Effects of cytoprotective antioxidants on lymphocytes from representative mitochondrial neurodegenerative diseases

Article abstract of DOI:10.1016/j.bmc.2012.11.051

Two new aza analogues of the neuroprotective agent idebenone have been synthesized and characterized. Their antioxidant activity, and ability to augment ATP levels have been evaluated in several different cell lines having suboptimal mitochondrial function. Both compounds were found to be good ROS scavengers, and to protect the cells from oxidative stress induced by glutathione depletion. The compounds were more effective than idebenone in neurodegenerative disease cells. These novel pyrimidinol derivatives were also shown to augment ATP levels in coenzyme Q₁₀-deficient human lymphocytes. The more lipophilic side chains attached to the pyrimidinol redox core in these compounds resulted in less inhibition of the electron transport chain and improved antioxidant activity.

Full text of DOI:10.1016/j.bmc.2012.11.051

Bioorganic & Medicinal Chemistry 21 (2013) 969–978
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Effects of cytoprotective antioxidants on lymphocytes from
representative mitochondrial neurodegenerative diseases
Ruth Goldschmidt, Pablo M. Arce, Omar M. Khdour, Valérie C. Collin, Sriloy Dey, Jennifer Jaruvangsanti,
⇑
David M. Fash, Sidney M. Hecht
Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new aza analogues of the neuroprotective agent idebenone have been synthesized and character-
ized. Their antioxidant activity, and ability to augment ATP levels have been evaluated in several different
cell lines having suboptimal mitochondrial function. Both compounds were found to be good ROS scav-
engers, and to protect the cells from oxidative stress induced by glutathione depletion. The compounds
were more effective than idebenone in neurodegenerative disease cells. These novel pyrimidinol deriva-
tives were also shown to augment ATP levels in coenzyme Q10-deficient human lymphocytes. The more
lipophilic side chains attached to the pyrimidinol redox core in these compounds resulted in less inhibi-
tion of the electron transport chain and improved antioxidant activity.
Received 11 October 2012
Revised 27 November 2012
Accepted 30 November 2012
Available online 12 December 2012
Keywords:
Adenosine triphosphate
Mitochondrial dysfunction
Antioxidants
Ó 2012 Elsevier Ltd. All rights reserved.
Reactive oxygen species
Electron transport chain
1. Introduction
Leigh’s syndrome, for instance, is a neurometabolic disorder
affecting the central nervous system. The most common causes of
Mitochondria perform numerous tasks in cells such as partici-
pating in controlling the cell cycle,1 neuronal cell signaling,2 apop-
tosis,1,3 and monitoring cell differentiation,4 growth and
development.5 Crucially, the mitochondria are the site of most cel-
lular energy production through the conversion of adenosine 50-
diphosphate (ADP) to adenosine 50-triphosphate (ATP).5–7 ATP is
produced as a consequence of electron flow through the electron
transport chain (ETC), which is located in the inner mitochondrial
membrane. The ETC consists of four protein complexes: NADH:ubi-
quinone oxidoreductase (complex I), succinate:ubiquinone oxido-
reductase (complex II), cytochrome bc1 complex (complex III),
and cytochrome c oxidase (complex IV). The flow of electrons
through these complexes results in the transport of protons from
the inner mitochondrial membrane into the intermembrane space.
These protons are used by a fifth complex, ATP synthase (complex
V), to convert ADP to ATP.8,9 Defects in any of the protein com-
plexes can cause mitochondrial disorders, including hereditary
mitochondrial cytopathies, and neurodegenerative diseases.10,11
Such diseases may be caused either by mutations, acquired or
inherited, in mitochondrial DNA (mtDNA) or in nuclear genes that
code for proteins required for proper mitochondrial function.12–14
Mitochondrial dysfunction can also result from the adverse effects
of drugs, infections, or other environmental causes.15,16
Leigh’s syndrome are either mutation of the SURF1 gene in nuclear
DNA,17,18 or mutation of the MT-ATP6 gene in mitochondrial
DNA.19,20 SURF1 reduces the formation of the normal cytochrome c
oxidase complex, while MT-ATP6 mutation damages the ATP syn-
thase protein complex and blocks ATP generation, both of which im-
pair mitochondrial energy production.21,22 Defects in the ETC are
believed to be accompanied by an impairment of electron flow
through the different mitochondrial complexes, causing increased
electron leakage, and increased production of reactive oxygen species
(ROS).23–25 Excessive amounts of ROS can damage DNA,26 RNA,27 lipid
membranes28 and proteins,29 and are likely to contribute to disease
progression.27,30 Accordingly, agents able to augment ATP production
and decrease redox stress should be highly beneficial.
Idebenone (CatenaÒ), an analogue of coenzyme Q10, is currently
undergoing clinical evaluation for the treatment of mitochondrial
and neurodegenerative diseases;31–33 it is approved provisionally
in Canada for the treatment of Friedreich’s ataxia.34 It has been
proposed that its neuroprotective effects can be attributed, at least
in part, to its ability to function as an antioxidant, involving redox
cycling between hydroquinone and quinone.35 However, biochem-
ical studies have indicated that idebenone is a significant inhibitor
of complex I of the mitochondrial respiratory chain,36–38 and thus
could potentially stimulate ROS production in mitochondria if used
at high doses.36–39
Recently, we described an aza analogue of idebenone
(compound 1, Fig. 1), having the same side chain as idebenone
⇑
Corresponding author. Tel.: +1 480 965 6625; fax: +1 480 965 0038.
E-mail address: sidney.hecht@asu.edu (S.M. Hecht).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.051

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R. Goldschmidt et al. / Bioorg. Med. Chem. 21 (2013) 969–978
but a different redox core.40 Compound 1 was more effective than
idebenone in suppressing ROS generation and lipid peroxidation,
following the induction of oxidative stress in cultured cells. The
compound was also effective in maintaining mitochondrial mem-
brane potential and cell viability following glutathione depletion.
Finally, the compound was found to be effective at rather low con-
centrations, consistent with the suggestion of catalytic function.
Presently, we describe the preparation and characterization of
two new pyrimidinol derivatives in protecting several different cell
lines from the effects of oxidative stress. These compounds, 2 and
3, have the same redox core as 1, but have modified side chains
lacking the hydroxyl group present in 1 (Fig. 1). By the use of cul-
tured cell lines derived from individuals with Leber’s disease,
Friedreich’s ataxia, Alzheimers disease, Leigh’s syndrome and Par-
kinson’s disease, the effects of the new compounds on ROS sup-
pression, cell viability and ATP levels have been studied. In
addition, the potential as have the potential inhibitory effects of
the compounds on ETC complexes I, III and IV have been evaluated.
2.2. Biochemical and biological evaluation of the pyrimidinol
analogues
2.2.1. Suppression of reactive oxygen species
The ability of the pyrimidinol analogues to suppress ROS in-
duced by depletion of glutathione with diethyl maleate (DEM)
was evaluated in three different cell lines (CEM leukemia cells, Alz-
heimer’s lymphocytes and Leber’s lymphocytes). ROS were mea-
sured
in
a
quantitative
FACS
experiment,
using
dichlorodihydrofluorescein diacetate (DCFH-DA) as a substrate for
determining intracellular oxidant production. DCFH-DA is hydro-
lyzed by esterases to afford dichlorodihydrofluorescein (DCFH),
the latter of which is trapped within the cell. This non-fluorescent
molecule is then oxidized to afford fluorescent dichlorofluorescein
(DCF) by the action of cellular oxidants. The results obtained in CEM
leukemia cells are presented in Figure 2, and show that analogues 1,
2, 3, and decylubiquinone were all more effective than idebenone in
suppressing ROS, and did so in a concentration dependent manner.
