Lipophilic methylene violet analogues as modulators of mitochondrial function and dysfunction

Article abstract of DOI:10.1016/j.bmc.2017.08.021

In an effort to identify methylene blue analogues having improved antioxidant activity, a series of new methylene violet analogues have been designed and synthesized. The analogues were prepared following a synthetic route that is more efficient than the

Full text of DOI:10.1016/j.bmc.2017.08.021

Accepted Manuscript
Lipophilic methylene violet analogues as modulators of mitochondrial function
and dysfunction
Sandipan Roy Chowdhury, Omar M. Khdour, Indrajit Bandyopadhyay, Sidney
M. Hecht
PII:
S0968-0896(17)31311-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.08.021
BMC 13924
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 June 2017
8 August 2017
10 August 2017
Please cite this article as: Roy Chowdhury, S., Khdour, O.M., Bandyopadhyay, I., Hecht, S.M., Lipophilic methylene
violet analogues as modulators of mitochondrial function and dysfunction, Bioorganic & Medicinal Chemistry
(2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.08.021
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Lipophilic methylene violet analogues as
modulators of mitochondrial function and
dysfunction
Sandipan Roy Chowdhury, Omar M. Khdour, Indrajit
Bandyopadhyay, Sidney M. Hecht*
Biodesign Center for BioEnergetics, and School of Molecular Sciences, Arizona State
University, Tempe, AZ 85287, United States
1

A R T I C L E I N F O
__________________________
Keywords:
Methylene blue
Methylene violet
Mitochondria
Reactive oxygen species
Friedreich’s ataxia
Cytoprotection
__________________________
__________
* Corresponding author. Tel.: +1 480 965 6625; fax: +1 480 965 0038.
E-mail address: sid.hecht@asu.edu (S.M. Hecht).
2

A B S T R A C T
________________________________________________________________________
In an effort to identify methylene blue analogues having improved antioxidant activity, a
series of new methylene violet analogues have been designed and synthesized. The
analogues were prepared following a synthetic route that is more efficient than the
previously reported methods, both in terms of yield and purity of the final products. The
route involves the Smiles rearrangement as one of the crucial steps. Smiles rearrangement
of suitably substituted diphenyl sulfide intermediates afforded the corresponding
phenothiazine analogues in high yields, which were subsequently converted to the final
products. The methylene violet analogues were evaluated for their ability to preserve
mitochondrial function in Friedreich’s ataxia (FRDA) lymphocytes. The analogues were
shown to be efficient ROS scavengers, and able to protect cultured FRDA lymphocytes
from oxidative stress resulting from inhibition of complex I. The analogues also
preserved mitochondrial membrane potential and augmented ATP production. The
analogues were found to be better antioxidants than the parent compounds methylene
blue and methylene violet.
______________________________________________________________________
3

1. Introduction
Mitochondrial dysfunction involving disruption of the electron transport chain has been
identified as an important factor in diseases ranging from neurodegenerative diseases,
including Alzheimer’s disease (AD), Parkinson’s disease (PD) and Friedreich’s ataxia
(FRDA), to diseases of the cardiovascular system, cancer, and diabetes.1-5 The broad
impact of mitochondria in so many diseases makes this organelle a target for therapeutic
intervention. In mammalian cells, mitochondria are the major site of ATP production via
oxidative phosphorylation (OXPHOS). This process involves four respiratory chain
complexes (I-IV) and ATP synthase (complex V), all of which are embedded in the inner
mitochondrial membrane.6-9 However, mitochondria are also a major source of reactive
oxygen species (ROS) even in fully functional mitochondria.10,11 Impaired oxidative
phosphorylation leads to enhanced production of ROS, which can overwhelm the
endogenous antioxidant systems and expose cellular macromolecules to oxidative
damage.12,13 Given the role of mitochondrial dysfunction in the development and
progression of metabolic disorders, strategies for preserving mitochondrial function are
of great interest for addressing mitochondrial and neurological diseases.14-19
Methylene blue (MB), a member of the phenothiazine family of compounds, was
originally of interest as a synthetic cationic dye, but has an extensive history of medical
uses for more than a century.20 MB is approved by the US Food and Drug Administration
for the treatment of methemoglobinemia and as an antidote to ifosfamide-induced
encephalopathy.21,22
MB can readily interconvert between its oxidized and reduced forms using specific
mitochondrial and cytosolic redox centers.18,19 This property of MB enables the
4

redirection of electron transfer across mitochondrial electron transfer complexes,
minimizing electron leakage and inhibiting superoxide production.18,19 Thus, MB
analogues may be able to act as alternative electron carriers, bypassing lesions in a
damaged mitochondrial respiratory chain. In fact, methylene blue itself is being evaluated
in Alzheimer’s disease.21,22
Methylene violet (MV) is a neutral phenothiazine dye; hydrolysis of MB under strongly
basic condition yields MV with increased hydrophobicity.23 Although MV is naturally
obtained in its oxidized (quinone) form, it can be reduced at the mitochondrial redox
centers, generating the phenolic (quinol) form. The reduced form of MV can act as a
phenolic antioxidant, similar to coenzyme Q10. The goal of the study was to design novel
MB/MV optimize MB analogues of potential utility as mitochondrial probes, leading to
the development of novel therapeutic agents.
In earlier studies involving other heterocyclic antioxidants as potential mitochondrial
therapeutic agents, we found that the presence of a lipophilic substituent was essential to
activity, presumably because interaction with mitochondrial respiratory complexes
embedded in the inner mitochondrial membrane represent the putative locus of
action.17,24-26 Presently we describe five lipophilic MV analogues capable of quenching
ROS and diminishing the degradation of cellular macromolecules, in addition to
supporting ATP synthesis in FRDA lymphocytes.
2. Results and discussion
2.1. Synthesis of the methylene violet analogues
The syntheses of MV analogues 1-5 having long alkyl substituents on the exocyclic
nitrogen atom (Figure 1) were carried out using the Smiles rearrangement27,28 as the key
5

