Amide linked redox-active naphthoquinones for the treatment of mitochondrial dysfunction

Article abstract of DOI:10.1039/c8md00582f

Naphthoquinones have been investigated as potential therapeutic molecules for neurodegenerative disorders, which is largely based on their anti-oxidative potential. However, a theoretical framework for the pleiotropic protective effects of naphthoquinone derivatives is largely missing. We synthesized a library of novel short chain 2,3-disubstituted naphthoquinone derivatives and measured their redox characteristics to identify a potential connection with their biological activity. Using two cell lines with different reducing potential, the compounds were tested for their inherent toxicity, acute rescue of ATP levels and cytoprotective activity. For the first time, a structure-activity-relationship for naphthoquinones has been established. Our results clearly demonstrate that it is the group on the alkyl side chain and not solely the redox characteristics of the naphthoquinone unit or lipophilicity that determines the extent of cytoprotection by individual compounds. From this, we developed a number of amide containing naphthoquinones with superior activity in ATP rescue and cell viability models compared to the clinically used benzoquinone idebenone.

Full text of DOI:10.1039/c8md00582f

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RESEARCH ARTICLE
Amide linked redox-active naphthoquinones for
the treatment of mitochondrial dysfunction†
Cite this: DOI: 10.1039/c8md00582f
Krystel L. Woolley,‡a Monila Nadikudi,‡b Mitra N. Koupaei,b Monika Corban,b
a
a
Paul McCartney,c Alex C. Bissember,
*
Trevor W. Lewis,
*
a
Nuri Gueven
b and Jason A. Smith
Naphthoquinones have been investigated as potential therapeutic molecules for neurodegenerative disor-
ders, which is largely based on their anti-oxidative potential. However, a theoretical framework for the
pleiotropic protective effects of naphthoquinone derivatives is largely missing. We synthesized a library of
novel short chain 2,3-disubstituted naphthoquinone derivatives and measured their redox characteristics to
identify a potential connection with their biological activity. Using two cell lines with different reducing po-
tential, the compounds were tested for their inherent toxicity, acute rescue of ATP levels and
cytoprotective activity. For the first time, a structure–activity-relationship for naphthoquinones has been
established. Our results clearly demonstrate that it is the group on the alkyl side chain and not solely the
redox characteristics of the naphthoquinone unit or lipophilicity that determines the extent of
cytoprotection by individual compounds. From this, we developed a number of amide containing naphtho-
quinones with superior activity in ATP rescue and cell viability models compared to the clinically used ben-
zoquinone idebenone.
Received 26th November 2018,
Accepted 20th December 2018
DOI: 10.1039/c8md00582f
rsc.li/medchemcomm
Mitochondrial membrane potential, which is essential for cel-
lular energy production is maintained by shuttling electrons
1. Introduction
Quinones are widely found in nature as co-factors, antioxi-
dants, signalling molecules and vitamins due to their reversible
redox-characteristics.1–4 Unfortunately, the use of naturally oc-
curring quinones is limited by their pharmacochemical charac-
teristics such as poor solubility, limited bioavailability and
their broad effects on a multitude of physiological processes.2
Furthermore, variation of their chemical and physiochemical
properties can lead to significant differences in pharmacologi-
cal effects often without clear structure–activity relationships
(SAR).1 Coenzyme Q10 (CoQ10) (1) is one of the best known
physiological quinones. CoQ10 (1) acts as a potent, physiologi-
cal antioxidant and is also essential for cellular energy produc-
tion due to its ability to undergo reversible redox reactions with
enzyme complexes of the mitochondrial respiratory chain.5,6
from complex I and II to complexes III and IV in the mitochon-
drial electron transport chain to support the production of
adenosine triphosphate (ATP).7–9
CoQ10 (1), which is responsible for the electron transport
between complexes I, II and III, features a benzoquinone
moiety and a large hydrocarbon side chain comprising ten
isoprenyl units. This highly lipophilic tail limits the effective-
ness of CoQ10 (1) as a potential therapeutic (Fig. 1). For exam-
ple, when administered orally, CoQ10 (1) displays extremely
low solubility in aqueous environments and is only poorly
absorbed.5,8,10 In light of its high lipophilicity, more hydro-
philic benzoquinones, such as idebenone (2) (Fig. 1), are un-
der investigation for indications that are associated with oxi-
dative stress and mitochondrial dysfunction.1–3 Clinical
studies using idebenone (2) reported some encouraging re-
sults in many neuromuscular and mitochondrial disorders
including Friedreich's ataxia (FRDA),11–13 Duchenne muscu-
lar dystrophy (DMD),14–17 multiple sclerosis (MS)18 and
Leber's hereditary optic neuropathy (LHON).1–3 Idebenone
was approved by the European Medicines Agency (EMA) as
treatment for LHON in 2015.19
a School of Natural Sciences – Chemistry, University of Tasmania, Hobart, TAS
7001, Australia. E-mail: Jason.Smith@utas.edu.au; Fax: +61 3 6226 2858;
Tel: +61 3 6226 2182
b School of Medicine – Pharmacy, University of Tasmania, Hobart, TAS 7001,
Australia. E-mail: Nuri.Guven@utas.edu.au; Fax: +61 3 6226 2870;
Tel: +61 3 6226 1715
c Royal Hobart Hospital, Hobart, TAS 7001, Australia
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8md00582f
Unfortunately, upon oral administration, short-chain qui-
nones such as idebenone (2) are subject to rapid liver metabo-
lism.20 Xenobiotics are typically detoxified in the liver by cyto-
chrome P450 enzymes (CytP450) that metabolize compounds
‡ These authors contributed equally.
