2d, p <.05; 1a versus 3, p <.05; 2a versus 3, p >.05; 1c versus 5, p <.05; 1c versus 6a,
p <0.05; 2c versus 5, p <.05; 1a versus 6a, p <.05. N.D., not detected.
The phenolic substituents on
x-(phenoxy)alkanoic acids, that is,
2-methylphenol, 2,6-dimethylphenol, 3,5-dimethylphenol, and
2,6-diisopropylphenol, were chosen because of their antioxidant

K. S. Roser et al. / Bioorg. Med. Chem. 18 (2010) 1441–1448
1443
of 5-(phenoxy)pentanoic acids were lower than those of similarly
substituted 3-(phenoxy)propanoic acids, except that the rates of
nitine.32 The formation of the 5-trans-tetradecenoylcarnitine is
attributed to the low rate of dehydrogenation of 5-trans-tetradec-
anoyl-CoA by the acyl-CoA dehydrogenases, which results in the
accumulation of the CoA thioester; the accumulated CoA thioester
is converted to the carnitine ester by carnitine palmitoyltransfer-
ase-II. Indeed, carnitine ester formation is favored over hydrolysis
biotransformation of
2b were similar. The rate of biotransformation of all
x
-(2-methylphenoxy)alkanoic acids 1b and
-(phen-
x
oxy)alkanoic acids studied was much lower than that of octanoic
acid (Table 1).
Rates of biotransformation of
2a decreased with increasing chain length, but this was not ob-
served with -(2-methylphenoxy)- [1b vs 2b], -(2,6-dimethyl-
phenoxy)- [1c vs 2c], or -(3,5-dimethylphenoxy)alkanoic acids
[1d vs 2d] (Table 1). Indeed, the rates of biotransformation of the
-(2,6-dimethylphenoxy)- [1c and 2c] and -(3,5-dimethylphen-
x
-(phenoxy)alkanoic acids 1a and
to the acid.32
were poor substrates for b-oxidation (Table 1). Hence, the observed
accumulation of -(2,6-dimethylphenoxy)alkanoyl-CoA esters
x-(2,6-Dimethylphenoxy)alkanoic acids 1c and 2c
x
x
x
x
may have been accompanied by the carnitine palmitoyltransfer-
ase-II-catalyzed conversion to the corresponding carnitine esters.
5-(Phenoxy)pentanoic acids must undergo two cycles of b-oxida-
tion to release a phenolic metabolite, whereas 3-(phenoxy)propanoic
acids release a phenolic metabolite after one cycle of b-oxidation
(Fig. 1). Moreover, 4-thiaalkanoic acids are better substrates for the
medium-chain acyl-CoA dehydrogenase than 4-oxaalkanoic acids.33
Hence, 3-([2,6-dimethylphenoxy]methylthio)propanoic acid 5 was
prepared to testthe hypothesis that theinsertion ofa methylthiolink-
ing group between the 2,6-dimethylphenoxy group and the propa-
noic acid moiety would result in less steric interaction between the
substrate and b-oxidation enzymes and, thereby, increase the rate
ofbiotransformation.Theintermediate(2,6-dimethylphenoxy)meth-
anethiol formed from acid 5 after one cycle of b-oxidation would be
expected to eliminate thioformaldehyde to afford 2,6-dimethylphe-
nol. Indeed, 4-thiaalkanoicacid5proved to be a much bettersubstrate
x
x
oxy)alkanoic acids [1d vs 2d] were low compared with the unsub-
stituted analogs.
3-(2-Naphthoxy)propanoic acid 3 was biotransformed at a rate
slower than that of 3-(phenoxy)propanoic acid 1a but similar to
that of the 5-(phenoxy)pentanoic acid 2a (Table 1).
Although the biotransformation of the alkanoic acids was stud-
ied in the presence of carnitine, the biotransformation of alkanoic
acids 1a and 5 was studied in both the presence and absence of
added carnitine, but no significant difference was observed (data
not shown), perhaps because sufficient carnitine concentrations
were present in isolated mitochondria.