step. Phenothiazines 1-5 were prepared by the intramolecular rearrangement of
functionalized diphenyl sulfides under basic conditions (Schemes 1 and 2). 2-Amino-5-
methoxythiophenol was prepared by the basic hydrolysis of 2-amino-6-
methoxybenzothiazole at reflux in aqueous KOH. The cooled reaction mixture was used
for the next step without isolation or further purification by admixture of the crude
N
N
N
S
O
N
S
O
2
1
N
S
N
S
N
O
N
O
4
3
N
S
N
O
5
Figure 1. Chemical structures of the newly synthesized methylene violet (MV)
analogues.
Scheme 1. Synthetic routes employed for compounds 1, 2 and 3.
O
NH2
S
O2N
Cl
N
NH2
KOH/H2O
reflux
NH NO2
NH2 NO2
NO2
NO2
SH
NO2
Ac2O/pyridine
DMF, r.t, 5 h
12 h
AcOH, EtOH, r.t, 3 h
81%
O
S
MeO
S
OMe
OMe
7
8
90%
6
O
O
O
O
H
N
S
N
N
NaH/Boc2O, DMF
0oC to r.t, 5 h
H2/Pd-C, EtOH
25 psi, r.t, 12 h
KOH/acetone
reflux, 2 h
H2N
OMe
O2N
S
OMe
O2N
S
O
90%
86%
11
9
10
O
O
N
S
Na2CO3 / alkyl-I
N
S
BBr3 (1M), DCM
-78oC to r.t, 12 h
R
N
R
O
CH3CN, 80o C, 48 h
R
N
R
OMe
47%
38%
44%
R = n-butyl
R = n-decyl
R= n-pentadecyl
1
2
R = n-butyl
R = n-decyl
R = n-pentadecyl 14
12
13
3
6

solution of 2-amino-5-methoxythiophenol to a solution of 2,4-dinitrochlorobenzene in a
mixture of ethanol and HOAc. Diphenyl sulfide intermediate 7 was prepared by the
coupling under mildly acidic conditions (Scheme 1). The high yield diphenyl sulfide 7
(81%) indicated the efficient formation of the thiol intermediate in situ. Compound 7
was treated with acetic anhydride in presence of triethylamine to convert the amine to
acetamide intermediate 8 in 90% yield. Smiles rearrangement of 8 under basic
conditions,27,28 followed by ring closure, yielded the functionalized phenothiazine 9 as a
violet solid in 90% yield. Compound 9 was then treated with NaH (60% in mineral oil)
followed by di-tert-butyl dicarbonate to obtain 10 in 86% yield as a bright yellow solid.
Subsequently, 10 was converted to the respective to aromatic amine 11 by hydrogenation
of the 7-nitro group over palladium-on-carbon. The crude amine was alkylated using
different alkyl iodides/bromides under basic conditions to obtain compounds 12-14
(Scheme 1). Compounds 12 and 13 were purified and fully characterized prior to
conversion to 1 and 2, respectively. However, alkylation of 11 with n-pentadecyl bromide
gave a mixture of mono- and di-N-alkylated products which could not be separated
readily, although unreacted starting material was removed by silica gel chromatography.
The crude mixture was converted to 3 directly and then purified by silica gel
chromatography. Removal of the Boc and methyl groups were carried out in each case by
treatment with BBr3, affording the MV analogues 1-3 as violet solids in yields that
ranged from 38% to 47% (Scheme 1).29
In order to synthesize compounds 4 and 5, compound 11 was first alkylated using 1-
bromopentadecane (Scheme 2). Mono-N-alkylated product 15 was purified on a silica gel
column and was subsequently alkylated using either methyl iodide or 1-bromobutane to
7

afford 16 and 17, respectively. Compounds 16 and 17 were then deprotected with 1 M
BBr3 to afford 4 and 5, respectively.
While the five new methylene violet analogues prepared were certainly insufficient in
number to define any structure‒activity relationships that might exist, by varying the
length of alkyl substituents on the exocyclic N-atom, we hoped to establish whether these
compounds interacted with the mitochondria in a fashion different from methylene violet
itself, and whether there were differences between the analogues in their biological
effects.
2.2. Biochemical and biological evaluation
2.2.1. Cytotoxicity
Although it is believed that methylene blue serves as an antioxidant, it has also been
noted to foster toxicity by exhibiting pro-oxidant properties under certain conditions.18 In
this regard, it seemed important to ensure that the prepared analogues were not cytotoxic.
Scheme 2. Synthetic routes employed for compounds 4 and 5.
O
O
N
N
BBr (1M), DCM
3
o
-78 C to r.t, 12 h
N
S
O
N
S
OMe
4
16
54%
Na CO / CH I
2
3
3
o
CH CN, 80 C, 16 h
3
O
O
O
O
Na CO / R-Br
2 3
N
N
o
R
CH CN, 80 C, 72 h
3
OMe
1
H N
2
S
N
H
S
OMe
15
11
R = n-pentadecyl
9%
Na CO / n-Bu-Br
3
2
o
CH CN, 80 C, 30 h
3
O
O
N
N
BBr (1M), DCM
3
o
-78 C to r.t, 12 h
N
S
O
N
S
OMe
5
25%
17
8

Therefore, the MV analogues were evaluated for their cytotoxicity and respiratory chain
effects by studying their effects on FRDA lymphocytes. These were grown on galactose
prior to assay to increase their sensitivity to respiratory chain inhibitors.30,31 The strong
cytotoxicity of MB itself in FRDA lymphocytes at 2.5 µM concentration was surprising,
given its long clinical use (Figure 2), but only one of the new analogues (1) exhibited any
significant cytotoxicity, and only at 2.5 µM concentration after a 48-hour incubation.
Measurement of cytotoxicity was carried out using the LIVE/ DEAD
Viability/Cytotoxicity Kit for mammalian cells (Molecular Probes, Eugene, OR). This
assay provides information about the functional status of the cell by detecting
cytoplasmic esterases activity and plasma membrane integrity.32
Figure 2. Methylene violet analogues (1-5) were evaluated for their cytotoxicity toward
cultured FRDA lymphocytes by incubation for 24 h or 48 h in glucose-free media
(galactose) to force the cells to rely on their mitochondria to produce ATP. Flow
cytometric determination of cell viability by fluorescence labeling was used
employing calcein acetoxy-methyl-ester and ethidium homodimer-1 as live and dead cell
stains, respectively. The actual percentages of live cells were extracted from a two-
dimensional color density dot plot. Results obtained were verified by running duplicates
and repeating experiments in two independent runs. The effects of the tested compounds
on the fluorescence intensities of the dyes never exceeded 0.1%.
From the results, it is evident that cytotoxicity decreased with increasing lipophilicity.
While MB itself was quite cytotoxic, the modified methylene violet analogues with
9

longer alkyl side chains (2-5) were not cytotoxic under any tested condition. Methylene
violet and short chain MV analogue 1 exhibited some relatively minor cytotoxicity.
2.2.2. Preservation of mitochondrial inner membrane potential (∆ψm)
Analogues 1-5 were next tested for their effects on inner mitochondrial membrane
potential (∆ψm) in FRDA lymphocytes using the JC-1 probe.33 This cationic fluorescent
dye, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodide (JC-1),
Figure 3. Flow cytometric analysis shows the effect of test compounds 1-5 on ∆ψm in
FRDA lymphocytes using the ratiometric fluorescent probe JC-1. This probe is a cell
penetrating dye that accumulates within mitochondria maintaining high ∆ψm. The red
regions represent intact mitochondrial membranes with JC-1 aggregates (red
fluorescence), whereas the gated regions shown in green depict cells with loss of ∆ψm.
The bar graph shows the percentage of cells with intact ∆ψm, calculated using
Accuri™ C6 software. For each analysis, 10000 events were recorded. Depolarization
with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), served as a
positive control. Results obtained were verified by running duplicates and repeating
experiments in two independent runs. The effects of the tested compounds on the
fluorescence intensity of JC-1 never exceeded 0.1%.
10