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Fig. 1 Structures of CoQ10 (1) and Idebenone (2).
by a one electron reduction. However, in the case of quinones,
this results in unstable semiquinones that give rise to the pro-
duction of reactive oxygen species (ROS).2,5 This ROS produc-
tion by semiquinones can damage cellular macromolecules
and can ultimately lead to cell death and tissue dysfunction.21
Most cells express a specialized enzyme, NADIJP)H:quinone ox-
idoreductase 1 (NQO1). Unlike CytP450 enzymes, NQO1 cata-
lyzes a two-electron dependent reduction, which produces the
of the alkyl side chain. These compounds were subjected to
two cellular models to evaluate their activity in ATP rescue
and cell viability after exposure to the mitochondrial Complex
I-inhibitor rotenone. It was demonstrated that the sidechain
had a dramatic effect on the activity in both of these assays,
particularly by incorporation of an amide bond into the
sidechain (Fig. 2) and the results are discussed herein.
hydroquinone.5 It is important to note that only the reduced 2. Results and discussion
hydroquinone form of the molecule is active as an antioxidant
2.1 Chemistry
and electron carrier.10,22 Consequently, it is hypothesized that
A series of short-chain-containing naphthoquinone analogues
the efficient two-electron reduction of quinones has to occur
to avoid cellular toxicity as well as for the compounds to be of
therapeutic value.21,23 However, inactivating NQO1 polymor-
phisms such as 609C > T are prevalent in the general popula-
tion, resulting in decreased or even absent enzymatic activ-
ity.21,23 Therefore, despite the potential of benzoquinones as
therapeutic agents, there is the very real possibility that these
compounds could be ineffective or even toxic in some
individuals.
Like benzoquinones, it has been demonstrated that
naphthoquinones are protective in pre-clinical disease
models by modulating mitochondrial function and reducing
oxidative stress.24–26 Unlike benzoquinones, naphthoquin-
ones, including vitamin K can be reduced to hydroquinones
by two different enzymes in addition to NQO1. Specifically,
vitamin K oxidoreductase 1 (VKORC1) and vitamin K oxidore-
ductase 1 like 1 (VKORC1L1).27–29 This suggests that in indi-
viduals carrying the inactivating NQO1 polymorphism,
naphthoquinones can still be efficiently reduced by VKORC1
and VKORC1L1, which should lower the risk of generating
unstable semiquinones as well as the associated elevated
ROS production and toxicity.
5–7, 9–11 and 18–34 were synthesized from menadione (4) in
a single step (Table 1). The installation of an alkyl side chain
at the 3-position of the naphthoquinone core was facilitated
via a silver-mediated radical decarboxylation.30 All analogues
were purified by flash column chromatography and charac-
terized. The structures of the substituted quinones were
1
supported by H NMR spectroscopy with the absence of a sin-
glet peak at 6.84 ppm indicating the loss of the proton at-
tached to C3 of 2-methyl-1,4-naphthoquinone (4). Additional
analogues 8, 12–17 were formed via standard functional
group manipulations, including epoxidation and esterifica-
tion reactions.
Another class of naphthoquinone analogues were synthe-
sized primarily by coupling acids 20–23 with either ester-
protected amino acids or primary amines. In this way, a li-
brary of amide-containing analogues 35–68 was prepared
(Table 2). The trifluoroacetic acid-mediated deprotection of
t-butyl esters 35–68 afforded acid derivatives 69–81 (Table 3).
2.2 Toxicity
Incomplete quinone reduction to the semiquinone is associ-
ated with ROS production and toxicity.5 Therefore, we
assessed the endogenous toxicity of the simple naphthoquin-
one derivatives 5–34 in two independent cell lines featuring
either high (HepG2) or low (RGC5) reducing capacity. In the
high reducing cell line, HepG2, the IC50 values for each com-
pound were at least 200% of the respective values in the low
reducing cell line, RGC5 (Fig. 3). This is indicative of lower
toxicity and supports the hypothesis that cellular reducing ca-
pacity determines cellular toxicity of naphthoquinones. As an
example, comparing idebenone (2) to the related
naphthoquinone derivative 5, which only differ in the qui-
none moiety, resulted in a doubling of the IC50 value in both
cell lines. (Fig. 3). This increase in the IC50 value illustrates
that in otherwise identical compounds, the naphthoquinone
moiety provides reduced cytotoxicity relative to the
In order to investigate this hypothesis, we synthesized 77
novel short-chain 3-methyl-naphthoquinone derivatives that
vary in the C2-position with differing length and functionality
Fig. 2 Original class of peptide-coupled, redox-active naphthoquin-
ones 3.