The mitochondrial biotransformation of 5-(2,6-dimethylphen-
oxy)pentanoic acid 2c was accompanied by the formation of the
carnitine ester of acid 2c (Fig. 3). In addition, acid 2c was biotrans-
formed to the carnitine ester of acid 1c, but 3-(2,6-dimethylphen-
oxy)propanoic acid 1c itself was not observed. The formation of the
product 2,6-dimethylphenol was, however, observed, indicating
that acid 2c underwent two cycles of b-oxidation to release prod-
uct (Fig. 1). This observation differs from the mitochondrial b-oxi-
dation of dietary fatty acids, which apparently proceeds to product
formation with no release of intermediates and is attributed to
intermediate channeling.30,31 The formation of carnitine esters
has, however, been observed with other xenobiotic alkanoic acids:
the mitochondrial b-oxidation of elaidic acid (9-trans-octadecenoic
acid) is accompanied by the formation of 5-trans-tetradecenoylcar-
for b-oxidation than
x-(2,6-dimethylphenoxy)alkanoic acids 1c and
2c (Table 1), affording a several-fold increase in reaction rate.
The mitochondrial biotransformation of endobiotic and xenobi-
otic alkanoic acids requires the medium-chain acyl-CoA dehydro-
genase-catalyzed formation of enoyl-CoA intermediates. (E)-3-
(2,6-Dimethylphenoxy)acrylic acid 6a was prepared to test the
hypothesis that circumventing the potential barrier formed by
the medium-chain acyl-CoA dehydrogenase might overcome the
apparent steric impediment to biotransformation observed with
x
-(2,6-dimethylphenoxy)alkanoic acids 1c and 2c (Table 1). The
rate of biotransformation of (E)-3-(2,6-dimethylphenoxy)acrylic
acid 6a was approximately 20-fold greater than that of the
Figure 3. Biotransformation of 5-(2,6-dimethylphenoxy)pentanoic acid 2c to carnitine esters. Acid 2c was incubated with rat liver mitochondria, and a sample of the reaction
mixture was analyzed by LC–MS, as described in Section 5. (A) 3-(2,6-Dimethylphenoxy)propanoylcarnitine; (B) 5-(2,6-dimethylphenoxy)pentanoylcarnitine; (C) 2,6-
dimethylphenol; (D) 5-(2,6-dimethylphenoxy)pentanoic acid 2c.

1444
K. S. Roser et al. / Bioorg. Med. Chem. 18 (2010) 1441–1448
saturated analog 3-(2,6-dimethylphenoxy)propanoic acid 1c, indi-
cating that steric effects play a larger role for the medium-chain
acyl-CoA dehydrogenase than for enoyl-CoA hydratase (Table 1).
Moreover, no biotransformation of (Z)-3-(2,6-dimethylphen-
oxy)acrylic acid was detected (data not shown); this is consistent
with the known selectivity of enoyl-CoA hydratase, which cata-
lyzes the conversion of (E)-2-alkenoyl-CoA thioesters, but not
(Z)-2-alkenoyl-CoA thioesters, to 3-hydroxyalkanoyl-CoA thioes-
ters.34 No quantifiable biotransformation was observed with the
bulky analog (E)-3-(2,6-diisopropylphenoxy)acrylic acid 6b
(Table 1), indicating that steric factors also affect enoyl-CoA hydra-
tase-catalyzed reactions. This is the first demonstration of the util-
ity of using
x-(phenoxy)acrylic acids as prodrugs for antioxidant
delivery to mitochondria.
Depolarization of the mitochondrial membrane potential with
carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP)
significantly decreased the biotransformation of
x-(phenoxy)alka-
noic acids 1a and 2a (1a: 1.69 0.19, 1a + FCCP: 0.41 0.12, p<.05;
2a: 1.17 0.27, 2a + FCCP: 0.64 0.10, p <.05; rates are expressed
as nmol product minÀ1 mg proteinÀ1 and are shown as mean SD,
n = 3). The mechanism by which FCCP decreased the biotransfor-
mation of
x-(phenoxy)alkanoic acids was not investigated, but
may be similar to that of Arochlor 1254, which, like FCCP,35 de-
creases both the mitochondrial membrane potential and the b-oxi-
dation of palmitate.36 The effect of Arochlor 1254 on b-oxidation
was attributed to Arochlor 1254-induced impairment of the elec-
tron transport chain.37 Several compounds, including perhexiline,
amiodarone, benzarone, and benzbromarone, also inhibit mito-
chondrial b-oxidation,38,39 but the mechanism by which these
drugs inhibit b-oxidation has not been elucidated. Although FCCP
may perturb the function of the carnitine shuttle and, hence, the
uptake of xenobiotic alkanoic acids by mitochondria, the effect of
FCCP on the carnitine shuttle has apparently not been reported.