Compounds 2 and 3 were the most potent, reducing ROS levels to
the same as controls not treated with DEM, when employed at
2. Results
0.25 and 2.5
none suppressed ROS completely when used at 2.5
tion, but were only partially effective at 0.25 M concentration.
l
M concentrations. Compounds 1 and decylubiqui-
l
M concentra-
2.1. Design and synthesis of pyrimidinol analogues
l
Similar results were obtained using Alzheimer’s and Leber’s lym-
phocytes (Supplementary data Figs. 1 and 2), although the basal le-
vel of ROS in these cells was found to be elevated even in the
absence of DEM treatment. Because the use of DCF fluorescence
as an endpoint has been reported to lack specificity under certain
circumstances,41,42 controls were run to determine whether the re-
sults obtained were reliable. Accordingly, CEM leukemia cells that
had been treated with DCF and DEM were additionally treated with
superoxide dismutase + catalase, or with 5 mM N-acetylcyste-
ine.40,43 As shown in Figure 3 of the Supplementary data, these
treatments reversed the increase in DCF fluorescence induced by
5 mM DEM treatment, suggesting that an increase in ROS was
responsible for the observed increase in DCF fluorescence.
Compounds 2 and 3 were synthesized in analogy with a previ-
ously reported procedure.40 First, 2-amino-4,6-dimethylpyrimi-
dine (4) was prepared in 95% yield by treating guanidine sulfate
with 2,4-pentanedione (Scheme 1). The pyrimidine ring was bro-
minated with N-bromosuccinimide in 83% yield. The exocyclic
amine was then protected as the respective 2,5-dimethyl-1H-pyr-
role derivative (6) by treatment with 2,5-hexanedione (81% yield).
Bromide 6 was then converted to the corresponding pyrimidinol
using KOH, tris(dibenzylideneacetone)palladium(0) (Pd2(dba)3)
and di-tert-butylphosphino-20,40,60-triisopropylbiphenyl (L1), fol-
lowed by O-benzylation with NaH and BnBr in 76% overall yield
for the two steps. Intermediate 7 was then converted to the two
desired products 2 and 3. The fully protected pyrimidinol was
monoalkylated on one of the methyl groups by generating the
carbanion with n-BuLi followed by treatment with nonyl bromide
and pentadecyl bromide to afford 8 and 9 in 53% and 24% yields,
respectively. The exocyclic amines were then deprotected by treat-
ment with hydroxylamine hydrochloride to afford 10 and 11 in
75% and 100% yields, respectively. The exocyclic amines were then
dimethylated using formalin and sodium cyanoborohydride to af-
ford 12 and 13 in 46% and 41% yields, respectively. Finally com-
pounds 2 and 3 were obtained quantitatively by hydrogenolysis
over Pd(OH)2/C.
2.2.2. Cellular ATP levels
Total cellular ATP was measured in a CoQ10-deficient human
lymphocyte cell line (GM17932). The cells were grown on glu-
cose-free media supplemented with galactose for two weeks to
maximize ATP production via oxidative phosphorylation. As shown
in Figure 4 of the Supplementary data, the CoQ10-deficient lympho-
cytes had about 20% less ATP than a normal human lymphocyte
cell line (GM15851). The CoQ10-deficient human lymphocytes
(GM17932) were verified to have a lower level of CoQ10 than
O
O
CH O
3
CH O
3
OH
CH O
3
CH O
3
O
O
idebenone
decylubiquinone
OH
OH
OH
N
N
N
N
N
N
1
2
OH
N
N
N
3
Figure 1. Chemical structures of compounds 1, 2, 3, decylubiquinone and idebenone.

R. Goldschmidt et al. / Bioorg. Med. Chem. 21 (2013) 969–978
971
O
Br
O
O
NH
NH2
·1/2 H2SO4
O
NBS
p-toluenesulfonic acid
H2N
N
N
N
N
acetonitrile
83%
toluene, reflux
81%
Na2CO3, H2O
95%
NH2
NH2
4
5
Br
OBn
OBn
1. KOH, Pd2(dba)3, L1
1:1 degased H2O/dioxane
HONH2-HCl
1. n-BuLi, anh THF
n
N
N
N
N
N
N
N
N
aq ethanol,
reflux
2. NaH, BnBr
anh. THF
76%
2. Br
N
n
8, n = 8 (53%)
9, n = 14 (24%)
6
7
OH
OBn
OBn
H2, Pd(OH)2/C
methanol
formalin, NaBH3CN
n
n
n
N
N
N
N
N
N
methanol
N
NH2
N
10, n = 8 (75%)
12, n = 8
2, n = 8
3, n =14
(46%)
(100%)
11, n = 14 (100%)
13, n = 14
(41%)
(100%)
O
P(t-Bu)2
i-Pr
i-Pr
L1
=
Pd2(dba)3 =
Pd2
3
i-Pr
Scheme 1.
normal lymphocytes (GM15851) by 18% (80.4
5
and
was studied. The inhibitory effects of the test compounds on bo-
vine heart mitochondrial complex I alone (NADH:ubiquinone oxi-
doreductase), or complexes I, III and IV together (NADH oxidase)
were evaluated using submitochondrial particles (SMP). The re-
sults, presented in Tables 2 and 3, show that compounds 1 and 2
were as inhibitory to respiratory chain function as idebenone.
Decylubiquinone was much less inhibitory to ETC function, as
was compound 3. Thus, the reduction in ATP levels in Table 1 likely
reflects the ability of the test compounds to inhibit one or more of
the complexes in the mitochondrial electron transport chain.
98.1 7 pmol/mg of protein (p = 0.02 compared with normal)).
Also studied were the effects of idebenone, decylubiquinone
and compounds 1–3 on cellular ATP levels. All the cells were
adapted to growth on galactose for at least one week prior to mea-
suring ATP levels. As shown in Table 1, idebenone strongly dimin-
ished ATP levels in a concentration dependent fashion in the CoQ10
deficient lymphocytes, as well as in Leber’s lymphocytes and
Friedreich’s ataxia lymphocytes. In the presence of 25 lM idebe-
none, only residual ATP concentrations were detected. Decylubiq-
uinone and compound 1 also produced somewhat lower ATP
concentrations, especially at the highest concentration studied,
although neither reduced ATP concentrations nearly as strongly
2.2.4. Cytoprotection
Cytoprotection was measured using six different lymphocyte
cell lines, including five from individuals with mitochondrial or
neurodegenerative diseases. The cultured cells were preincubated
with the test compounds for 15 h, and treated with diethyl maleate
to induce oxidative stress through depletion of glutathione. The ex-
as idebenone. When used at 5 lM concentration, compound 2 re-
sulted in a slight increase in ATP concentrations in the CoQ10 defi-
cient lymphocytes, and had essentially no effect on the ATP level in
Leber’s lymphocytes. This pyrimidinol analogue suppressed ATP
levels strongly only when used at 25
lM concentration in Leber’s
tent of cytoprotection afforded by 0.5 lM idebenone, decylubiqui-
and Friedreich’s ataxia lymphocytes. Finally, compound 3 in-
creased ATP levels in the CoQ10 deficient lymphocytes when used
at 5 lM concentration, and lowered ATP levels minimally at any
of the tested concentrations in the three cell lines studied.