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Table 1 Synthesized naphthoquinone analogues from menadione
Compound
R1 =
Compound
R1 =
5
20
6
7
8
9
21
22
23
24
10
11
25
26
12
13
14
15
16
17
18
27
28
29
30
31
32
33
19
34
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Table 2 Analogues formed through the peptide coupling
Compound
n =
Amino acid moeity
Compound
n =
Amino acid moeity
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
2
2
2
2
2
2
2
2
2
2
2
1
3
2
2
4
2
L-Phenylalanine methyl ester
L-Phenylalanine t-butyl ester
L-Proline t-butyl ester
L-Norvaline t-butyl ester
Glycine t-butyl ester
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
2
2
2
2
3
4
2
2
3
3
4
4
2
2
2
2
2
D-Phenylalaninol
D-Phenylglycinol
L-Phenylglycinol
Phenethylamine
L-Phenylalaninol
L-Phenylalaninol
L-Threonine methyl ester
L-Serine methyl ester
3,4-Dimethoxyphenylethylamine
Tyramine
Tyramine
3,4-Dimethoxyphenylethylamine
Butylamine
R-2-Hydroxy-4-amino-butyric acid
trans-4-Hydroxy-L-proline
(S)-Phenylglycine methyl ester
(R)-Phenylglycine methyl ester
DMT-tic-Gly-Benz
L-Leucine t-butyl ester
L-Tyrosine t-butyl ester
L-Tyrosine t-butyl ester dimer
L-Prolinol
L-Phenylalaninol
L-Phenylalanine t-butyl ester
L-Phenylalanine t-butyl ester
Tryptamine
Tyramine
L-Phenylalanine t-butyl ester
3,4-Dimethoxyphenylethylamine
Table 3 Synthesis of amido acid containing naphthoquinones
Compound
n =
R′
Compound
n =
R′
69
70
71
72
73
74
75
2
2
2
2
2
2
2
L-Phenylalanine
L-Proline
L-Norvaline
L-Glycine
L-Leucine
L-Tyrosine
L-Phe-L-pro
76
77
78
1
3
4
2
2
2
L-Phenylalanine
L-Phenylalanine
L-Phenylalanine
D-Phenylalanine
(S)-Phenylglycine
(R)-Phenylglycine
79
80a
81a
a
Amino acid was formed by the Hydrolysis of the methyl ester, see ESI.
benzoquinone moiety. This effect is potentially explained by
the aforementioned more efficient reduction (bioactivation)
of the naphthoquinone by both vitamin K oxidoreductase
(VKORC1) as well as NQO1 in comparison to the benzoqui-
none, which is exclusively reduced by NQO1.1 Although it is
known that HepG2 cells express high levels of NQO1,1 expres-
sion of VKORC1 as the second reductase is unknown for both
cell lines at present.
drial dysfunction in two different cell lines, RGC5 and HepG2.
As rotenone inhibits mitochondrial complex I and therefore
the production of ATP by oxidative phosphorylation it is a pre-
liminary model for diseases associated with dysfunctional
complex I such as LOHN.3 In both cell lines, several novel
naphthoquinones provided increased cytoprotection com-
pared to the reference compound idebenone (2)
(Fig. 4a and b). However, consistent with previous studies fea-
turing benzoquinones,2 no clear structure activity relationship
(SAR) with various functionalities within the initial suite of
analogues 5–9, 11–21, 24–30, 32–35 could be established. Nev-
ertheless, the idebenone-like analogue 5 induced a high level
of cytoprotection, which indicated that this was influenced by
2.3 Cytoprotection against mitochondrial dysfunction
Naphthoquinones 5–9, 11–21, 24–30, 32–35 were evaluated for
their ability to protect against rotenone-induced mitochon-
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Fig. 3 Toxicity of individual naphthoquinones 5–34 measured in RGC5 and HepG2 cell lines. Concentrations are given as [μM]. IC50: Defined as
drug concentration that reduces cellular survival by 50%. Note: Maximal concentration tested: 200 μM.