The decreased mitochondrial membrane potential that may
accompany pathological mitochondrial dysfunction may, however,
reduce the rate of biotransformation of alkanoic-acid-based anti-
oxidant prodrugs.
Figure 4. Cytoprotective effects of
acids 4a or 4b and 3-(2,6-dimethylphenoxy)propanoic acid 1c and 3-(2,6-dimeth-
ylphenoxy)acrylic acid 6a in isolated rat cardiomyocytes. Upper panel: Alkanoic
x-(1-methyl-1H-imidazol-2-ylthio)alkanoic
acids 4a and 4b (1 lM), methimazole (1 lM), and etomoxir (20 lM) were incubated
with cardiomyocytes in a hypoxia-reoxygenation protocol, as described in Section
5. CON, control; HR, hypoxia-reoxygenation; Eto, etomoxir. Statistical analysis (one-
way ANOVA, n = 3): CON versus HR, p <.05; HR versus HR + 4a, p <.05; CON versus
HR + 4a, NS; HR + 4a versus HR + 4a + Eto, p <.05; HR versus HR + 4b, p <.05;
HR + 4b versus HR + 4b + Eto, p <.05; HR versus Methimazole, NS. Lower panel:
The cytoprotective potential of the thiol-based antioxidant pro-
drugs 4a and 4b and of 2,6-dimethylphenol prodrugs 1c and 6a
was tested in a hypoxia-reoxygenation protocol with isolated
cardiomyocytes, which is a model system relevant to ischemia/
reperfusion injury. Incubation of cardiomyocytes with either
x-
Alkanoic acids 1c and 6a (1
lM), 2,6-dimethylphenol (1
lM), and etomoxir (20 lM)
were incubated with cardiomyocytes in
a
hypoxia-reoxygenation protocol, as
(1-methyl-1H-imidazol-2-ylthio)alkanoic acids 4a or 4b conferred
significant cytoprotection in cardiomyocytes incubated in a hypox-
described in Section 5. CON, control; HR, hypoxia-reoxygenation; Eto, etoximir.
Statistical analysis (one-way ANOVA, n = 3): CON versus HR, p <.05; HR versus
HR + 1c, p <.05; CON versus HR + 1c, p <.05; HR + 1c versus HR + 1c + Eto, p <.05; HR
ia-reoxygenation protocol (Fig. 4). The cytoprotective effect of
x-
(1-methyl-1H-imidazol-2-ylthio)alkanoic acids 4a or 4b was
blocked by etomoxir, which inhibits carnitine palmitoyl transfer-
ase-I and, thereby, prevents their delivery to mitochondria.40 Incu-
bation of cardiomyocytes under the hypoxia-reoxygenation
protocol with etomoxir alone exerted no cytoprotective effect. Sig-
nificantly, incubation of cardiomyocytes under the hypoxia-reoxy-
genation protocol with methimazole alone failed to provide
versus HR + 6a,
dimethylphenol, NS.
p <.05; HR + 6a versus HR + 6a + Eto, p <.05; HR versus 2,6-
antioxidants for the management of cardiac ischemia/reperfusion
injury.44 A range of phenols, particularly ortho-substituted phenols,
are chain-breaking antioxidants that react with a range of free-rad-
ical species.45,24 2,6-Dimethylphenol, which is released from pro-
drugs 1c and 6a, is a chain-breaking antioxidant.26 Methimazole,
which is released from prodrugs 4a and 4b, scavenges superoxide,
hydrogen peroxide, hydroxyl radical, and free radicals.46–48,29
Hence, the cytoprotective actions of prodrugs 1c, 4a, 4b, and 6a
are consistent with the pathophysiology of ischemia/reperfusion
injury and with the known antioxidant chemistry of 2,6-dimethyl-
phenol and methimazole.
cytoprotection. Hence, the methimazole prodrugs
x-(1-methyl-
1H-imidazol-2-ylthio)alkanoic acids 4a or 4b allow the delivery
and release of methimazole to mitochondria and provide cytopro-
tection that is superior to that afforded by the methimazole itself.