none, compound 2 and compound 3 is shown in Figure 3. It is clear
from the data that compounds 2 and 3, in addition to decylubiqui-
none, conferred very efficient cytoprotection in all of the cell lines,
as has already been reported for compound 1.40 This was in sharp
contrast to idebenone, which failed to confer complete protection
to any of the cell lines. As might have been expected, idebenone
2.2.3. Mitochondrial electron transport chain function
In order to understand the basis for the diminished levels of ATP
noted in Table 1, the effects of the test compounds on ETC function
was more effective when employed at 2.5
plementary data, Fig. 5).
lM concentration (Sup-

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R. Goldschmidt et al. / Bioorg. Med. Chem. 21 (2013) 969–978
Figure 2. Flow cytometric analysis of CCRF CEM leukemia cells stained with dichlorofluorescein diacetate (DCFH-DA) for 25 min, following pretreatment with the test
compounds at 2.5, 0.25 and 0.025 M concentrations for 18 h, and subsequent treatment with diethyl maleate (DEM) for 1 h to induce the production of ROS. Data shown
l
represent the mean S.E.M. of three different experiments run as duplicates.
Table 1
Total ATP concentration in CoQ10 deficient cells, Leber’s disease and Friedreich’s ataxia lymphocytes, upon incubation with the treated compounds for 48 h
Compound
Total ATP concentration (% of control)
Leber’s lymphocytes
CoQ10 deficient lymphocytes
10 25
100 100
Friedreich’s ataxia lymphocytes
10 25
100 100
5
l
M
lM
lM
5
lM
10
lM
25
l
M
5
l
M
l
M
lM
Untreated control
100
100
100
100
100
Idebenone
Decylubiquinone
1
2
3
79.8 2.9
100.0 5.6
98.8 3.3
105.2 1.4
108.2 3.1
45.7 6.6
86.2 7.0
86.1 1.6
97.1 1.4
103.0 3.4
1.6 0.5
55.7 0.7
75.1 1.0
75.5 4.2
95.6 5.8
102.7 4.3
29.9 5.9
63.4 3.4
45.6 4.7
75.0 6.4
95.6 2.3
3.0 1.0
73.0 6.6
92.8 3.5
77.2 5.1
83.3 6.8
95.3 5.8
45.6 3.7
76.4 1.5
46.3 4.2
74.4 2.6
90.3 3.4
2.6 0.8
71.1 6.9
46.4 3.5
72.4 2.6
95.0 5.8
47.0 3.0
20.7 1.5
40.6 4.4
83.4 4.1
62.7 6.4
27.7 2.3
42.2 3.5
74.7 7.1
Table 2
also in reducing complex III.44 The latter property suggests the po-
tential utility of such compounds in augmenting ATP production in
cells having partially dysfunctional mitochondria. However, the re-
ports that idebenone inhibits complex I at fairly modest concentra-
tions36–38 suggested the need for analogues lacking this
unfavorable property.
The inhibitory effect of compounds 1, 2, 3, idebenone, and decylubiquinone on bovine
heart mitochondrial NADH oxidase activity (complexes I, III and IV)
Compound
NADH oxidase activitya (%)
1
l
M
5
l
M
10 lM
Idebenone
Decylubiquinone
1
2
3
57.4 4.6
93.0 6.9
50.0 5.0
35.4 1.4
96.2 2.6
19.3 2.7
82.4 2.2
22.0 4.7
17.7 1.1
88.6 5.0
13.2 1.1
61.5 6.6
15.0 3.2
3.9 0.3
Decylubiquinone is closely related to idebenone, differing in
structure only in that it lacks the side chain OH group present in
idebenone. The extensive use of decylubiquinone experimentally
as a coenzyme Q surrogate suggested that it might be informative
to characterize the properties of decylubiquinone in several assays
that measure different facets of mitochondrial function. In fact, as
shown in Tables 1–3, decylubiquinone was found to lack signifi-
cant inhibition of the mitochondrial ETC, but to retain the ROS sup-
pressive properties noted for idebenone (Fig. 2), and to have
cytoprotective effects on several cultured cell lines superior to that
of idebenone (Fig. 3). Accordingly, we prepared two pyrimidinol
analogues of compound 140 based on our results with decylubiqui-
none and idebenone. These new derivatives 2 and 3 lack the side
chain OH group present in (idebenone and) 1. Compound 2 has
the same side chain as decylubiquinone while compound 3 has a
longer (16-carbon) side chain. Both compounds were synthesized
using a strategy similar to that used for the preparation of com-
pound 1 (Scheme 1).40 The biochemical effects of 2 and 3 on mito-
chondria and their ability to suppress ROS in cells have been tested
and compared to those of idebenone and decylubiquinone. Surpris-
ingly, the removal of the OH group from compound 1 did not
diminish the inhibition of the ETC complexes by the new deriva-
tive. Compound 2 was as inhibitory to mitochondrial complex I
76.9 4.4
a
Relative to an untreated control.
Table 3
The inhibitory effect of compounds 1, 2, 3, idebenone, and decylubiquinone on bovine
heart mitochondrial complex I
a
Compound
IC50
(l
M)
Imax (%)
KI (lM)
Idebenone
Decylubiquinone
1
2
3
6
>200
1
2
3
84
N/A
85
88
84
2
3
2
1
4
2
>200
0.7
1.4 0.7
141
0
1
0
198 12
9
a
Maximal inhibition achievable.
3. Discussion
Idebenone has been studied extensively over a period of years,
and has been found to be effective both as an antioxidant37 and

R. Goldschmidt et al. / Bioorg. Med. Chem. 21 (2013) 969–978
973
Leber's disease
Friedreich's ataxia
Alzheimer's disease
Leigh's syndrome
Parkinson's disease
CEM leukemia
Figure 3. Cell viability of Leber’s disease lymphocytes, Friedreich’s ataxia lymphocytes, Alzheimer’s disease lymphocytes, Leigh’s syndrome lymphocytes, Parkinson’s disease
lymphocytes and CCRF CEM leukemia lymphocytes following pretreatment with compounds 1, 2, 3, decylubiquinone and idebenone at 0.5 lM concentration for 15 h and
then treatment with 5 mM DEM for 6 h. Data shown represent the average S.E.M. of three different experiments run as triplicates.
as compound 1 and slightly more inhibitory to NADH oxidase
activity (Tables 2 and 3). However, compound 3, having a longer
side chain, was dramatically improved in this regard, having an
bearing a mitochondrial DNA mutation in the NADH dehydroge-
nase subunit 4 gene (MTND4), in which the arginine at amino acid
position 340 is replaced by a histidine (R340H). This mutation ac-
counts for ꢀ50% of LHON cases among Caucasians and >90% of the
cases among Asians.45 The second cell line was Alzheimer’s disease
(familial, type 3) lymphocytes. The cells carry a missense mutation
[Ala246Glu (A246E)] in the AD3 [presenilin 1 (PSEN1)] gene. This
mutation has been linked to early onset Alzheimer’s disease by
several investigators.46–48 The third cell line involved the use of
Parkinson’s disease (familial, type 1) lymphocytes, identified with
whole gene triplication, including amplification of SNCA exons.