Fig. 4 Cytoprotection by quinones (5–9, 11–21, 24–30, 32–35) at 10 μM given as a relative percentage cell survival against rotenone induced
complex I dysfunction in HepG2 (a) and RGC5 (b) cell lines.
the introduction of oxygen into the alkyl side chain. Com-
pound 27, which does not contain a terminal alcohol substitu-
ent exhibited little to no protective activity. However, reducing
the alkyl chain length at the 2-position provided an increase
in cytoprotective activity (i.e., molecules 24–26). Following this
observation, derivatives containing shorter, oxygenated alkyl
side-chains (i.e., ≤C10) were synthesised and a number of
these analogues (6 and 7) provided high levels of
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Fig. 5 Cytoprotection by naphthoquinones (10 μM) versus their redox characteristics as determined by cyclic voltammetry in two cell lines RGC5
(a and b) and HepG2 (c and d). each data point is the average of 3 independent experiments with 6 replicates within each experiment for viability
and 1 experiment for cyclic voltammetry (blue dots are for idebenone as a reference). Error bars were omitted for clarity.
cytoprotection (Fig. 4a). Cytoprotection was also observed to
be cell line dependent, as demonstrated for RGC5 cells, which
afforded the highest degree of cytoprotection by three-carbon
ethyl ester 12 (Fig. 4b). In comparison, HepG2 cells showed
Fig. 6 Biological evaluation of polar, aliphatic and acid derivatives in two assays (i) Cytoprotection against rotenone induced complex I
dysfunction by quinones at 10 μM given as a relative percentage of cell survival compared to untreated HepG2 cells (ii) ATP rescue by quinones at
10 μM in the presence of rotenone-induced complex I dysfunction as percentage of untreated HepG2 cells. Data represents the mean of n = 3 in-
dependent experiments with 6 replicates each. Note: Analogues had to provide at least 30% of normal function in either assay to be included in
the above figure. Aliphatic esters 12–17 are not present in this graph as they showed minimal activity in both assays.
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little cytoprotection from this particular analogue, while
idebenone-like structure 5 and hydroxybutyl analogue 6
achieved the highest protection (Fig. 4a). This is surprising in
the light of previous data for benzoquinones that demon-
strated no species or tissue dependence for biological
activity.2
the cytoprotective effects of compounds with their redox
characteristics. Intriguingly, despite significant differences
in the cytoprotective potential of these compounds, no cor-
relation between the redox potentials of each analogue to
its cytoprotective capacity was observed in either cell lines
(Fig. 5a–d). Any major difference of the redox profile for
the compounds was attributed to the functional groups in
the side chain and not the quinone. For example, carboxylic
acids show non-reversible oxidation consistent with electro-
chemical oxidative decarboxylation.31
As no significant difference in redox characteristics was
observed across naphthoquinone analogues 5–9, 11–21,
24–30, and 32–35, this strongly suggests that they do not in-
fluence the cytoprotective activity in the series of
naphthoquinone compounds. This is reinforced by compar-
ing the benzoquinone-containing idebenone (2) (Eox = −0.637
2.4 Cyclic voltammetry
In agreement with previous studies,1 the results from the
two cell lines with different reduction capacity could be an
indication that the reduction of the quinone to the hydro-
quinone is essential for their antioxidant function. This is
hypothesized due to their ability to donate electrons to the
mitochondrial electron transport chain which is believed to
be responsible for cytoprotection.4 Therefore, we correlated
Fig. 7 Biological evaluation of amido ester (a) and amido acid (b) derivatives in two assays (i) Cytoprotection against rotenone induced complex I
dysfunction by quinones at 10 μM given as a relative percentage of cell survival compared to untreated HepG2 cells (ii) ATP rescue by quinones at
10 μM in the presence of rotenone-induced complex I dysfunction as percentage of untreated HepG2 cells. Data represents the mean of n = 3 in-
dependent experiments with 6 replicates each.
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Fig. 8 Biological evaluation of amino alcohol derivatives in two assays (i) Cytoprotection against rotenone induced complex I dysfunction by
quinones at 10 μM given as a relative percentage of cell survival compared to untreated HepG2 cells (ii) ATP rescue by quinones at 10 μM in the
presence of rotenone-induced complex I dysfunction as percentage of untreated HepG2 cells. Data represents the mean of n = 3 independent ex-
periments with 6 replicates each.
V vs. SCE; Ered = −0.714 V vs. SCE) and naphthoquinone-
containing analogue 5 (Eox = −0.768 V vs. SCE; Ered = −0.845 V
vs. SCE). When taken together, these data also indicate that
the specific functionality of the alkyl side chain has a greater
effect on cytoprotective activity than the specific redox-
characteristics of the quinones. This conclusion is in agree-
ment with earlier studies using a range of benzoquinones.2,10
(Fig. 6). A general grouping was observed for aliphatic ana-
logues, which were generally unable to rescue ATP levels in
the presence of the mitochondrial inhibitor rotenone, whilst
having a varied ability to protect cell viability. However, the
analogues containing an acid functionality and the slightly
polar analogues showed varied responses in both assays
(Fig. 6). The polarity of the acid functionality appeared to
show an increase in cytoprotective ability and the capacity to
rescue ATP levels, however, both were still below 100% of
normal function (∼60–90%) (Fig. 6).