selectively accumulates in mitochondria due to the negative potential across the inner
mitochondrial membrane.34 The dye exists as a monomer at low concentrations giving a
green fluorescence. At higher concentrations the dye forms J-aggregates, which exhibit
red fluorescence.33 Therefore, mitochondrial depolarization is indicated by a decrease in
the red/green fluorescence ratio. FACS analysis of ∆ψm showed that MB significantly
depolarized ∆ψm (Figure 3) while MV had a lesser effect. Again, analogue 1 also
produced significant reduction in mitochondrial membrane potential comparable to MV.
In comparison, analogues 2-5 having 10-15 carbon side chains did not significantly affect
mitochondrial membrane potential, even at 5 µM concentration. Carbonyl cyanide 4-
(trifluoromethoxy)phenylhydrazone (FCCP), a commonly used uncoupler of oxidative
phosphorylation in mitochondria, was used as a negative control to dissipate the
chemiosmotic proton gradient, which results in depolarization of mitochondrial
membrane potential.
2.2.3. Suppression of reactive oxygen species
The ability of MV analogues 1-5 to suppress ROS induced by the depletion of cellular
glutathione was evaluated in FRDA lymphocytes in a quantitative FACS experiment
using dichlorodihydrofluorescein diacetate (DCFH-DA) as an indicator of intracellular
ROS levels, as described previously.35 The results are shown in Figure 4, and
demonstrated that compounds 4 and 5 were more potent and effective in protecting
FRDA lymphocytes than the parent compound MV and did so in a concentration
dependent manner. In comparison, MV analogues 1-3 exhibited behavior similar to MV
itself.
11

Figure 4. Flow cytometric analysis of FRDA lymphocytes stained with
dichlorodihydrofluorescein diacetate (DCFH-DA) for 20 min, following pretreatment
with compounds 1-5 for 16 h, and subsequent treatment with diethyl maleate (DEM) for
80 min to induce the production of ROS by depleting glutathione. Results were expressed
as a percentage of the median mean fluorescence intensity of DCF fluorescence relative
to a DEM-treated control. Results obtained were verified by running duplicates and
repeating experiments in two independent runs. The effects of the tested compounds on
the fluorescence intensity of DCF never exceeded 0.1%. The actual fluorescence intensity
data is shown in Figure S1 of the Supplementary Data.
2.2.4. Cytoprotection
The ability of the test compounds 1-5 to confer cytoprotection to buthionine
sulfoximine (BSO)-treated FRDA fibroblasts and to rotenone-treated FRDA lymphocytes
from oxidative damage-induced death was determined by using simultaneous staining
with a two-color fluorescence assay, the Live/Dead® Viability/Cytotoxicity Kit
(Molecular Probes, Eugene, OR). The cells were subjected to L-buthionine (S,R)-
sulfoximine, an inhibitor of de novo glutathione (GSH) biosynthesis.36 Oxidative
damage-induced death of FRDA fibroblasts was blocked by the test compounds in a
concentration dependent manner (Figure 5). Compounds 4 and 5, each having a single
longer alkyl substituent, exhibited better potency than 1 or 2 and were by far the most
efficient when tested at lower concentrations. Again, in this assay the optimal side chain
12

length was 15 carbon atoms. Interestingly, compound 3, having two 15-carbon
substituents, was the least effective in this assay.
Figure 5. Cytoprotective effect of compounds 1-5 in (BSO)-treated FRDA fibroblasts
following preincubation with test compounds and subsequent treatment with BSO (5
mM) for 48 h. Results obtained were verified by running triplicates and repeating
experiments in two independent runs. The effects of the tested compounds on the
fluorescence intensities of the dyes never exceeded 0.1%. The actual fluorescence
intensity data is shown in Figure S2 of the Supplementary Data.
Figure 6. Cytoprotective effect of the compounds 1-5 in FRDA lymphocytes following
preincubation with the test compounds for 12 h in glucose-free media and subsequent
treatment with rotenone (50 nM) for 24 h to inhibit complex I. Cell viability was
expressed as the percentage of cells relative to control. The actual percentages of live
cells were extracted from a two-dimensional color density dot plot. Results are an
average of two independent trials run in duplicate. Statistically significant differences,
where observed, are also indicated. Statistical analyses were performed with a two-tailed
Student’s t-test, and data are mean values SD (n = 2). * P > 0.05, as compared to
rotenone treated control.
13

The compounds were also tested for their ability to confer cytoprotection to rotenone-
treated FRDA lymphocytes. The cells were treated with rotenone to induce cytotoxicity
by inhibiting complex I. As shown in Figure 6, the results closely paralleled those found
for BSO-treated FRDA fibroblasts. Compounds 4 and 5 were able to protect the cells
against rotenone-induced cytotoxicity more efficiently than MB, MV or analogues 1-3.
2.2.5. NADH: ubiquinone oxidoreductase activity (complex I activity)
An important characteristic of any CoQ10 surrogate is its ability to act as an electron
carrier between the individual mitochondrial complexes in the respiratory chain.
Accordingly, we evaluated MB, MV, and compound 5 for their effects on complex I
function in presence and absence of rotenone (Figure 7). Both MB and MV were able to
Figure 7. Effect of compound 5 on bovine heart mitochondrial complex I (SMP) activity
in the presence of rotenone. In common with methylene blue and methylene violet,
analogue 5 was able redirect electron flow from complex I resulting from NADH
oxidation, even when the normal electron flow from complex I was completely blocked
by (50 nM) rotenone. Results obtained were verified by running duplicates and repeating
experiments in two independent runs.
14