Similarly, both 3-(2,6-dimethylphenoxy)propanoic acid 1c and
3-(2,6-dimethylphenoxy)acrylic acid 6a were cytoprotective in a
hypoxia-reoxygenation protocol, and the cytoprotective effects
were blocked by etomoxir (Fig. 4).
The observed cytoprotective effects of prodrugs 1c, 4a, 4b, and
6a may be attributed to their antioxidant actions. Cardiac ische-
mia/reperfusion injury is characterized by the formation of reac-
tive oxygen species, including superoxide, hydrogen peroxide,
and oxygen- and carbon-centered free radicals.41–43 Accordingly,
there is interest in the development of mitochondrial-targeted
4. Summary
The data presented herein demonstrate that the mitochondrial
biotransformation of xenobiotic alkanoic acids can be exploited
for the delivery of phenol- and thiol-based antioxidant prodrugs

K. S. Roser et al. / Bioorg. Med. Chem. 18 (2010) 1441–1448
1445
to mitochondria. The rates of biotransformation of
oxy)alkanoic acids were highly dependent on the number and
position of methyl groups on the phenoxy moiety. The finding that
x
-(phen-
dimethylbenzene with methyl 3-mercaptopropanoate followed by
hydrolysis of the ester. 2-(Chloromethoxy)-1,3-dimethylbenzene: A
three-necked, 500-mL flask equipped with thermometer, reflux con-
denser, argon inlet, and a magnetic stirring bar was charged with
6.12 g (0.05 mol) of 2,6-dimethylphenol, 100 mL dry tetrahydrofu-
ran, and 4.0 g (0.1 mol) sodium hydroxide powder. The mixture
was heated at 66 °C for 1 h. The mixture was cooled to room temper-
ature, and 98 mL (1.5 mol) of bromochloromethane was added. The
suspension that formed was cooled to room temperature, filtered
through CeliteÒ, and the solvent was removed under reduced
pressure. The oily residue was purified by vacuum distillation (1–
1.5 torr, bp, 66 °C) to afford an overall yield of 6.50 g (76%) of 2-(chlo-
romethoxy)-1,3-dimethylbenzene. 1H NMR (400 MHz, CDCl3): 2.33
(s, 6H), 5.80 (s, 2H), 6.99–7.06 (m, 3H).
x
x
-(phenoxy)alkanoic acid 1c, 3-(phenoxy)acrylic acids 6a, and
-(1-methyl-1H-imidazol-2-ylthio)alkanoic acids 4a and 4b
undergo mitochondrial b-oxidation and afford protection
against hypoxia-reoxygenation injury in cardiomyocytes dem-
onstrate the potential utility of the use of antioxidant-based
xenobiotic alkanoic acids as a mitochondrial delivery system.
This is the first investigation of the structural requirements for
the mitochondrial b-oxidation of
x-(phenoxy)alkanoic acids
and the first demonstration that alkanoic acid-based antioxidant
prodrugs are cytoprotective.
5. Experimental
Methyl 3-[(2,6-dimethylphenoxy)methylthio]propanoate: A mix-
ture of 2.40 g (20 mmol) of methyl 3-mercaptopropionate, 2.58 g
(20 mmol) of diisopropylethylamine, and 1.70 g (100 mmol) of
2-(chloromethoxy)-1,3 dimethylbenzene in 40 mL of dry tetrahy-
drofuran under argon was heated at reflux for 52 h. The reaction
mixture was cooled, diluted with 60 mL methylene chloride, and
extracted with 5% HCl. The organic phase was extracted twice with
30 mL of distilled water, then with 40 mL of saturated sodium
chloride solution, and dried with anhydrous magnesium sulfate.
The solvent was removed under reduced pressure, and 3.20 g of
yellowish oil was obtained. The product was purified by normal-
phase chromatography (CombiFlash, Teledyne Isco, Inc., Lincoln,
NE) with a hexane/ethyl acetate gradient (100:0, 5 min, 95:5,
15 min) to afford 2.20 g (87%) of methyl 3-[(2,6-dimethylphen-
oxy)methylthio]propanoate. 1H NMR (400 MHz, CDCl3): 2.32 (s,
6H), 2.78–2.82 (t, 2H, J = 7.2 Hz), 3.03–3.07 (t, 2H, J = 7.2 Hz), 3.70
(s, 3H), 4.07 (s, 2H), 6.92–7.02 (m, 3H).