This was suggested as the cause of Parkinson’s disease in a family
from which the cells were derived, formerly classified as PARK.49,50
Another cell line employed was Leigh’s syndrome lymphocytes,
bearing an homoplasmic mutation of ATPase 6 (MTATP6). Holt
et al. found a heteroplasmic T to G transversion at nucleotide pair
8993 in a maternal pedigree which resulted in the change of a
hydrophobic leucine to a hydrophilic arginine at position 156 in
subunit 6 of mitochondrial H(+)-ATPase.51 The insertion of an argi-
nine in the hydrophobic sequence of ATPase 6 probably interferes
with the hydrogen ion channel formed by subunits 6 and 9 of the
ATPase, thus causing failure of ATP synthesis. Tatuch et al. and
Shoffner et al. demonstrated that the nucleotide 8993 mutation
can cause Leigh’s disease.52,53 Also studied were Friedreich’s ataxia
lymphocytes having an identified mutation (GAA)n expansion. GAA
triplet repeat expansions between 200 and 900 copies in the first
intron of the frataxin gene occurred in 71 out of 74 FRDA patients
studied by Campuzano et al.54
IC50 value of about 200 lM for complex I inhibition, and exhibiting
only modest inhibition of NADH oxidase activity. This was fully
consistent with the observation that compound 3 was also better
than 2 in preserving ATP levels at all tested concentrations in the
three cell lines studied (Table 1). Nonetheless, compound 2 was
superior both to idebenone and compound 1, both in maintenance
of ATP levels and ROS suppression (Table 1 and Fig. 2). Importantly,
compound 3 actually increased total ATP above the basal level,
when used at low doses in CoQ10-deficient lymphocytes, and did
not materially affect ATP levels in the other cell lines at 5 and
10 lM concentrations. This makes compound 3 the best analogue
identified to date, exhibiting little inhibition of the ETC, and an
ability to augment ATP production. The results in Table 1 for the ef-
fects of 3 on CoQ10 deficient lymphocytes are fully consistent with
the belief that 3 can reduce complex III within the ETC. The basis
for the lack of inhibitory effects noted for compound 3 on complex
I function are currently not well understood. Nonetheless, the fact
that it is possible to identify a CoQ10 analogue that functions in
complex III without inhibiting complex I is significant and suggests
the promise of the current strategy in identifying CoQ10 analogues
of potential utility for therapeutic intervention.
As noted in Figure 2, as well as Figures 1 and 2 of Supplemen-
tary data, compounds 2 and 3 were more efficient in suppressing
ROS than the other compounds tested, implying that the structural
changes to the side chain of 1 did not decrease the antioxidant
properties of these pyrimidinols.40
Finally, we have studied the ability of these compounds to pro-
tect a variety of mitochondrial disease cells from the cytotoxic ef-
fects of diethyl maleate (DEM), a depletor of glutathione that
exposes the cells to oxidative stress. DEM was utilized because it
induces oxidative stress at least as great as that likely to be
encountered physiologically, permitting the identification of com-
pounds anticipated to function well under pathophysiological
conditions.
As is clear from Figure 3, compounds 2 and 3 both exhibited
good cytoprotective activity in all of the cell lines, even when used
at the low concentration of 0.5 lM. Their cytoprotective activity
was clearly greater than that of idebenone, and slightly better than
decylubiquinone in some of the disease cells (e.g., CEM leukemia
and Parkinson’s disease lymphocytes).
4. Conclusions
Two new pyrimidinol derivatives, 2 and 3, have been synthe-
sized and characterized biochemically. They both exhibited im-
proved antioxidant activity in comparison with 1, and the ability
For this analysis, we used several cell lines derived from pa-
tients with mitochondrial neurodegeneratic diseases. These in-
cluded Leber’s hereditary optic neuropathy (LHON) lymphocytes

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R. Goldschmidt et al. / Bioorg. Med. Chem. 21 (2013) 969–978
to maintain ATP levels in cellular mitochondrial disease models. In
addition, compound 3 was shown to have a much lower inhibitory
effect on ETC function than the other compounds tested. These
findings suggest the importance of side chain optimization to
achieve the best compounds suitable for potential therapeutic
use. This is in agreement with the work of Fato et al.36 who con-
cluded that hydrophilic quinones may enhance oxidative stress
by interaction with a different site on complex I than the physio-
logical quinone reducing site used by more hydrophobic quinones.
It is interesting that side chains optimal for CoQ10 analogues
having a benzoquinone redox core were not optimal for the pyri-
midinol analogues of primary interest in the current study. This
observation indicates that interaction with the mitochondrial com-
plexes of the respiratory chain must involve recognition both of the
redox core and the side chain of individual analogues, and suggests
that recognition of individual types of redox cores may differ
qualitatively.
183–185 °C; silica gel TLC Rf 0.15 (2:1 ethyl acetate/hexanes); 1H
NMR (CDCl3) d 2.44 (s, 6H) and 5.19 (br s, 2H); 13C NMR (CDCl3)
d
24.7, 109.6, 160.7 and 166.3; mass spectrum (APCI), m/z
201.9982 (M+H)+ (C6H9N3Br requires 201.9980).
5.1.3. 5-Bromo-2-(2,5-dimethyl-1H-pyrrol-1-yl)-4,6-
dimethylpyrimidine (6)
To a stirred solution containing 2.00 g (9.89 mmol) of 2-amino-
5-bromo-4,6-dimethylpyrimidine (5) in 16 mL of toluene was
added 1.36 mL (1.32 g; 11.5 mmol) of 2,5-hexanedione followed
by 96 mg (0.50 mmol) of p-toluenesulfonic acid. The reaction mix-
ture was heated and stirred at reflux for 12 h. The reaction mixture
was poured into 150 mL of water and then extracted with 200 mL
of ethyl acetate. The organic solution was washed with 150 mL of
brine, dried (MgSO4) and concentrated under diminished pressure.
The residue was purified by chromatography on a silica gel column
(15 ꢁ 5 cm). Elution with 5:1 hexanes/ethyl acetate afforded 6 as
light yellow crystals: yield 2.23 g (81%); mp 64–65 °C; silica gel
TLC Rf 0.65 (6:1 hexanes/ethyl acetate); 1H NMR (CDCl3) d 2.34
(s, 6H), 2.67 (s, 6H) and 5.89 (s, 2H); 13C NMR (CDCl3) d 14.5,
24.9, 108.7, 118.6, 129.5, 155.3 and 166.9; mass spectrum (APCI),
m/z 280.0458 (M+H)+ (C12H15N3Br requires 280.0449).
5. Experimental section
5.1. Chemistry
1H NMR spectra were recorded on a Varian Inova 400 MHz,
using chloroform-d or methanol-d4 as solvents. 1H NMR chemical
shifts were reported relative to residual chloroform at 7.24 ppm
or to residual methanol at 3.31 ppm. 13C NMR chemical shifts were
reported relative to residual chloroform at 77.1 ppm or to residual
methanol at 49.0 ppm. All solvents were of analytical grade and
were used without further purification. All chemicals were pur-
chased from Sigma–Aldrich Chemical Co. and were used without
further purification. Reactions were carried out under an argon
atmosphere. Column chromatography was carried out using silica
gel (60 Å particle size, 230–240 mesh, Silicycle). Analytical thin
layer chromatography separations were carried out on glass plates
coated with silica gel (60 Å particle size, F-254, E. Merck). The TLC
chromatograms were visualized by UV irradiation or by immersing
the plates in 2.5% potassium permanganate in ethanol, or 2% anis-
aldehyde + 5% sulfuric acid + 1.5% glacial acetic acid in ethanol,
both applications being followed by heating of the TLC plates.