maintain electron flow in the respiratory chain resulting from NADH oxidation even if
complex I was blocked by rotenone. While compound 5 was not as efficient as MV or
MB in the presence or absence of rotenone, it did support the flow of electrons. These
results are in agreement with earlier studies which demonstrated that MB-mediated
electron transfer is insensitive to either rotenone or antimycin A inhibition, consistent
with the suggestion that MB provides an alternative route for electron transfer,18,19,37
especially involving cytochrome c. MB may accept electrons from NADH at
mitochondrial complex I and transfer them to cytochrome c, thus maintaining aerobic
respiration. The alternative electron transfer through MB prevents electron leakage
induced by complex I/III inhibition, thus consistent with a potential role in
neuroprotection. The apparent ability of MV analogue 5 to participate in the alternative
electron transfer mechanism, coupled with its lesser intrinsic cytotoxicity than MB and
MV (Figure 2), and its superior properties in maintaining mitochondrial membrane
potential (Figure 3), suppressing ROS (Figure 4) and conferring cytoprotection (Figures 5
and 6) seems fully consistent with its possible utility in maintaining the function of
partially dysfunctional mitochondria. Indeed, a recent study demonstrated that MB itself
rescues heart defect in a frataxin-depleted drosophila model of FRDA.38
2.2.6. Determination of ATP levels
Since the transfer of electrons directly from complex I to cytochrome c would logically
involve the concomitant pumping of fewer protons into the intermembrane space, it
might be anticipated that this pathway would result in the production of less ATP.
Accordingly, MV analogues 1–5 were evaluated for their ability to support ATP
production in FRDA lymphocytes. As shown in Figure 8, MB strongly diminished ATP
15

levels in a concentration dependent fashion (0.5 and 5 µM) in Friedreich’s ataxia
lymphocytes and had no effect at 100 nM concentration. Similar results were observed
Figure 8. Total ATP level in FRDA lymphocytes following incubation with compounds
1-5 for 24 h in glucose free media (25 mM galactose). Results are expressed as the
percentage of total ATP relative to an untreated control. Results obtained were verified
by running triplicates and repeating experiments in three independent runs. The actual
chemiluminescence intensity data is shown in Figure S3 of the Supplementary Data.
for MV and analogue 1, the latter having a short alkyl substituent chain. Compounds 2
and 3 had little effect on the ATP level. In comparisons, compound 4 and 5 clearly
increased the ATP level when employed at 0.1 and 0.5 µM concentrations, although they
suppressed ATP levels when used at 5 µM concentration. The mechanism by which ATP
levels were increased is presently unclear, but could plausibly reflect more efficient redox
cycling by the analogues between complex I and cytochrome c, at a level sufficient to
compensate for reduced proton pumping in a single cycle.
3. Conclusions
In conclusion, five lipophilic MV analogues were designed and synthesized. The
synthetic route involved Smiles rearrangement as one of the key steps to obtain a
substituted phenothiazine analogue. The final compounds were obtained in a good state
16

of purity. While the number of analogues studied was too small to allow detailed
conclusions to be reached regarding structure‒activity relationships, the specific side
chain modifications did affected the biological activity of individual MV derivatives.
Compounds 4 and 5 were more effective than MB and MV in almost all of the assays,
and exhibited better antioxidant activity than the parent compounds. Surprisingly,
compounds 4 and 5 were able to increase ATP levels in cultured FRDA lymphocytes,
consistent with their potential utility for therapeutic intervention in mitochondrial
disorders. Compounds 4 and 5 were also superior to 1 – 3 in most of the bioassays,
suggesting that more detailed investigation may reveal more systematic structure‒activity
relationships.
4. Experimental section
4.1. General experimental procedures
Reagent grade chemicals and solvents were purchased from Sigma-Aldrich Chemicals
and were used without further purification. All the reactions were performed under an
argon atmosphere, unless otherwise specified. Thin layer chromatography (TLC) plates
(precoated glass plates with silica gel 60 F254, 0.25 mm thickness) were used for
analytical TLC and were visualized by UV irradiation (254 nm). Flash chromatography
was carried out using Silicycle 200−400 mesh silica gel. 1H and 13C NMR spectra were
obtained using a Varian 400 MHz NMR spectrometer. Chemical shifts (δ) are reported in
parts per million (ppm) and are referenced to residual CHCl3 (δ 7.26 ppm for 1H NMR
and δ 77.16 for 13C NMR) as the internal standard. Splitting patterns are designated as s,
singlet; d, doublet; m, multiplet. High resolution mass spectra were obtained at the
17

Facility or the Arizona State University CLAS High Resolution Mass Spectrometry
Facility.
Human mitochondrial disease cell lines, Friedreich’s ataxia lymphocytes (GM15850),
and Friedreich’s ataxia skin fibroblast (GM04078), were obtained from Coriell Cell
Repositories (Camden, NJ).
4.1.1. 2-(2,4-Dinitrophenyl)thio)-4-methoxyaniline (7). A sample containing 2.00 g
(11.1 mmol) of 2-amino-6-methoxybenzothiazole (6) was suspended in 40 mL of water
and treated with 9.30 g (167 mmol) of solid KOH. The resulting suspension was heated at
reflux for 12 h. The cooled reaction mixture was added dropwise to a solution of 2.25 g
(11.1 mmol) of 2, 4-dinitrochlorobenzene in a mixture of 30 mL of ethanol and 20 mL of
HOAc in an ice‒water bath. The reaction mixture was then stirred at room temperature
for an additional 3 h. The precipitate was filtered, washed with 1:1 water‒ethanol and
dried to afford 7 as an orange solid: yield 2.90 g (81%); silica gel TLC Rf 0.7 (3:7 ethyl
acetate‒hexanes); 1H NMR (acetone-d6) δ 3.76 (s, 3H), 6.85 (d, 1H, J = 8.8 Hz), 6.97 (d,
1H, J = 2.8 Hz), 7.00-7.06 (m, 2H), 8.18 (dd, 1H, J = 9.2 and 2.6 Hz) and 9.12 (d, 1H, J =
2.4 Hz); 13C NMR (DMSO-d6) δ 55.9, 111.0, 117.5, 120.4, 120.6, 121.6, 127.1, 128.6,
143.3, 144.4, 144.6, 145.8 and 153.0; mass spectrum (APCI), m/z 322.0499 (M+H)+
(C13H12N3O5S requires m/z 322.0498).
4.1.2. N-(2-(2,4-Dinitrophenyl)thio-4-methoxyphenyl)acetamide (8). To a solution of
2.90 g (9.03 mmol) of 7 in 10 mL of anhydrous DMF was added 3.66 mL (2.74 g, 27.1
mmol) of anhydrous triethylamine followed by 4.30 mL (4.64 g, 45.5 mmol) of acetic
anhydride. The reaction mixture was stirred for 12 h at room temperature under an argon
atmosphere and then quenched by pouring into ice-cold water. The aqueous layer was
18