5.1. General
HEPES, EGTA (Sigma E4378), sucrose, ATP disodium salt (Sigma
A7699), CoA (Sigma C3144), KCl, MgCl2Á6H2O, and
purchased from Sigma–Aldrich (St. Louis, MO). KH2PO4 was ob-
tained from JT Baker; and -dithiothreitol was purchased from
TCI America (Boston, MA). Chemicals for organic synthesis were
obtained from Aldrich (Milwaukee, WI).
L-carnitine were
D,L
5.2. Chemicals and syntheses (the structures are shown in
Fig. 2)
3-(Phenoxy)propanoic acid 1a was purchased from Aldrich.
3-(2-Methylphenoxy)propanoic acid 1b was obtained from
UkrOrgSynthesis (Kiev, Ukraine).
3-(2,6-Dimethylphenoxy)propanoic acid 1c was prepared as
3-[(2,6-Dimethylphenoxy)methylthio]propanoic acid 5: A 50-mL
flask equipped with reflux condenser and a magnetic stirring bar
was charged with 0.254 g (1 mmol) of methyl 3-[(2,6-dimethyl-
phenoxy)methylthio]propanoate and 0.362 g (2 mmol) of trimeth-
yltin hydroxide dissolved in 25 mL of 1,2-dichloroethane and was
refluxed overnight under an argon atmosphere. The solvent was re-
moved under reduced pressure, the organic residue was dissolved
in 40 mL of ethyl acetate, extracted three times with 10 mL 5% HCl,
then with 10 mL brine, and dried with anhydrous magnesium sul-
fate. The solvent was removed under reduced pressure, and the
reddish residue was purified by normal-phase (CombiFlash) chro-
matography with a hexane/ethyl acetate (95:5, 7 min; 95:40,
15 min) to afford 0.2 g (83%) of 3-[(2,6-dimethylphenoxy)methyl-
thio]propanoic acid 5. 1H NMR (400 MHz, CDCl3): 2.33 (s, 6H),
2.78–2.82 (t, 2H, J = 7.2 Hz), 3.03–3.07 (t, 2H, J = 7.2 H), 4.99 (s,
2H), 6.94–7.04 (m, 3H), 10.98 (br, OH). MS (ESI): 241 [M+H]+;
263 [M+Na]+.
The new compound (E)-3-(2,6-dimethylphenoxy)acrylic acid
6a was prepared by the general method of Fan et al.:53 ethyl (E/
Z)-3-(2,6-dimethylphenoxy)acrylate: 2,6-Dimethylphenol (1.5 g,
12.3 mmol) and 1,4-diazabicyclo[2.2.2]octane (100 mg, 0.9 mmol)
and 2 mL of methylene chloride were combined with magnetic
stirring. Ethyl propiolate (1.2 mL, 12.6 mmol) in 3 mL of methylene
chloride was added drop wise over 5–10 min. Thin-layer chroma-
tography of the reaction mixture (silica gel, hexane) showed the
formation of two products. The reaction mixture was diluted with
ꢂ70 mL of ethyl acetate and extracted with 10% potassium hydrox-
ide, and then with saturated sodium chloride solution. The mixture
was then extracted with 10% hydrochloric acid solution and then
again with saturated sodium chloride solution. The ethyl acetate
extract was dried with anhydrous magnesium sulfate, filtered,
and evaporated to dryness to afford 2.9 g (100%) of product. A
sample of this material (1.0 g) was purified by normal-phase
chromatography (CombiFlash) to afford 650 mg of ethyl (E)-3-
(2,6-dimethylphenoxy)acrylate [1H NMR (400 MHz, CDCl3): 1.24
described by Lichtenberger and Geyer.49
3-(3,5-Dimethylphenoxy)propanoic acid 1d was purchased
from UkrOrgSynthesis.
5-(Phenoxy)pentanoic acid 2a was obtained from TCI America
(Boston, MA).
The new compound 5-(2-methylphenoxy)pentanoic acid 2b
was prepared from 2-methylphenol and methyl 5-bromopentano-
ate by the method described by Sanders et al.50 1H NMR: (400 MHz,
CDCl3) 1.83–1.90 (m, 4H), 2.22 (s, 3H), 2.44–2.48 (t, 2H, J = 7.0 Hz),
3.96–3.99 (t, 2H, J = 5.8 Hz), 6.78–7.15 (m, 4H), 11 (br s, 1H). MS
(ESI): 231 [M+Na]+.