Melting points were recorded on a MelTemp apparatus and are
uncorrected. High resolution mass spectra were obtained in the
Arizona State University CLAS High Resolution Mass Spectrometry
Laboratory.
5.1.4. 5-Benzyloxy-2-(2,5-dimethyl-1H-pyrrol-1-yl)-4,6-
dimethylpyrimidine (7)
To a stirred solution containing 4.87 g (17.4 mmol) of 5-bromo-
2-(2,5-dimethyl-1H-pyrrol-1-yl)-4,6-dimethylpyrimidine (6) in
50 mL of 1:1 dioxane/degassed water was added 632 mg
(0.69 mmol) of Pd2(dba)3 followed by 293 mg (0.69 mmol) of 2-
di-tert-butylphosphino-20,40,60-triisopropylbiphenyl
(L1)
and
2.92 g (52.1 mmol) of KOH. The reaction mixture was stirred at
100 °C for 3 h. The cooled reaction mixture was poured into
200 mL of water and extracted with 100 mL of ethyl acetate. The
aqueous layer was acidified with HCl (pH 2–3) and then extracted
with two 150-mL portions of ethyl acetate. The combined organic
layer was washed with 150 mL of brine, dried (MgSO4) and concen-
trated under diminished pressure. The residue was dissolved in
50 mL of anhyd THF and treated with 3.10 mL (4.46 g; 26.0 mmol)
of benzyl bromide followed by 1.40 g (34.8 mmol) of a 60% suspen-
sion of NaH in mineral oil. The reaction mixture was stirred at
23 °C for 48 h. The reaction mixture was quenched with satd aq so-
dium bicarbonate, poured into 150 mL of water and extracted with
two 150-mL portions of ether. The combined organic layer was
washed with 150 mL of brine, dried (MgSO4) and concentrated un-
der diminished pressure. The residue was purified by chromatogra-
phy on a silica gel column (20 ꢁ 6 cm). Elution with 9:1 hexanes/
ethyl acetate afforded 7 as a light yellow oil: yield 4.09 g (76%); sil-
ica gel TLC Rf 0.6 (6:1 hexanes/ethyl acetate); 1H NMR (CDCl3) d
2.29 (s, 6H), 2.47 (s, 6H), 4.92 (s, 2H), 5.86 (s, 2H) and 7.42 m,
5H); 13C NMR (CDCl3) d 14.1, 19.1, 75.3, 107.9, 128.3, 128.6,
129.0, 129.2, 136.0, 147.7, 152.3 and 161.6; mass spectrum (APCI),
m/z 307.1675 (M)+ (C19H21N3O requires 307.1685).
5.1.1. 2-Amino-4,6-dimethylpyrimidine (4)
To a stirred solution containing 4.00 g (37.0 mmol) of guanidine
sulfate and 8.40 g (79.3 mmol) of sodium carbonate in 25 mL of
water was added 6.00 mL (5.88 g; 58.1 mmol) of 2,4-pentanedione.
The reaction mixture was stirred at 100 °C for 24 h. The cooled
reaction mixture was poured into 150 mL of water and then ex-
tracted with two 150-mL portions of dichloromethane. The com-
bined organic phase was washed with 150 mL of brine, dried
(MgSO4) and then concentrated under diminished pressure to af-
ford 4 as a colorless solid: yield 4.31 g (95%); mp 152–153 °C; silica
gel TLC Rf 0.50 (9:1 dichloromethane/methanol); 1H NMR (CDCl3) d
2.24 (s, 6H), 5.39 (br s, 2H) and 6.33 (s, 1H); 13C NMR (CDCl3) d
23.7, 110.5, 162.9 and 167.7; mass spectrum (APCI), m/z
124.0869 (M+H)+ (C6H10N3 requires 124.0875).
5.1.5. 5-Benzyloxy-4-decyl-2-(2,5-dimethyl-1H-pyrrol-1-yl)-6-
methylpyrimidine (8)
To a stirred solution at ꢂ78 °C containing 1.00 g (3.25 mmol) of
5-benzyloxy-2-(2,5-dimethyl-1H-pyrrol-1-yl)-4,6-dimethylpyrim-
idine (7) and 415 lL (452 mg; 2.16 mmol) of 1-bromononane in
30 mL of anhyd THF was added 2.70 mL (4.32 mmol) of a 1.60 M
solution of n-BuLi in pentane. The reaction mixture was stirred at
23 °C for 30 min. The reaction was quenched with satd aq ammo-
nium chloride and then poured into 70 mL of water. The mixture
was extracted with two 70-mL portions of ether. The combined or-
ganic layer was then washed with 100 mL of brine, dried (MgSO4)
and concentrated under diminished pressure. The residue was
purified by chromatography on a silica gel column (15 ꢁ 5 cm).
5.1.2. 2-Amino-5-bromo-4,6-dimethylpyrimidine (5)
To a stirred solution containing 4.31 g (34.8 mmol) of 2-amino-
4,6-dimethylpyrimidine (4) in 150 mL of acetonitrile was added
6.15 g (52.1 mmol) of N-bromosuccinimide. The reaction mixture
was stirred at 23 °C for 3 h. The formed precipitate was filtered
and dried to afford 5 as a colorless solid: yield 5.93 g (83%); mp

R. Goldschmidt et al. / Bioorg. Med. Chem. 21 (2013) 969–978
975
Elution with 4:1 hexanes/ether afforded 8 as a colorless oil: yield
502 mg (53%); silica gel TLC Rf 0.60 (4:1 hexanes/ether); 1H NMR
(CDCl3) d 0.89 (t, 3H, J = 7.2 Hz), 1.20–1.30 (m, 14H), 1.75 (m,
2H), 2.31 (s, 6H), 2.48 (s, 3H), 2.77 (dd, 2H, J = 7.6, 7.6 Hz), 4.90
(s, 2H), 5.86 (s, 2H) and 7.40–7.44 (m, 5H); 13C NMR (CDCl3) d
14.1, 14.3, 19.3, 22.7 27.7, 29.3, 29.4, 29.51, 29.57, 31.62, 31.9,
75.7, 107.9, 128.0, 128.6, 128.7, 129.3, 136.2, 147.4, 152.6, 161.6
and 165.1; mass spectrum (APCI), m/z 434.3172 (M+H)+
(C28H40N3O requires 434.3171).
poured into 70 mL of water and then adjusted to pH 9–10 with
1 N aq NaOH. The mixture was extracted with two 70 mL portions
of ethyl acetate. The combined organic layer was washed with
70 mL of brine, dried (MgSO4) and concentrated under diminished
pressure. The residue was purified by chromatography on a silica
gel column (10 ꢁ 3 cm). Elution with 3:2 hexanes/ethyl acetate
afforded 11 as a colorless oil: yield 231 mg (100%); silica gel TLC
Rf 0.25 (1:1 hexanes/ethyl acetate); 1H NMR (CDCl3) d 0.85 (t, 3H,
J = 6.8 Hz), 1.26 (m, 26H), 1.61 (quint, 2H, J = 7.2 Hz), 2.28 (s, 3H),
2.56 (dd, 2H, J = 7.6, 7.6 Hz), 4.72 (s, 2H), 5.03 (br s, 2H) and
7.35–7.40 m, 5H); 13C NMR (CDCl3) d 14.1, 18.9, 22.7, 28.4, 29.3,
29.4, 29.5, 29.62, 29.63, 29.67, 29.73, 31.9, 75.9, 127.9, 128.2,
128.6, 136.8, 142.9, 158.8, 161.1 and 164.9; mass spectrum (APCI),
m/z 440.3646 (M+H)+ (C28H46N3O requires 440.3641).