extracted with four 25-mLportions of ethyl acetate. The combined organic layer was
dried over anhydrous MgSO4, then filtered and concentrated under diminished pressure.
The residue was purified on a silica gel column (8 × 4 cm). Elution with 3:7 ethyl
acetate‒hexanes afforded 8 as a bright yellow solid: yield 2.95 g (90%); silica gel TLC Rf
0.57 (3:7 ethyl acetate‒hexanes); 1H NMR (DMSO-d6) δ 1.86 (s, 3H), 3.77 (s, 3H), 6.99
(d, 1H, J = 8.8 Hz), 7.18 (dd, 1H, J = 8.8 and 2.4 Hz), 7.23 (d, 1H, J = 2.8 Hz), 7.66 (d,
1H, J = 8.8 Hz), 8.32 (dd, 1H, J = 9.0 and 2.6 Hz), 8.88 (d, 1H, J = 2.4 Hz) and 9.43 (br
s, 1H); 13C NMR (DMSO-d6) δ 22.8, 55.6, 117.7, 120.5, 121.0, 123.8, 127.5, 128.1,
129.2, 133.7, 144.05, 144.07, 145.6, 157.2 and 168.7; mass spectrum (APCI), m/z
364.0609 (M+H)+ (C15H14N3O6S requires m/z 364.0603).
4.1.3. 3-Methoxy-7-nitro-10H-phenothiazine (9). To a stirred solution of 2.95 g (8.13
mmol) of 8 in 20 mL of acetone at reflux was added portionwise 0.91 g (16.2 mmol) of
KOH in 10 mL of ethanol. The reaction mixture was maintained at reflux for an
additional 3 h and poured into ice-cold water. The aqueous layer was extracted with four
25-mLportions of ethyl acetate. The combined organic layer was dried over anhydrous
MgSO4, then filtered and concentrated under diminished pressure. The residue was
purified on a silica gel column (8 × 2 cm). Elution with 1:1 ethyl acetate‒hexanes gave 9
as a violet solid: yield 2.00 g (90%); silica gel TLC Rf 0.43 (3:7 ethyl acetate‒hexanes);
1H NMR (acetone-d6) δ 3.70 (s, 3H), 6.57-6.70 (m, 4H), 7.73 (d, 1H, J = 2.4 Hz), 7.81-
7.84 (m, 1H) and 8.47 (br s, 1H); 13C NMR (acetone-d6) δ 55.0, 111.7, 113.0, 113.3,
116.1, 117.0, 117.2, 121.6, 124.2, 132.4, 141.5, 148.3 and 156.6; mass spectrum (APCI),
m/z 275.0488 (M+H)+ (C13H11N2O3S requires m/z 275.0490).
19

4.1.4. tert-Butyl 3-Methoxy-7-nitro-10H-phenothiazin-10-carboxylate (10). To a
solution of 1.42 g (5.18 mmol) of 9 in 20 mL of anhydrous DMF at 0 oC was added 0.55
g (13.7 mmol) of NaH (60% in mineral oil). The reaction mixture was stirred at 0 oC for
another 15 min and 2.40 g (11.0 mmol) of di-tert-butyl dicarbonate was added. The
reaction mixture was stirred at room temperature for 4 h under an argon atmosphere, and
was then quenched with 30 mL of water. The aqueous layer was extracted with three 20-
mL portions of ethyl acetate. The combined organic layer was washed with 20 mL of
brine, dried over anhydrous MgSO4 and concentrated under diminished pressure. The
residue was purified on a silica gel column (8 × 4 cm). Elution with 3:7 ethyl
acetate‒hexanes afforded 10 as a bright yellow solid: yield 1.66 g (86%); silica gel TLC
Rf 0.54 (3:7 ethyl acetate‒hexanes); 1H NMR (acetone-d6) δ 1.54 (s, 9H), 3.86 (s, 3H),
6.97 (dd, 1H, J = 9.2 and 2.8 Hz), 7.01 (d, 1H, J = 2.8 Hz), 7.47 (d, 1H, J = 9.2 Hz), 7.84
(d, 1H, J = 8.8 Hz), 8.20 (dd, 1H, J = 9.0 and 2.6 Hz) and 8.24 (d, 1H, J = 2.4 Hz); 13C
NMR (acetone-d6) δ 29.2, 57.1, 84.3, 113.3, 115.6, 123.7, 124.1, 129.8, 130.0, 132.3,
133.1, 135.3, 146.9, 147.1, 153.2 and 159.9; mass spectrum (APCI), m/z 374.0932
(M+H)+ (C18H19N2O5S requires m/z 374.0936).
4.1.5. tert-Butyl 3-Amino-7-methoxy-10H-phenothiazin-10-carboxylate (11). To a
suspension of 0.42 g (1.12 mmol) of 10 in 10 mL of ethanol was added 10 mg of 10%
palladium-on-carbon. The reaction mixture was stirred at room temperature under a
hydrogen atmosphere (25 psi) overnight. The reaction mixture was filtered through a
Celite pad and the filtrate was concentrated under diminished pressure. The crude product
(11) was used for the next reaction without further purification.
20

4.1.6. tert-Butyl 3-(N,N-Dibutylamino)-7-methoxy-10H-phenothiazin-10-carboxylate
(12). To a solution of crude 11 in 3 mL of acetonitrile was added 1.40 g (12.8 mmol) of
Na2CO3 followed by 0.94 g (5.12 mmol) of 1-iodobutane. The reaction mixture was
sealed under a nitrogen atmosphere and stirred at 80 oC overnight. The cooled reaction
mixture was filtered and the filtrate was concentrated under diminished pressure and
purified on a silica gel column (10 × 2 cm). Elution with 1:9 ethyl acetate‒hexanes gave
compound 12 as a pale yellow solid: yield 0.31 g (61%); silica gel TLC Rf 0.43 (1:9 ethyl
acetate‒hexanes); 1H NMR (CDCl3) δ 0.95 (t, 6H, J = 7.4 Hz), 1.32-1.37 (m, 4H), 1.49
(s, 9H), 1.51-1.57 (m, 4H), 3.23 (t, 4H, J = 7.6 Hz), 3.78 (s, 3H), 6.52-6.55 (m, 2H), 6.78
(dd, 1H, J = 8.8 and 2.8 Hz), 6.86 (d, 1H, J = 2.8 Hz), 7.31 (d, 1H, J = 8.4 Hz) and 7.39
(d, 1H, J = 8.8 Hz); 13C NMR (CDCl3) δ 13.9, 20.2, 28.1, 29.2, 50.8, 55.5, 81.2, 109.1,
110.2, 111.5, 112.6, 126.8, 127.2, 127.5, 132.3, 132.5, 133.2, 146.2, 153.2 and 157.0;
mass spectrum (APCI), m/z 457.2530 (M+H)+ (C26H37N2O3S requires m/z 457.2525).
4.1.7. 7-(N,N-Dibutylamino)-3H-phenothiazin-3-one (1). To a solution of 86.0 mg
(0.19 mmol) of 12 in 2 mL of anhydrous CH2Cl2 was added dropwise 0.76 mL (0.76
mmol) of 1 M BBr3 in CH2Cl2 at ‒78 oC. The reaction mixture was stirred overnight at
room temperature under an argon atmosphere and was then quenched with 10 mL of
water. The aqueous layer was extracted with two 10-mL portions of ethyl acetate. The
combined organic layer was washed with 10 mL of brine, dried over anhydrous MgSO4
and concentrated under diminished pressure. The residue was purified on a silica gel
column (7 × 2 cm). Elution with 1:1 ethyl acetate‒hexanes afforded 1 as a violet solid:
yield 30 mg (47%); silica gel TLC Rf 0.42 (1:1 ethyl acetate‒hexanes); 1H NMR (CDCl3)
δ 0.95 (t, 6H, J = 7.4 Hz), 1.26-1.37 (m, 4H), 1.51-1.57 (m, 4H), 3.23 (m, 4H), 6.52-6.55
21