The new compound 5-(2,6-dimethylphenoxy)pentanoic acid 2c
was prepared in the same manner as 2b,50 except that 2,6-dimethyl-
phenol was used as the starting material. 1H NMR: (400 MHz,
CDCl3): 7.0–7.1 (m, 3H), 3.83 (t, 2H, J = 6 Hz), 2.37 (t, 2H, J = 6 Hz),
2.73 (s. 6H), 1.92 (m, 4H). MS (ESI): 223 [M+H]+; 245 [M+Na]+.
The new compound 5-(3,5-dimethylphenoxy)pentanoic acid
2d was prepared in the same manner as 2b,50 except that 3,5-
dimethylphenol was used as the starting material. 1H NMR (CDCl3,
400 MHz): 6.58 (s, 1H), 6.51 (s, 2H), 3.94 (t, 2H, J = 5.6 Hz), 2.44 (t,
2H, J = 7.2 Hz), 2.27 (s, 6H), 1.83 (m, 4H). MS (ESI): 245 [M+Na]+.
3-(2-Naphthoxy)propanoic acid 3 was prepared as described by
Bel and Duewel.51
The new compound 3-(1-methyl-1H-imidazol-2-ylthio)propa-
noic acid 4a was prepared as described for 5-(1-methyl-1H-imida-
zol-2-ylthio)pentanoic acid,52 except that 3-bromopropanoic acid
was used as the starting material. 1H NMR (400 MHz, CDCl3):
2.62–2.65 (t, 2H, J = 6.4 Hz), 3.13–3.16 (t, 2H, J = 6.4 Hz), 3.73 (s,
3H), 7.34–7.35 (d, 1H, J = 2.1 Hz), 7.38–7.39 (d, 1H, J = 2.1 Hz), 11
(br s, 1H). MS (ESI): 187 [M+H]+.
5-(1-Methyl-1H-imidazol-2-ylthio)pentanoic acid 4b was syn-
thesized as described by Tweit et al.52
The new compound 3-([2,6-dimethylphenoxy]methylthio)prop-
anoic acid 5 was prepared by reaction of 2-(chloromethoxy)-1,3-

1446
K. S. Roser et al. / Bioorg. Med. Chem. 18 (2010) 1441–1448
(t, 3H, J = 7.2 Hz), 2.17 (s, 6H), 4.13 (q, 2H, J = 7.2 Hz), 5.00 (d, 1H,
J = 12.4 Hz), 7.04 (m, 3H), 7.74 (d, 1H, J = 12.4 Hz], and 300 mg of
the ethyl (Z)-3-(2,6-dimethylphenoxy)acrylate [1H NMR
(400 MHz, CDCl3): 1.31 (t, 3H, J = 7.2 Hz), 2.24 (s, 6H), 4.23 (q,
2H, J = 7.2 Hz), 5.02 (d, 1H, J = 6.8 Hz), 6.46 (d, 1H, J = 6.8 Hz),
7.02 (m, 3H)]. (E)-3-(2,6-Dimethylphenoxy)acrylic acid 6a. Three
hundred milligrams of ethyl (E)-3-(2,6-dimethylphenoxy)acrylate
was combined with 0.5 g of KOH in 10 mL of a 20:80 mixture of
water/ethanol and heated at reflux for 2.5 h. The progress of the
reaction was monitored by thin-layer chromatography (silica gel,
hexane/ethyl acetate, 95:5). When the reaction had gone to com-
pletion, the reaction mixture was removed from heat and the eth-
anol was removed under reduced pressure. The reaction mixture
was acidified with 5% hydrochloric acid solution and extracted
with three 30-mL portions of ethyl acetate. The ethyl acetate solu-
tion was extracted with saturated sodium chloride solution, dried
with anhydrous sodium sulfate, and the solvent was removed un-
der reduced pressure. The solid that formed was titurated with
hexane and collected by vacuum filtration to afford 100 mg of
(E)-3-(2,6-dimethylphenoxy)acrylic acid 6a. NMR (400 MHz,
CDCl3): 2.17 (s, 6H), 5.00 (d, 1H, J = 12.4 Hz), 7.05 (s, 3H), 7.80 (d,
1H, J = 12.4 Hz), 11.0 (br s, 1H). MS (ESI): 215 [M+Na]+.