5.1.6. 5-Benzyloxy-2-(2,5-dimethyl-1H-pyrrol-1-yl)-4-
hexadecyl-6-methylpyrimidine (9)
To a stirred solution at ꢂ78 °C containing 1.00 g (3.25 mmol) of
5-benzyloxy-2-(2,5-dimethyl-1H-pyrrol-1-yl)-4,6-dimethylpyrim-
idine (7) and 630 lL (630 mg; 2.16 mmol) of 1-bromopentadecane
in 20 mL of anhyd THF was added 2.70 mL (4.32 mmol) of a 1.60 M
solution of n-BuLi in pentane. The reaction mixture was stirred at
23 °C for 30 min. The reaction was quenched with satd aq ammo-
nium chloride and then poured into 70 mL of water. The mixture
was then extracted with two 70-mL portions of ether. The com-
bined organic layer was washed with 100 mL of brine, dried
(MgSO4) and concentrated under diminished pressure. The residue
5.1.9. 5-Benzyloxy-4-decyl-2-(N,N-dimethylamino)-6-
methylpyrimidine (12)
To a stirred solution containing 305 mg (0.86 mmol) of 2-ami-
no-5-benzyloxy-4-decyl-6-methylpyrimidine (10) in 3 mL of
methanol was added 3 mL of 35% aq formaldehyde, followed by
271 mg (4.30 mmol) of NaCNBH3. The reaction mixture was stirred
at 23 °C for 3 h. The reaction was quenched with acetic acid until
bubbling ceased, then poured into 20 mL of water and extracted
with two 40-mL portions of ethyl acetate. The combined organic
layer was washed with 40 mL of satd aq NaHCO3 and 40 mL of
brine. The organic solution was dried (MgSO4) and concentrated
under diminished pressure. The residue was purified by chroma-
tography on a silica gel column (8 ꢁ 3 cm). Elution with 2:1 hex-
anes/ethyl acetate afforded 12 as a colorless oil: yield 154 mg
(46%); silica gel TLC Rf 0.8 (2:1 hexanes/ethyl acetate); 1H NMR
(CDCl3) d 0.87 (t, 3H, J = 6.4 Hz), 1.33 (m, 14H), 1.69 (quint, 2H,
J = 7.2 Hz), 2.32 (s, 3H), 2.61 (dd, 2H, J = 7.6, 7.6 Hz), 3.14 (s, 6H),
4.70 (s, 2H) and 7.33–7.44 (m, 5H); 13C NMR (CDCl3) d 14.1, 19.3,
22.7, 27.9, 29.3, 29.50, 29.55, 29.59, 29.6, 31.7, 31.9, 37.2, 75.7,
127.9, 128.1, 128.5, 137.2, 141.3, 158.6, 159.0 and 163.5; mass
spectrum (APCI), m/z 384.3003 (M+H)+ (C24H38N3O requires
384.3015).
was purified by chromatography on
a silica gel column
(15 ꢁ 5 cm). Elution with 4:1 hexanes/ether afforded 9 as a color-
less oil: yield 269 mg (24%); silica gel TLC Rf 0.5 (4:1 hexanes/
ether); 1H NMR (CDCl3) d 0.89 (t, 3H, J = 7.2 Hz), 1.20–1.30 (m,
26H), 1.75 (quint, 2H, J = 7.2 Hz), 2.29 (s, 6H), 2.47 (s, 3H), 2.76
(dd, 2H, J = 8.0, 8.0 Hz), 4.89 (s, 2H), 5.85 (s, 2H) and 7.30–
7.42 m, 5H); 13C NMR (CDCl3) d 14.1, 14.30, 14.30, 19.3, 22.7,
27.7, 29.3, 29.4, 29.50, 29.55, 29.63, 29.67, 31.6, 31.9, 75.7, 107.9,
128.0, 128.5, 128.7, 129.3, 136.2, 147.4, 152.6, 161.6 and 165.1;
mass spectrum (APCI), m/z 518.4113 (M+H)+ (C34H52N3O requires
518.4110).
5.1.7. 2-Amino-5-benzyloxy-4-decyl-6-methylpyrimidine (10)
To a stirred solution containing 502 mg (1.15 mmol) of 5-ben-
zyloxy-4-decyl-2-(2,5-dimethyl-1H-pyrrol-1-yl)-6-methylpyrimi-
dine (8) in 10 mL of 9:1 ethanol/water was added 800 mg
(11.5 mmol) of hydroxylamine hydrochloride. The reaction mix-
ture was stirred at reflux for 5 h. A second portion of 800 mg
(11.5 mmol) of hydroxylamine hydrochloride was added and the
reaction mixture was stirred at reflux for 16 h. The cooled reaction
mixture was then poured into 70 mL of water and then adjusted to
pH 9–10 with 1 N aq NaOH. The mixture was extracted with two
70-mL portions of ethyl acetate. The combined organic layer was
washed with 70 mL of brine, dried (MgSO4) and concentrated un-
der diminished pressure. The residue was purified by chromatogra-
phy on a silica gel column (10 ꢁ 3 cm). Elution with 3:2 hexanes/
ethyl acetate afforded 10 as a colorless oil: yield 305 mg (75%); sil-
ica gel TLC Rf 0.25 (3:2 hexanes/ethyl acetate); 1H NMR (CDCl3) d
0.85 (t, 3H, J = 6.4 Hz), 1.26 (m, 14H), 1.61 (m, 2H), 2.27 (s, 3H),
2.56 (dd, 2H, J = 8.0, 8.0 Hz), 4.71 (s, 2H), 5.24 (br s, 2H) and
7.35–7.40 m, 5H); 13C NMR (CDCl3) d 13.9, 18.9, 22.6, 28.4, 29.3,
29.4, 29.5, 29.6, 29.7, 31.87, 31.88, 75.9, 127.9, 128.2, 128.6,
136.8, 142.8, 158.9, 161.1 and 162.8; mass spectrum (APCI), m/z
356.2704 (M+H)+ (C22H34N3O requires 356.2702).
5.1.10. 5-Benzyloxy-2-(N,N-dimethylamino)-4-hexadecyl-6-
methylpyrimidine (13)
To a stirred solution containing 230 mg (0.52 mmol) of 2-ami-
no-5-benzyloxy-4-hexadecyl-6-methylpyrimidine (11) in 4 mL of
methanol was added 4 mL of 35% aq formaldehyde, followed by
263 mg (4.18 mmol) of NaCNBH3. The reaction mixture was stirred
at 23 °C for 3 h. The reaction was quenched with acetic acid until
bubbling ceased, then poured into 20 mL of water and extracted
with two 40-mL portions of ethyl acetate. The combined organic
layer was washed with 40 mL of satd aq NaHCO3 and 40 mL of
brine. The organic solution was dried (MgSO4) and concentrated
under diminished pressure. The residue was purified by chroma-
tography on a silica gel column (8 ꢁ 3 cm). Elution with 4:1 hex-
anes/ethyl acetate afforded 13 as a colorless oil: yield 100 mg
(41%); silica gel TLC Rf 0.75 (4:1 hexanes/ethyl acetate); 1H NMR
(CDCl3) d 0.86 (t, 3H, J = 6.8 Hz), 1.23–1.28 (br m, 26H), 1.68 (quint,
2H, J = 6.8 Hz), 2.31 (s, 3H), 2.61 (dd, 2H, J = 7.6, 7.6 Hz), 3.14 (s,
6H), 4.69 (s, 2H) and 7.40–7.46 (m, 5H); 13C NMR (CDCl3) d 14.1,
19.3, 22.6, 27.8, 29.3, 29.50, 29.55, 29.62, 29.64, 29.66, 31.7, 31.9,
37.2, 75.7, 127.9, 128.2, 128.5, 137.2, 141.3, 158.6, 159.0 and
163.6; mass spectrum (APCI), m/z 468.3950 (M+H)+ (C30H50N3O re-
quires 468.3954).