(m, 2H), 6.76-6.87 (m, 2H), 7.31 (d, 1H, J = 8.8 Hz) and 7.38 (d, 1H, J = 8.8 Hz); mass
spectrum (APCI), m/z 341.1690 (M+H)+ (C20H25N2OS requires m/z 341.1688).
4.1.8. tert-Butyl 3-(N,N-Didecylamino)-7-methoxy-10H-phenothiazin-10-carboxylate
(13). A sample containing 0.74 g (2.90 mmol) of 1-iododecane in 2 mL of acetonitrile
was added to a mixture of 0.40 g (~ 1.16 mmol) of crude 11 in 2 mL of acetonitrile and
1.23 g (11.6 mmol) of Na2CO3. The reaction mixture was sealed under a nitrogen
atmosphere and stirred at 80 oC for 48 h. The cooled reaction mixture was filtered and the
filtrate was concentrated under diminished pressure and purified on a silica gel column (8
× 2 cm). Elution with 1:19 ethyl acetate‒hexanes gave compound 13 as a pale yellow
solid: yield 30 mg (5%); silica gel TLC Rf 0.41 (1:19 ethyl acetate‒hexanes); 1H NMR
(CDCl3) δ 0.81 (t, 6H, J = 6.8 Hz), 1.19 ( m, 28H), 1.22 (s, 9H), 1.40-1.46 (m, 4H), 3.13
(t, 4H, J = 7.6 Hz), 3.69 (s, 3H), 6.42-6.44 (m, 2H), 6.90 (dd, 1H, J = 8.6 and 2.6 Hz),
6.70 (d, 1H, J = 2.8 Hz), 7.21 (d, 1H, J = 8.4 Hz) and 7.30 (d, 1H, J = 8.8 Hz); 13C NMR
(CDCl3) δ 14.1, 22.7, 27.1, 28.2, 29.3, 29.5, 29.6, 31.9, 51.2, 55.5, 81.2, 109.1, 110.2,
111.6, 112.6, 126.8, 127.3, 127.6, 132.4, 132.6, 133.2, 146.3, 153.3 and 157.1; mass
spectrum (APCI), m/z 625.4390 (M+H)+ (C38H61N2O3S requires m/z 625.4403).
4.1.9. 7-(N,N-Didecylamino)-3H-phenothiazin-3-one (2). A sample containing 0.20 mL
(0.20 mmol) of 1 M BBr3 in CH2Cl2 was added dropwise to a solution containing 30.0
mg (0.05 mmol) of compound 13 in 4 mL of CH2Cl2 at ‒78 oC. The reaction mixture was
stirred overnight at ambient temperature and was then quenched with 10 mL of water.
The product was extracted with two 10-mL portions of ethyl acetate. The violet combined
organic phase was washed with 20 mL of brine, dried over anhydrous Na2SO4 and
concentrated under diminished pressure. The resulting violet solid was purified on a silica
22

gel column (7 × 2 cm). Elution with 1:2 ethyl acetate‒hexanes afforded 2 as a violet
solid: yield 9 mg (38%); silica gel TLC Rf 0.50 (1:2 ethyl acetate‒hexanes); 1H NMR
(CDCl3) δ 0.87 (t, 6H, J = 6.8 Hz), 1.24-1.34 (m, 28H), 1.63 (br s, 4H), 3.36 (t, 4H, J =
7.8 Hz), 6.51 (d, 1H, J = 2.8 Hz), 6.66 (d, 1H, J = 2.0 Hz), 6.75-6.82 (m, 2H), 7.53 (d,
1H, J = 9.6 Hz) and 7.46 (d, 1H, J = 9.2 Hz); 13C NMR (CDCl3) δ 14.1, 22.6, 27.0, 27.3,
29.3, 29.4, 29.50, 29.54, 29.7, 31.8, 51.4, 104.7, 113.2, 118.4, 128.6, 130.9, 132.1, 134.8,
135.9, 139.4, 139.6, 149.8 and 182.1; mass spectrum (APCI), m/z 509.3553 (M+H)+
(C32H49N2OS requires m/z 509.3566).
4.1.10. tert-Butyl 3-(N,N-Dipentadecylamino)-7-methoxy-10H-phenothiazin-10-
carboxylate (14). A sample containing 0.98 g (2.90 mmol) of 1-iodopentadecane in 2 mL
of acetonitrile was added to a mixture of 0.40 g (~ 1.16 mmol) of crude 11 in 2 mL of
acetonitrile and 1.23 g (11.6 mmol) of Na2CO3. The reaction mixture was sealed under a
nitrogen atmosphere and stirred at 80 oC for ~ 48 h. The cooled reaction mixture was
filtered and the filtrate was concentrated under diminished pressure. The crude product
was purified on a silica gel column (8 × 2 cm) to separate unreacted starting material.
Elution with 1:19 ethyl acetate_hexanes gave a mixture of mono and dialkylated products
as a yellow oil. The crude product (14) was used for the next reaction without further
purification.
4.1.11. 7-(N,N-Dipentadecylamino)-3H-phenothiazin-3-one (3). A sample containing
0.28 mL (0.28 mmol) of 1 M BBr3 in CH2Cl2 was added dropwise to a solution of 50.0
mg of crude compound 14 in 2 mL of CH2Cl2 at ‒78 oC. The reaction mixture was stirred
overnight at ambient temperature and was then quenched with 10 mL of water. The
product was extracted with two 10-mL portions of ethyl acetate. The combined organic
23