2.5 Structure–activity relationships
For this study, protection of viability and acute rescue of ATP
levels were used to establish SAR as both were deemed to in-
dicate normal cell function. Initially, using a limited number
of compounds, no clear structure activity could be
established. The compounds were grouped based on the side
chain functionalities of increasing polarity (aliphatic hydro-
carbons, slight polarity i.e. alcohols, ketones and ethers, car-
boxylic acid and aliphatic esters) to determine whether SAR
based on the functional groups could be established. Within
the initial suite of compounds (5–34), no clear SAR in rela-
tion to either cytoprotection or ATP rescue was established
These results led to the optimization of the current suite
of analogues to generate compounds with high levels of
cytoprotection and the ability to efficiently rescue ATP levels.
In order to increase polarity and potential solubility, an
amino acid fragment was introduced onto the carboxylic acid
of the alkyl side chain. Due to synthetic processes, both the
amido acid and amido ester intermediate were evaluated for
biological activity and resulted in a noticeable trend (Fig. 7).
Analogues 69, 70 and 73, containing amido acid moiety,
showed high levels of cytoprotection ∼90–100% (Fig. 7a) but
no compounds performed well in the ATP rescue assay ∼5–
Fig. 9 Structures of the top five analogues 36, 45, 49, 51 and 69 which were selected to perform further structure activity relationship (SAR)
studies.
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70% (Fig. 7b). However, the less polar amido ester analogues
exhibited the reverse effect, with analogues 41 and 42 show-
ing high levels of activity in the ATP rescue (∼90%), while
not showing high levels of cytoprotection (Fig. 7a). This result
indicates that amido acids may be too polar to provide activ-
ity that increases both ability to restore ATP levels and pro-
vide cytoprotection. From this observation, the synthesis of
amido alcohol derivatives or equivalent amide derivatives to
provide analogues with an intermediate level of polarity was
performed and their activity evaluated (Fig. 8).
In general, these analogues exhibited both very high levels
of cytoprotection (∼90–100%) and also efficiently rescued
ATP levels (80–100%) (Fig. 8), which were all significantly
more active than idebenone (2). Specifically, compounds 45
(L-phenylalaninol), 48 (tryptamine), 49 (tyramine) and 51 (3,4-
dimethylphenylethylamine) provided exceptional results with
these analogues exhibiting normal cell function (∼100%) in
both assays (Fig. 8). The measure of ATP levels after acute,
short term exposure to determine protective activity of a com-
pound is not a reliable indicator for its potential therapeutic
Fig. 10 A comparison of linker length of top five analogues in assays (a) cytoprotection against rotenone induced complex I dysfunction by
quinones at 10 μM given as a relative percentage of cell survival compared to untreated HepG2 cells (b) ATP rescue by quinones at 10 μM in the
presence of rotenone-induced complex I dysfunction as percentage of untreated HepG2 cells. Data represents the mean of n = 3 independent ex-
periments with 6 replicates each.
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use if cells are not viable over a longer period of time. There-
fore a combination of both ATP rescue and cell viability was
used as a representative model to screen potential therapeu-
tics to protect mitochondrial function. Five derivatives, L-
phenylalanine t-butyl ester (36), L-phenylalaninol (45), tyra-
mine (49), 3,4-dimethoxyphenethylamine (51) and L-
phenylalanine (69) derivatives were identified as analogues
utilised for further SAR studies (Fig. 9).
Using these amido moieties, further investigation into
other structural requirements were investigated. From the
toxicity data it was proposed that the naphthoquinone core
was ideal, providing significant less toxicity than the benzo-
quinone core. However, between the core and the amido moi-
eties was a four-carbon linker. The impact the length of the
linker had on the ability of the analogue to provide protec-
tion was unknown. For this reason analogues containing a
three-, five- and six-carbon linkers were synthesised. There-
fore, carboxylic acid analogues 20, 22 and 23 were coupled to
these five amido fragments and subjected to biological
evaulation (Fig. 10).
There was no significant difference between the 3-, 4- and
6-carbon linkers with respect to the 4-carbon linker (Fig. 10).
In general, with exception of the free carboxylic acid deriva-
tives, the 4-carbon linker did provide the highest level of ac-
tivity (Fig. 10). Surprisingly, the free carboxylic acids showed
significant differences in activity. The 5- and 6-carbon carbox-
ylic acid derivatives 22 and 23 featured much improved
activity in both ATP rescue and cytoprotection when com-
pared to the three- and four- carbon carboxylic acid deriva-
tives 20 and 21. In fact, addition of the amine derivative to
the side chain barely had any effect on activity in these cases.
However, these straight-chain carboxylic acid derivatives
would be susceptible to the oxidative degradation observed
for idebenone (2)20 and are still not quite as active as the
amido derivatives. This result combined with the fact that all
initial analogues were sythesised from the four-carbon linker,
suggested that there was no benefit to changing the linker
length to explore any further structure activity relationships
(SARs). Therefore incorprating an amide or peptide bond into
the sidechain has the effect of increasing both the activity of
in ATP rescure and cell viability assays which is optimal for
terating mitochrondrial disorders that inactivate complex I.