The new compound (E)-3-(2,6-diisopropylphenoxy)acrylic acid
6b was also prepared by the general method of Fan et al.:53 ethyl (E/
Z)-3-(2,6-diisopropylphenoxy)acrylate. 2,6-Diisopropylphenol (2.0 g,
11.3 mmol), 1,4-diazabicyclo[2.2.2]octane (122 mg, 1 mmol), and
10 mL of methylene chloride were combined with magnetic stir-
ring. Ethyl propiolate (1.20 mL, 116 mmol) in 4 mL of methylene
chloride was added drop wise over 10 min. The reaction mixture
was refluxed overnight and was then was diluted with 85 mL of
ethyl acetate and extracted with 10% potassium hydroxide solu-
tion, with 10% hydrochloric acid solution, and then with saturated
sodium chloride solution. The ethyl acetate layer was dried with
anhydrous magnesium sulfate and filtered. The solvent was re-
moved under reduced pressure to afford 2.72 g (87% yield) of a
mixture of E- and Z-isomers of the product. The isomeric mixture
(500 mg) was separated and purified by normal-phase chromatog-
raphy (CombiFlash) with a hexane/ethyl acetate gradient (95:5,
6 min; 80:20, 10 min) to afford 325 mg (65%) of ethyl (E)-3-(2,6-
diisopropylphenoxy)acrylate. 1H NMR (400 MHz, CDCl3): 1.17–
1.19 (d, 12H), 1.22–1.26 (t, 3H, J = 7.2), 3.00–3.07 (m, 2H), 4.11–
4.17 (q, 2H), 5.06–5.09 (d, 1H, J = 12.4 Hz), 7.14–7.22 (m, 3H),
7.74–7.77 (d, 1H, J = 12.4 Hz).
homogenized with a glass Dounce homogenizer with loose-fitting
pestle in liver mitochondria isolation medium that contained
0.25 M sucrose, 2.0 mM HEPES, 1.0 mM EGTA (pH 7.4) at 4 °C.
The homogenate was centrifuged once at 1000g for 3 min at 4 °C
to remove cellular debris. After removal of visible surface fat, the
resulting supernatant was centrifuged at 10,000g for 10 min at
4 °C. The supernatant was discarded after removing visible surface
fat, and the pellet was suspended in ice-cold liver mitochondria
isolation medium and centrifuged again at 10,000g for 10 min at
4 °C. The mitochondrial pellet thus obtained was suspended in
Ca2+-depletion buffer (1 mM EGTA, 10 mM NaCl, 5 mM succinate,
195 mM mannitol, 25 mM sucrose, 40 mM HEPES, pH 7.2) at room
temperature) and incubated with gentle stirring first at room tem-
perature for 10 min and then in an ice bath for 5 min. Ca2+-Deple-
tion buffer serves to chelate calcium, activate mitochondrial
respiration, and activate the Na+/Ca2+ exchanger. The mitochon-
drial suspension was then centrifuged at 10,000g for 10 min at
4 °C, and the pellet was suspended in liver swelling buffer that con-
tained 195 mM mannitol, 25 mM sucrose, 40 mM HEPES (pH 7.2)
at 4 °C; liver swelling buffer allows for ionic/osmotic equilibrium
through the remainder of the preparation. The suspension was
centrifuged at 10,000g for 10 min at 4 °C, followed by the removal
of light mitochondria and a final centrifugation at 10,000g for
10 min at 4 °C. The mitochondrial pellet was suspended in liver
swelling buffer at 4 °C. Protein concentrations were determined
with the Lowry reagent.56
The quality of mitochondrial preparations was determined by
estimating the respiration control ratio (RCR, i.e., the ratio of state
3 vs state 4 respiration rates) with a Clark-type oxygen electrode
(Oxygraph, Hansatech Instruments, Norfolk, England). The incuba-
tion mixtures contained 120 mM KCl, 25 mM sucrose, 5 mM MgCl2,
5 mM KH2PO4, 1 mM EGTA, 10 mM HEPES, 10 mM succinate,
2.5 mg/mL fat-free bovine serum albumin, 0.5–0.8 mg/mL mito-
chondrial protein, 5 lM rotenone, and 100 lM ADP (pH 7.4) and
were incubated at 37 °C. RCR ranged from 4.5 to 6.5.