layer was washed with 20 mL of brine, dried over anhydrous Na2SO4 and concentrated
under diminished pressure. The resulting violet solid was purified on a silica gel column.
Elution with 1:4 ethyl acetate‒hexanes afforded 3 as a violet solid: yield 20 mg (44%);
silica gel TLC Rf 0.50 (1:4 ethyl acetate‒hexanes); 1H NMR (CDCl3) δ 0.88 (t, 6H, J =
6.8 Hz), 1.26-1.35 (m, 48H), 1.62-1.64 (m, 4H), 3.37 (t, 4H, J = 7.6 Hz), 6.53 (d, 1H, J =
2.8 Hz), 6.67 (d, 1H, J = 2.0 Hz), 6.76-6.84 (m, 2H), 7.55 (d, 1H, J = 9.6 Hz) and 7.66 (d,
1H, J = 9.2 Hz); 13C NMR (CDCl3) δ 14.3, 22.8, 27.1, 27.5, 29.50, 29.53, 29.6, 29.7,
29.77, 29.79, 29.81, 29.83, 32.1, 51.5, 104.8, 113.3, 118.7, 128.7, 131.0, 132.4, 134.9,
136.0, 139.6, 140.0, 150.0 and 182.4; mass spectrum (APCI), m/z 648.5144 (M+H)+
(C42H69N2OS requires m/z 649.5131).
4.1.14. tert-Butyl 3-(N-Pentadecylamino)-7-methoxy-10H-phenothiazin-10-
carboxylate (15). A sample containing 2.11 g (7.25 mmol) of 1-bromopentadecane in 3
mL of acetonitrile was added to a mixture of 0.50 g (~ 1.45 mmol) of crude compound 11
in 2 mL of acetonitrile and 1.55 g (14.6 mmol) of Na2CO3. The reaction mixture was
sealed under a nitrogen atmosphere and stirred at 80 oC for 3 days. The cooled reaction
mixture was filtered and the filtrate was concentrated under diminished pressure and
purified on a silica gel column. Elution with 1:9 ethyl acetate‒hexanes gave compound
15 as a pale yellow solid: yield 0.14 g (9%); silica gel TLC Rf 0.31 (1:9 ethyl
acetate‒hexanes); 1H NMR (CDCl3) δ 0.87 (t, 3H, J = 6.6 Hz), 1.25 ( br s, 24H), 1.45 (s,
9H), 1.52-1.57 (m, 2H), 3.04 (t, 2H, J = 7.2 Hz), 3.76 (s, 3H), 6.45 (dd, 1H, J = 8.6 and
2.6 Hz), 6.50 (d, 1H, J = 2.8 Hz), 6.76 (dd, 1H, J = 8.8 and 2.8 Hz), 6.83 (d, 1H, J = 2.8
Hz), 7.24 (d, 1H, J = 8.4 Hz) and 7.36 (d, 1H, J = 8.8 Hz); 13C NMR (CDCl3) δ 14.2,
22.8, 27.2, 28.20, 28.27, 28.31, 28.4, 29.46, 29.5, 29.69, 29.70, 29.75, 29.8, 32.0, 44.2,
24

55.7, 81.4, 109.8, 111.65, 111.7, 112.8, 127.6, 127.8, 128.7, 132.5, 132.7, 133.3, 146.7,
153.3 and 157.3; mass spectrum (APCI), m/z 555.3629 (M+H)+ (C33H51N2O3S requires
m/z 555.3620).
4.1.15. tert-Butyl 3-(N-Methyl-N-pentadecylamino)-7-methoxy-10H-phenothiazin-
10-carboxylate (16). A sample containing 7.00 µL (16.0 mg, 0.11 mmol) of iodomethane
was added to a mixture of 53.0 mg (0.10 mmol) of compound 15 in 2 mL of acetonitrile
and 16.0 mg (0.15 mmol) of Na2CO3. The reaction mixture was sealed under a nitrogen
atmosphere and stirred at 80 oC for 16 h. The cooled reaction mixture was filtered, and
the filtrate was concentrated under diminished pressure. The crude product was used in
the next step without further purification.
4.1.16. 7-(N-Methyl-N-pentadecylamino)-3H-phenothiazin-3-one (4). A sample
containing 0.27 mL (0.27 mmol) of 1 M BBr3 in CH2Cl2 was added dropwise to a
solution of 50.0 mg of crude 16 in 2 mL of CH2Cl2 at ‒78 oC. The reaction mixture was
stirred overnight at room temperature and was then quenched with 10 mL of water. The
product was extracted with two 10-mL portions of ethyl acetate. The combined organic
extract was washed with 20 mL of brine, dried over anhydrous Na2SO4 and concentrated
under diminished pressure. The resulting violet solid was purified on a silica gel column
(7 × 2 cm). Elution with 3:7 ethyl acetate‒hexanes afforded 5 as a violet solid: yield 22
mg (54%); silica gel TLC Rf 0.50 (1:4 ethyl acetate‒hexanes); 1H NMR (CDCl3) δ 0.86
(t, 3H, J = 6.4 Hz), 1.24-1.32 (m, 24H), 1.60-1.65 (m, 2H), 3.09 (s, 3H), 3.37 (t, 2H, J =
7.6 Hz), 6.55 (d, 1H, J = 2.8 Hz), 6.68 (d, 1H, J = 2.4 Hz), 6.80-6.84 (m, 2H), 7.54 (d,
1H, J = 9.6 Hz) and 7.67 (d, 1H, J = 9.2 Hz); 13C NMR (CDCl3) δ 14.2, 22.6, 27.1, 27.4,
29.50, 29.55, 29.6, 29.7, 29.77, 29.78, 29.81, 29.83, 51.5, 104.8, 113.3, 118.7, 128.7,
25

131.0, 132.4, 134.8, 136.1, 139.6, 140.2, 150.0 and 182.4; mass spectrum (APCI), m/z
453.2947 (M+H)+ (C28H41N2OS requires m/z 453.2940).
4.1.17. tert-Butyl 3-(N-Butyl-N-pentadecylamino)-7-methoxy-10H-phenothiazin-10-
carboxylate (17). A sample containing 0.11 mL (0.14 g, 1.00 mmol) of 1-bromobutane
was added to a mixture of 90.0 mg (0.16 mmol) of compound 15 in 2 mL of acetonitrile
and 0.17 g (1.60 mmol) of Na2CO3. The reaction mixture was sealed under a nitrogen
atmosphere and stirred at 80 oC for 30 h. The cooled reaction mixture was filtered and the
filtrate was concentrated under diminished pressure. The crude product was used in the
next step without further purification.
4.1.18. 7-(N-Butyl-N-pentadecylamino)-3H-phenothiazin-3-one (5). A sample
containing 0.48 mL (0.48 mmol) of 1 M BBr3 in CH2Cl2 was added dropwise to a
solution of 100 mg (~ 0.16 mmol) of crude 17 in 2 mL of CH2Cl2 at ‒78 oC. The reaction
mixture was stirred overnight at room temperature and was then quenched with 10 mL of
water. The crude product was extracted with two 10-mLportions of ethyl acetate. The
combined organic extract was washed with 20 mL of brine, dried over anhydrous Na2SO4
and concentrated under diminished pressure. The resulting violet solid was purified on a
silica gel column (7 × 2 cm). Elution with 3:7 ethyl acetate‒hexanes afforded 6 as a violet
solid: yield 20 mg (25%); silica gel TLC Rf 0.50 (1:4 ethyl acetate‒hexanes); 1H NMR
(CDCl3) δ 0.88 (t, 3H, J = 6.6 Hz), 1.00 (t, 3H, J = 7.2 Hz), 1.26-1.43 (m, 26H), 1.62-
1.64 (m, 4H), 3.36-3.41 (m, 4H), 6.55 (d, 1H, J = 2.4 Hz), 6.71 (s, 1H), 6.79-6.85 (m,
2H), 7.57 (d, 1H, J = 9.6 Hz) and 7.68 (d, 1H, J = 9.2 Hz); 13C NMR (CDCl3) δ 14.3,
22.8, 27.1, 27.5, 29.50, 29.53, 29.6, 29.7, 29.77, 29.79, 29.81, 29.83, 32.1, 51.5, 104.8,
26