However, the role of this structural feature remains unclear
and further investigation is required.
recognized. From the analogues investigated, a variety of im-
portant factors were identified. Through modification of the
linker and the amide functionality, 30 analogues provided
higher levels of cytoprotection than that of the lead com-
pound idebenone (2) which is approved for treatment of
LOHN in Europe. Development of SAR has given a clear indi-
cation that although the quinone core is essential to be able
to shuttle electrons through the electron transport chain, it
does not influence the analogue's ability to provide
cytoprotection or restore ATP levels. This was also supported
by Erb et al. when investigating idebenone derivatives.2
Similarly, a 4-, 5- and 6-carbon linkers had little effect on
activity. The most significant outcome has been the effect
that groups in the side-chain have on the ability for an ana-
logue to provide both cytoprotection and restore ATP levels. A
balanced level of polarity in the side chain resulted in signifi-
cantly increased cytoprotection when compared to ideben-
one. Although previous literature indicates the importance of
an analogue's ability to restore ATP levels as the key factor,
little emphasis was placed on cytoprotection as it was as-
sumed that increased ATP would result in cytoprotection.
However, this study suggests that this is not the case. While
ATP rescue is a rapid one-hour assay, it is not a realistic mea-
sure if the cells do not survive over time with cytoprotection
being a five-day assay. This study has demonstrated the im-
portance of the side-chain to tune/modify activity indepen-
dently of the quinone core, with numerous examples demon-
strating increased ATP but no increase in cytoprotection.
Therefore, our results can direct the rational development of
novel short-chain quinone compounds for the treatment of
disorders associated with mitochondrial dysfunction.
4. Experimental
4.1 General
A representative sample of compounds are reported in the ex-
perimental section. The synthesis and spectral data for all
other compounds is available in the supplementary
information.
NMR experiments were performed either on a Bruker
Avance III NMR spectrometer operating at 400 MHz (1H) or
100 MHz (13C) or on a Bruker Avance III NMR spectrometer
operating at 600 MHz (1H) or 150 MHz (13C). The deuterated
solvent used was CDCl3 unless otherwise specified. Chemical
shifts were recorded in ppm. Spectra were calibrated by as-
signment of the residual solvent peak to δH 7.26 and δC 77.16
for CDCl3. Coupling constants (J) were recorded in Hz. Infra-
red spectrometry was performed on a Shimadzu FTIR 8400 s
spectrometer, with samples analyzed either as thin films on
NaCl plates or using an ATR attachment. ESIMS analyses
were conducted on a Thermo-Scientific LTQ-Orbitrap mass
spectrometer. EIMS analyses were performed using a Kratos
Analytical Concept ISQ hybrid magnetic sector quadrupole
tandem or Shimadzu GCMS-QP2010 mass spectrometers.
TLC was performed using Merck silica gel 60-F254 plates.
Developed TLC plates were visualized by UV absorbance (254
3. Conclusion
Our results indicate that the increased protective activity and
reduced toxicity of some naphthoquinones are likely the re-
sult of enhanced bioactivation of naphthoquinones by both
VKOR enzymes and NQO1, which is a highly cell-type depen-
dent process. For the first time, we report a SAR of naphtho-
quinones for cell viability and ATP rescue for mitochondrial
dysfunction. Our results indicate that the functionality of the
alkyl side chain determines the cytoprotective potential of in-
dividual compounds to a much larger extent than previously
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nm) or through application of heat to a plate stained with ce-
rium molybdate {CeIJNH4)2IJNO3)6, (NH4)6Mo7O24·4H2O, H2SO4,
H2O}. Flash column chromatography was performed with
flash grade silica gel (60 μm) and the indicated eluent in ac-
cordance with standard techniques. Unless otherwise speci-
fied, reactions were conducted with magnetic stirring under
N2. Unless otherwise specified all reagents employed in these
studies were used as received from Sigma-Aldrich, AK Scien-
tific, combi-blocks, and oakwood and were used without
purification.
28.9, 29.5, 29.6, 29.7, 30.1, 32.9, 63.2, 126.3, 126.4, 132.3,
132.4, 133.4, 133.5, 143.3, 147.7, 184.9, 185.5 (one carbon
overlapping); νmax/cm−1: 3525, 2917, 2848, 1658, 1618, 1593,
1459, 1738, 1327, 1297, 717 cm−1; HRMS: for C21H28O3, pre-
dicted 328.20384, found 328.20383, m/z 328 (M+, 62%), 310
(5), 211 (10), 187 (100), 174 (12) 158 (18).
4-(3-Methyl-naphthoquione-2-yl)butanoic
acid
(21).