Reaction mixtures were prepared in disposable glass culture
tubes and contained 20 mM HEPES, 1 mM EGTA, 100 mM KCl,
5 mM KH2PO4, 10 mM MgCl2, 25 mM sucrose, 2 mM L-carnitine
(if added), 5 mM ATP, 10 mg/mL bovine serum albumin, 1.4 mM
dithiothreitol, 0.13 mM CoA, and 1 mM substrate (pH 7.4). Reac-
tion mixtures were incubated at 37 °C with gentle stirring. Mito-
chondrial protein (approximately 5 mg/mL) was added, and the
samples were incubated for 5 min with gentle mixing, which was
maintained during the incubation period, to keep the homogenate
suspended. After 5 min of incubation, the substrates were added.
The substrates were dissolved in methanol and added to the
reaction mixtures to a final concentration of 1 mM, unless other-
wise stated; the final methanol concentration was ꢃ0.5%.
(E)-3-(2,6-Diisopropylphenoxy)acrylic acid 6b. Ethyl (E)-3-(2,6-
diisopropylphenoxy)acrylate (250 mg, 0.91 mmol) was combined
with 0.5 g of solid KOH in 10 mL of a mixture of water/ethanol
(20:80), and the mixture was refluxed overnight. The solvent was
removed under reduced pressure. The residue was acidified with
5% HCl solution and then extracted with three 25 mL portions of
ethyl acetate. The ethyl acetate solution was extracted with satu-
rated sodium chloride solution and dried with anhydrous sodium
sulfate. The solvent was removed under reduced pressure to yield
150 mg (60%) of (E)-3-(2,6-diisopropylphenoxy)acrylic acid 6b. 1H
NMR (400 MHz, CDCl3) 1.17–1.19 (d, 12H), 2.98–3.05 (m, 2H),
5.05–5.08 (d, 1H, J = 12.4 Hz), 7.14–7.22 (m, 3H), 7.81–7.85 (d,
1H, J = 12.4 Hz). MS (ESI): 271 [M+Na]+.
x
-(1-Methyl-1H-imidazol-2-ylthio)alkanoic acids 4a and 4b were
dissolved in distilled water and added to the reaction mixtures to
a final concentration of 1 mM. Samples were incubated at 37 °C
for 30 min with constant gentle mixing, and samples were col-
lected for analysis at t = 0, 10, 20, and 30 min.
5.4. Analyses
Phenolic metabolite formation, that is, phenol, 2-methylphenol,
2,6-dimethylphenol, 3,5-dimethylphenol, and 2,6-diisopropylphe-
5.3. Mitochondrial biotransformation of alkanoic-acid-based
prodrugs
nol, from
ysis. Two hundred microliters of the reaction mixture was added to
200 L of acetonitrile; the mixtures were mixed and then soni-
x-(phenoxy)alkanoic acids, was quantified by HPLC anal-
Rat liver mitochondria were isolated from livers freshly col-
lected from adult male Sprague-Dawley rats (190–225 g) and were
used as soon as possible after isolation.54,55 The animal-use proto-
col was reviewed and approved by the University of Rochester Uni-
versity Committee on Animal Resources. Livers were removed
immediately after CO2 euthanasia of the animal, minced, and
l
cated in a Branson ultrasonic water bath (Danbury, CT) for 5–
10 min. The denatured sample mixtures were centrifuged at
15,000–16,000g for 5 min. Supernatants were analyzed with an
Agilent 1100 series HPLC (Santa Clara, CA) with UV detection at
265 and 272 nm. A 5 lL sample of the supernatant was injected

K. S. Roser et al. / Bioorg. Med. Chem. 18 (2010) 1441–1448
1447
onto
a
Phenomenex Synergi Hydro-RP analytical column
m particle size, 80 Å pore, 30 °C). The col-
Acknowledgements
(150 mm  2.0 mm, 4
l
umn was eluted at a flow rate of 0.4 mL/min with 0.1% (v/v) formic
acid (solvent A) and acetonitrile (solvent B) with this gradient: 15%
B for 5 min, increased to 95% B over 20 min, held at 95% B for
10 min, and returned to 15% B in 2 min. The column was equili-
brated with 15% B for 10 min before the next sample was analyzed.
The authors thank James L. Robotham for support of this re-
search and Horst Schulz for helpful discussions.
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x-(1-methyl-1H-imidazol-2-
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x-(1-
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