5.1.8. 2-Amino-5-benzyloxy-4-hexadecyl-6-methylpyrimidine
(11)
To a stirred solution containing 269 mg (0.52 mmol) of 5-ben-
zyloxy-2-(2,5-dimethyl-1H-pyrrol-1-yl)-4-hexadecyl-6-methyl-
pyrimidine (9) in 10 mL of ethanol was added 723 mg (10.4 mmol)
of hydroxylamine hydrochloride. The reaction mixture was stirred
at reflux for 5 h. A second portion of 723 mg (10.4 mmol) of
hydroxylamine hydrochloride was added and the reaction mixture
was stirred at reflux for 16 h. The reaction mixture was then
5.1.11. 4-Decyl-2-(N,N-dimethylamino)-6-methylpyrimidin-5-ol
(2)
To a stirred solution containing 150 mg (0.48 mmol) of 5-ben-
zyloxy-4-decyl-(2-N,N-dimethylamino)-6-methylpyrimidine (12)
in 5 mL of methanol was added 5 mg of 20% palladium hydroxide

976
R. Goldschmidt et al. / Bioorg. Med. Chem. 21 (2013) 969–978
on carbon (Degussa type E101 NE/E). The reaction mixture was
stirred at 23 °C under a hydrogen atmosphere for 15 min. The reac-
tion mixture was filtered through Celite and the filtrate was con-
centrated under diminished pressure to afford 2 as a colorless
solid: yield 140 mg (100%). An analytical sample was obtained by
chromatography on a silica gel column (8 ꢁ 1 cm). Elution with
4:1 hexanes/ethyl acetate afforded 2 as a colorless solid; mp 71–
72 °C; silica gel TLC Rf 0.25 (4:1 hexanes/ethyl acetate); 1H NMR
(methanol-d4) d 0.87 (t, 3H, J = 6.8 Hz), 1.21–1.31 (m, 14H), 1.65
(quint, 2H, J = 7.2 Hz), 2.26 (s, 3H), 2.62 (dd, 2H, J = 7.2, 7.2 Hz)
and 3.05 (s, 6H); 13C NMR (methanol-d4) d 13.0, 17.6, 22.3, 27.3,
29.0, 29.15, 29.17, 29.26, 29.29, 31.2, 31.6, 36.52, 36.52, 138.0,
155.6, 157.3 and 159.4; mass spectrum (APCI), m/z 294.2554
(M+H)+ (C17H32N3O requires 294.2545).
diacetate (DCFH-DA) (Molecular Probes, Eugene, OR). One milliliter
of cells (2.5 ꢁ 105 cells) were plated in a 24-well plate, treated with
the test compounds and incubated at 37 °C for 18 h in a humidified
atmosphere containing 5% CO2 in air. Cells were treated with 5 mM
diethyl maleate (DEM) (97%, Sigma–Aldrich, St. Louis, MO) for 1 h,
collected by centrifugation at 300ꢁg for 3 min and then washed
twice with phosphate buffered saline (PBS) (Invitrogen, Grand Is-
land, NY). Cells were resuspended in PBS containing 10 mM glu-
cose and incubated at 37 °C in the dark for 25 min with 10 lM
DCFH-DA. Cells were collected by centrifugation at 300ꢁg for
3 min and then washed twice with PBS. The samples were ana-
lyzed immediately by flow cytometry (C6 Accuri, BD Biosciences,
San Jose, CA), using a 488 nm excitation laser and the FL1-H chan-
nel 530 15 nm emission filter. The generation of ROS, mainly per-
oxides, was detected as a result of the oxidation of DCFH (kex
488 nm; kem 515–540 nm). In each analysis, 10,000 events were re-
corded after cell debris were electronically gated out. The results
obtained were verified by running duplicates and repeating exper-
iments in three independent experiments. Hydrogen peroxide was
5.1.12. 2-(N,N-Dimethylamino)-4-hexadecyl-6-
methylpyrimidin-5-ol (3)
To a stirred solution containing 100 mg (0.21 mmol) 5-benzyl-
oxy-2-(N,N-dimethylamino)-4-hexadecyl-6-methylpyrimidine
(13) in 5 mL of methanol was added 5 mg of 20% palladium
hydroxide on carbon (Degussa type E101 NE/E). The reaction mix-
ture was stirred at 23 °C under hydrogen atmosphere for 15 min.
The reaction mixture was filtered through Celite and the filtrate
was concentrated under diminished pressure to afford 3 as a color-
less solid: yield 79 mg (100%). An analytical sample was obtained
by chromatography on a silica gel column (8 ꢁ 1 cm). Elution with
4:1 hexanes/ethyl acetate afforded 3 as a colorless solid; mp 86–
87 °C; silica gel TLC Rf 0.45 (4:1 hexanes/ethyl acetate); 1H NMR
(methanol-d4) d 0.89 (t, 3H, J = 6.8 Hz), 1.28–1.34 (m, 26H), 1.67
(quint, 2H, J = 7.2 Hz), 2.28 (s, 3H), 2.63 (dd, 2H, J = 7.6, 7.6 Hz)
and 3.07 (s, 6H); 13C NMR (methanol-d4) d 13.0, 17.5, 22.3, 27.2,
29.0, 29.10, 29.12, 29.20, 29.29, 29.30, 31.2, 31.6, 36.50, 138.0,
155.6, 157.3 and 159.4; mass spectrum (APCI), m/z 378.3491
(M+H)+ (C23H44N3O requires 378.3484).
used to produce
a positive control. Data are reported as
means S.E.M. (n = 3).
5.2.3. Cell viability analysis
A hemocytometer-based assay was used to determine the num-
ber of viable cells present in a cell suspension. The assay is based
on the principle that live cells possess intact cell membranes that
exclude certain dyes, such as trypan blue, whereas dead cells do
not. This technique was used to assess the cytoprotective effects
of the test compounds in cultured CEM leukemia cells, Friedreich’s
ataxia lymphocytes, Leber’s optic atrophy lymphocytes, Alzhei-
mers disease lymphocytes, Parkinson’s disease lymphocytes, or
Leigh’s syndrome lymphocytes treated with DEM to induce oxida-
tive cell stress by glutathione depletion. Briefly, cells were seeded
at a density of 5 ꢁ 105 cells/mL and incubated with 0.5
lM or
2.5 lM of the indicated compounds at 37 °C for 16 h under a 5%
5.2. Biochemical and biological evaluation of the pyrimidinol
analogues
CO2 in air atmosphere. After preincubation, the cells were treated
with 5 mM DEM for 6 h. Cells not treated with DEM exhibited
>90% cell viability, whereas DEM treatment reduced cell viability
to ꢀ30%. The ability of the test compounds to protect the cells
against the effects of DEM was determined. Cell viability was
expressed as the percentage of untreated control, normalized to
100%. The results obtained were run in three independent
experiments as triplicates. Data are expressed as means S.E.M.