Naphthoquinone 21 was prepared according to general
procedure A from menadione (4) (5.998 g, 34.83 mmol) and
glutaric acid (4.611 g, 34.89 mmol) and the product purified
by flash chromatography (100% dichloromethane followed by
100% ethyl acetate) to give 21 as a bright yellow crystalline
solid in 62% yield (5.579 g, 21.67 mmol). whose spectral data
were consistent with that reported previously; (ref. 32) δH
(400 MHz; CDCl3): 1.82 (quin, J = 7.4 Hz, 2H), 2.20 (s, 3H),
2.46 (t, J = 7.4 Hz, 2H), 2.69 (t, J = 7.4 Hz, 2H), 7.66–7.69 (m,
2H), 8.04–8.06 (m, 2H); δC (100 MHz, CDCl3): 12.8, 23.5, 26.4,
33.8, 126.4, 126.5, 132.2, 132.28, 133.61, 133.62, 144.1, 146.2,
179.3, 184.7, 185.3; νmax/cm−1: 3064, 2938, 1706 (CO), 1658
(CO), 1616, 1595, 1412, 1379, 1295, 1260, 717, 660 cm−1
(S)-tert-Butyl-2-(4-(3-methyl-1,4-naphthoquinone-2-
yl)butanamido)-3-phenylpropanoate (36). Naphthoquinone 36
4.2 Chemistry
General Procedure A: Silver-mediated radical decarboxyl-
ation general method. Carboxylic acid (2 equiv.) was added to
a magnetically stirred solution of menadione (1 equiv.) in
CH3CN/H2O (3 : 1) and the ensuing mixture was heated to 75
°C. AgNO3 (0.1 equiv.) was then added, followed by the slow
addition of (NH4)2S2O8 (2.5 equiv.) in H2O (5 mL) over 10
min. The resulting mixture was stirred for a further 1 h. The
mixture was cooled to room temperature, extracted with
CH2Cl2 and the organic extract washed with H2O. The organic
layer was dried (MgSO4), filtered and the solvent removed un-
der reduced pressure to give the crude product, which was
purified by flash column chromatography (silica gel).
General procedure B: quinone amide coupling general
method. Quinone acid (1 equiv.) was added to anhydrous
CH2Cl2 (5–10 ml) under an atmosphere of N2 and cooled to
0 °C. Amine derivative (1 equiv.), DMAP (0.1 equiv.), triethyl-
amine (Et3N, 2.5 equiv.) and either EDCI, BOP or PyBOP (1.4
equiv.) were added successively and the reaction mixture
warmed slowly to room temperature before leaving overnight.
The reaction was quenched with H2O (20 mL) and the or-
ganic layer was washed with KHSO4 (saturated aqueous solu-
tion), NaHCO3 (saturated aqueous solution) and H2O. The or-
ganic layer was dried (MgSO4), filtered and the solvent
removed under reduced pressure to give the crude product,
which was purified by flash column chromatography (silica
gel) to give the desired amide.
was prepared according to general procedure
B from
4-(3-methyl-1,4-naphthalen-2-yl)-butanoic acid (21) (504.2 mg,
1.9522 mmol) and L-phenylalanine t-butyl ester. HCl (489.4
mg, 1.9023 mmol) and the product purified by flash chroma-
tography (40% ethyl acetate/hexanes) to give 36 as yellow oil
in 36% yield (317 mg, 0.687 mmol).
δH (400 MHz; CDCl3): 1.40 (s, 9H), 1.78 (quin, J = 7.6 Hz,
2H), 2.17 (s, 3H), 2.27 (t, J = 7.6 Hz, 2H), 2.60–2.62 (m, 2H),
3.04–3.13 (m, 2H), 4.74–4.79 (m, 1H), 6.09 (d, J = 7.8 Hz, 1H),
7.14–7.27 (m, 5H), 7.66–7.69 (m, 2H), 8.04–8.07 (m, 2H); δC
(100 MHz, CDCl3): 12.8, 24.3, 26.3, 28.0, 36.1, 38.2, 53.5, 82.4,
126.3, 126.4, 127.0, 128.4 (2 × C), 129.5 (2 × C), 132.21,
132.26, 133.4, 133.5, 136.3, 144.0, 146.4, 170.9, 171.8, 184.8,
185.3; [α]D:20 +36.24° (c 0.91, CHCl3); νmax/cm−1: 3420 (N–H),
2978, 1732 (CO), 1658 (CO), 1595, 1525, 1367, 1329,
1294, 1257, 1226, 1155, 700 cm−1; HRMS [M + Na]: for
C28H31N1O5Na, predicted 484.2100, found 484.2110
General procedure C: t-butyl ester deprotection method.
The t-butyl esters were added to 10% TFA in CH2Cl2 (5.0 mL)
and the reaction mixture was stirred at room temperature
overnight. After this time, the solvent removed under reduced
pressure. The crude product was purified by flash column
chromatography (silica gel) to give the carboxylic acid
derivative.
(S)-2-(4-(3-Methyl-1,4-naphthoquinone-2-yl)butanamido)-3-
phenylpropanoic acid (7a). Naphthoquinone 69 was prepared
from the deprotection of 36 (317.3 mg, 0.6875 mmol), using
general procedure C. The product was purified by flash
chromatography (5% methanol/ethyl acetate) to give 69 as
brown viscous oil in 79% yield (220 mg, 0.542 mmol).