113.3, 118.7, 128.7, 131.0, 132.4, 134.9, 136.0, 139.6, 140.0, 150.0 and 182.4; mass
spectrum (APCI), m/z 495.3330 (M+H)+ (C31H47N2OS requires m/z 495.3331).
4.2. Biochemical and biological evaluation of the MV analogues
4.2.1. Cytotoxicity Assay. Evaluation of methylene blue analogues for their cytotoxicity
and their ability to function within the mitochondrial respiratory chain was carried out by
incubation of the prepared compounds for 24 or 48 h with FRDA lymphocytes. We have
used a nutrient-sensitized screening strategy by culturing FRDA cells in galactose as the
sole sugar source which forces mammalian cells to rely on mitochondrial oxidative
phosphorylation (OXPHOS) to produce their ATP; they also become more sensitive to
mitochondrial respiratory chain inhibitors than cells grown in glucose medium.30,31
Methylene blue analogues were tested for their cytotoxicity in FRDA lymphocytes using
a simultaneous staining with a two-color fluorescence assay, the Live/ Dead®
Viability/Cytotoxicity Kit (Molecular Probes). This assay is used to measure two
recognized parameters of cell viability, intracellular esterase activity and plasma
integrity. The membrane-impermeant DNA dye ethidium homodimer-1 (EthD-1) was
used to identify dead cells whose plasma membrane integrity was disrupted. The
membrane-permeant dye calcein-AM was used to label live cells. It penetrates into the
cells where it is metabolized by cytoplasmic esterases and becomes a fluorescent but
membrane-impermeant probe which is retained in viable cells. One mL of FRDA
lymphocyte cells (5 × 105 cells) was plated in a 24-well plate in glucose free media
(galactose 25 mM), treated with the test compounds and incubated at 37 °C for 24 h or 48
h in a humidified atmosphere containing 5% CO2 in air. Cells were collected by
centrifugation at 300 × g for 3 min and washed with phosphate buffered saline. Cells
27

were resuspended in phosphate buffered saline containing 25 mM galactose. Cell
suspension was stained with 0.1 µM calcein AM and 0.2 µM EthD-1 and incubated in the
dark at 37 °C for 15 minutes. Cells were collected by centrifugation at 300 × g for 3 min
and then washed with PBS. The samples were analyzed immediately by flow cytometry
(C6 Accuri, BD Biosciences, San Jose, CA), using a 488 nm excitation laser and the and
the FL1-H channel 530 15 nm emission filter and the FL2-H channel 585 15 nm. For
each analysis 10,000 events were recorded and analyzed using C6 Accuri software (BD
Biosciences). Results obtained were verified by running duplicates and repeating
experiments in two independent runs.
4.2.2. JC-1 Mitochondrial Membrane Potential (∆ψm) Assay. The ability of the test
compounds to depolarize or maintains mitochondrial inner membrane potential (∆ψm)
was assessed using the JC-1 probe. JC-1 is a cationic dye which exhibits potential-
dependent accumulation in mitochondria. JC-1 is a dual stain, which can identify high
membrane potential through J-aggregates (red fluorescence) and low membrane potential
through J-monomers (green fluorescence). When the ∆ψm collapses, the reagent (JC-1) no
longer accumulates inside the mitochondria; instead, it is diffuses throughout the cell
cytosol in the monomeric form which fluoresces green. The detection of mitochondrial
depolarization using JC-1was accomplished by flow cytometry as described before.24
Briefly, FRDA lymphocytes cells (5 × 105 cells) were pre-treated with or without the test
compounds for 16 h. The cells were incubated at 37 °C in the dark for 20 min with 1 µM
JC-1. Cells were collected by centrifugation at 300 × g for 3 min and washed with
phosphate buffered saline. Cells were resuspended in phosphate buffered saline
supplemented with 20 mM glucose and were analyzed immediately by FACS (C6 Accuri,
28

BD Biosciences, San Jose, CA), using a 488 nm excitation laser and the FL1-H channel
530 15 nm emission filter and the FL2-H channel 585 15 nm. For each analysis 10,000
events were recorded and analyzed using C6 Accuri software (BD Biosciences). FCCP
(carbonyl cyanide p-trifluoromethoxyphenyl hydrazone), a mitochondrial uncouple, was
used to produce a negative control. Results expressed as the percentage of cells with
intact ∆ψm were verified by running duplicates and repeating the experiments in two
independent runs.
4.2.3. Reactive Oxygen Species (ROS). Intracellular ROS production was measured in
FRDA lymphocyte cells (GM15850, Coriell Cell Repositories, Camden, NJ) using the
oxidant sensitive fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA)
(Molecular Probes) as described previously.17,35 One mL of FRDA lymphocyte cells (5 ×
105 cells) was plated in a 24-well plate, treated with the test compounds and incubated at
37 °C for 16 h in a humidified atmosphere containing 5% CO2 in air. Cells were treated
with or without 5 mM diethyl maleate (DEM) for 80 min, collected by centrifugation at
300 × g for 3 min and then washed with phosphate buffered saline (PBS) (Life
Technologies). Cells were resuspended in PBS containing 20 mM glucose and incubated
at 37 °C in the dark for 25 min with 10 µM DCFH-DA. Cells were collected by
centrifugation at 300 × g for 3 min and then washed with PBS. The samples were
analyzed immediately by flow cytometry (C6 Accuri, BD Biosciences, San Jose, CA),
using a 488 nm excitation laser and the FL1-H channel 530 15 nm emission filter for
DCF. The generation of ROS, mainly peroxides, was detected as a result of the oxidation
of DCFH. In each analysis, 10,000 events were recorded after cell debris was
electronically gated out. Results obtained were verified by running duplicates and
29