(n = 3).
5.2.1. Cell culture
A CoQ10-deficient lymphocyte cell line (GM17932) was ob-
tained from Coriell Cell Repositories (Camden, NJ). The lympho-
cytes were cultured in glucose-free media supplemented with
galactose for two weeks to force energy production predominantly
through oxidative phosphorylation rather than glycolysis.55,56 The
lymphocytes were cultured in RPMI 1640 glucose-free medium
(Gibco, Grand Island, NY) supplemented with 25 mM galactose,
2 mM glutamine and 1% penicillin–streptomycin (Cellgro, Manas-
5.2.4. Mitochondria isolation and preparation of
submitochondrial particles
sas, VA), and 10% dialyzed fetal bovine serum (FBS) (<0.5
(Gemini Bio-Product, West Sacramento, CA).
l
g/mL)
Mitochondria were prepared as described.57,58 Briefly, one beef
heart was ground and blended in sucrose buffer (0.25 M sucrose,
10 mM Tris–HCl, pH 7.8, containing 0.2 mM EDTA) at 4 °C. Cell deb-
ris were removed by centrifugation at 1200ꢁg for 20 min. The
supernatant was filtered through two layers of cheesecloth. Mito-
chondria were harvested by centrifugation at 26,000ꢁg for
15 min and then homogenized in the same buffer with a Dounce
homogenizer. Mitochondria were harvested by centrifugation at
12,000ꢁg for 30 min, and stored at ꢂ80 °C in sucrose buffer. Submi-
tochondrial particles (SMPs) were prepared as described by Linnane
and Titchener.59 Briefly, mitochondria were sonicated with a sonic
dismembrator (Fisher Scientific) in 10 mM Tris–HCl, pH 7.8, con-
taining 0.25 M sucrose, 5 mM MgCl2,1 mM ATP, 10 mM MnCl2,
and 1 mM sodium succinate at 4 °C. Intact mitochondria were pel-
leted by centrifugation at 20,000ꢁg at 4 °C for 7 min. SMPs were
harvested by centrifugation at 152,000ꢁg at 4 °C for 30 min and
stored at ꢂ80 °C in 10 mM Tris–HCl, pH 7.5, containing 0.25 M su-
crose, 5 mM MgCl2, 2 mM ATP, 2 mM glutathione and 1 mM sodium
succinate. The protein concentration was determined by BCA
Friedreich’s ataxia lymphocytes (GM15850), normal lympho-
cytes (GM15851), Leber’s optic atrophy lymphocytes (GM10744),
Alzheimers disease lymphocytes (AG06849), Parkinson’s disease
lymphocytes (GM15010), and Leigh’s syndrome lymphocytes
(GM13740) were obtained from Coriell, and were cultured in RPMI
medium with 15% FBS (Fisher Scientific, TX), 2 mM glutamine (Hy-
Clone, South Logan, UT) and 1% penicillin–streptomycin mix anti-
biotics. Cells were maintained in log phase at a concentration
between 1 ꢁ 105 and 1 ꢁ 106 cells/mL.
CEM leukemia cells (ATCC, CRL-2264) were cultured in RPMI
medium with 10% FBS (Fisher Scientific), 2 mM glutamine and 1%
penicillin–streptomycin mix antibiotics. Cells were maintained in
log phase at a concentration between 1 ꢁ 105 and 1 ꢁ 106 cells/mL.
5.2.2. Suppression of reactive oxygen species (ROS)
Intracellular ROS production was measured using the oxidant
sensitive
fluorescent
probe
2,7-dichlorodihydrofluorescein

R. Goldschmidt et al. / Bioorg. Med. Chem. 21 (2013) 969–978
977
titration (Pierce, Rockford, IL) and the sample was diluted as de-
scribed below.
the precipitate and transferred to a glass vial. The supernatant
layer was concentrated following the lipid extraction. Prior to
injection on HPLC, lipids were dissolved in 100
lL of n-propanol
5.2.5. NADH oxidase activity
and then 25 L of a 2 mg/mL solution of 1,4-benzoquinone was
l
The inhibitory effects of the test compounds on bovine heart
mitochondrial complexes I, III and IV were evaluated. The com-
pounds were dissolved in dimethylsulfoxide (DMSO), and then
used to make serial dilutions. Maximal DMSO concentrations never
exceeded 2% and were shown to have no influence on the enzy-
matic activity. Bovine heart SMPs were diluted to 0.5 mg/mL.
Activity was assayed at 30 °C and monitored spectrophotometri-
added, vortexed and left to stand for 10 min. The addition of 1,4-
benzoquinone oxidizes the CoQ10, allowing for determination of
total CoQ10 in each sample.63 Samples were analyzed by HPLC on
a reversed phase Zorbax SB-Phenyl (150 mm ꢁ 4.6 mm, 5
lm),
using a mobile phase consisting of 50% aq methanol ? 100% meth-
anol from 0 to 15 min, 100% aq methanol from 15 to 23 min, and
100% methanol ? 50% aq methanol from 23 to 28 min at a flow
rate of 1.5 mL/min with UV detection at 275 nm. Prior to injection
on HPLC, the CoQ10 concentration was estimated by comparison of
the peak area with those of a standard solution of known concen-
tration and normalized to protein concentration. The protein con-
tent was determined by the bicinchoninic acid assay method
(micro-BCA kit, Pierce) using bovine serum albumin (BSA) as a ref-
erence. Data are reported as the mean of at least three independent
experiments.
cally using
a
Beckman Coulter DU-530 (340 nm,
e
6.22 mMꢂ1 cmꢂ1). NADH oxidase activity was determined in a
reaction medium (2.5 mL total volume) containing 50 mM Hepes,
pH 7.5, and 5 mM MgCl2. The final mitochondrial protein concen-
tration was 30 lg. After the pre-equilibration of SMP with inhibitor
for 5 min, the initial rates were calculated from the linear portion
of the traces. Data are reported as the mean of three independent
experiments each run in triplicate.
5.2.6. NADH:ubiquinone oxidoreductase activity
Acknowledgments
The inhibition of NADH:ubiquinone oxidoreductase activity was
determined using the same experimental conditions described pre-
viously.60,61 Twelve micrograms of SMPs were incubated at 39 °C for
5 min with the test compound in 1 mL of 50 mM phosphate buffer,
We thank Dr. Stuart Stein for helpful comments at the outset of
this study. Fresh bovine heart was generously provided by JBS
Tolleson, Inc. This work was supported by a research grant from
the Friedreich’s Ataxia Research Alliance.
pH 7.4, containing 0.25 M sucrose, 1 mM MgCl2, 2
and 2 mM KCN. The reaction was initiated by the addition of
50 M NADH and 50 M of coenzyme Q1. Enzymatic activity, mea-
lM antimycin A
l
l
Supplementary data
sured by the loss of NADH absorbance, was monitored at 340 nm.
Data are reported as the mean of three independent experiments
each run in triplicate. The KI values were determined as described.62
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.11.051.
5.2.7. Cellular ATP concentration
Briefly, lymphocytes (2 ꢁ 105 cell/mL) were plated (1 mL in 12-
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