δH (400 MHz; CDCl3): 1.72–1.79 (m, 2H), 2.14 (s, 3H), 2.29
(t, J = 7.2 Hz, 2H), 2.58 (t, J = 7.8 Hz, 2H), 3.12 (dd, J = 14.0,
7.0 Hz, 1H), 3.26 (dd, J = 14.0, 5.4 Hz, 1H), 4.90 (m, 1H), 6.54
(d, J = 7.7 Hz, 1H), 7.17–7.28 (m, 5H), 7.66–7.69 (m, 2H),
8.02–8.05 (m, 2H), 8.92 (bs, 1H); δC (100 MHz, CDCl3): 12.8,
24.2, 26.2, 35.8, 37.3, 53.4, 126.4, 127.2, 128.7 (2 × C), 129.4
(2 × C), 132.0, 132.2, 133.5, 133.6, 135.9, 144.3, 146.2, 173.5,
174.7, 185.1, 185.2; [α]D:20 +35.83° (c 0.24, CHCl3); νmax/cm−1:
3491 (N–H), 2931, 1716 (CO), 1660 (CO), 1616, 1595,
1521, 1456, 1332, 1296, 1267, 1217, 702 cm−1; HRMS [M +
Na]: for C24H23N1O5Na, predicted 428.1474, found 428.1484.
2-(10-Hydroxydecyl)-3
methyl-1,4-naphthoquinone
(5).
Naphthoquinone was prepared according to general
5
procedure A from menadione (201 mg, 1.17 mmol) and 11-
hydroxyundecanoic acid (467 mg, 2.30 mmol) and the
product purified by flash chromatography (40% ethyl acetate/
hexanes) to give 5 as a pale yellow solid in 19% yield (168
mg, 0.511 mmol). mp 74–75 °C.
δH (400 MHz; CDCl3): 1.24–1.57 (m, 16H), 2.17 (s, 3H),
2.61 (t, J = 7.0 Hz, 2H), 3.62 (t, J = 6.6 Hz, 2H), 7.65–7.69 (m,
2H), 8.04–8.07 (m, 2H); δC (100 MHz, CDCl3): 12.8, 25.8, 27.2,
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(S)-N-(1-Hydroxy-3-phenylpropan-2-yl)-4-(3-methyl-1,4-
4.3 Cell culture
naphthyoquinone-2-yl)butanamide (45). Naphthoquinone 45
Human hepatic carcinoma cells (HepG2, ECACC) and rodent
retinal precursor cells (RGC5, ATCC) were used for this study.
Cells were cultivated under standard conditions (95% humid-
ity, 5% CO2, 37 °C) in DMEM media containing 5% FCS (for
RGC5) or 10% FCS (for HepG2) to achieve comparable growth
rates. Both cell lines were selected based on their high
(HepG2) and low reducing capacity (RGC5), which is associ-
ated with high and low expression of NQO1 (data not
shown).1,3
was prepared according to general procedure
B from
4-(3-methyl-1,4-naphthalen-2-yl)-butanoic acid (21) (133.5 mg,
5169 mmol) and L-phenyl analinol (76.4 mg, 0.5053 mmol)
and the product purified by flash chromatography (100%
ethyl acetate) to give 45 as yellow/orange oil in 49% yield
(97.7 mg, 0.2496 mmol).
δH (400 MHz; CDCl3): 1.70–1.78 (m, 2H), 2.15 (s, 3H), 2.25 (t, J
= 7.2 Hz, 2H), 2.55 (t, J = 8.0 Hz, 2H), 2.83–2.94 (m, 2H), 3.03 (bs,
1H), 3.59 (dd, J = 11.2, 5.4 Hz, 1H), 3.71 (dd, J = 11.2, 3.8 Hz,
1H), 4.21–4.29 (m, 1H), 6.31 (d, J = 8.0 Hz, 1H), 7.15–7.27 (m,
5H), 7.64–7.69 (m, 2H), 8.00–8.05 (m, 2H); δC (100 MHz, CDCl3):
12.7, 24.3, 26.2, 36.2, 37.0, 52.9, 64.1, 126.35, 126.36, 126.6, 128.6
(2 × C), 129.2 (2 × C), 132.0, 132.1, 133.5, 133.6, 137.9, 144.2,
4.4 Compound toxicity
To assess the toxicity inherent to these compounds, IC50
values (defined as the concentration necessary to reduce cell
viability by 50%) were generated using a commercially avail-
able viability assay according to the manufacturers' recom-
mendations (WST-1, Roche Diagnostics).
146.2, 173.1, 185.12, 185.13; [α]D:20 −21.33° (c 1.57, CHCl3); νmax
/
cm−1: 3369 (N–H), 3296 (–OH), 2933, 1658 (CO), 1595, 1539,
1456, 1377, 1330, 1296, 1043, 717, 702 cm−1; HRMS [M + Na]: for
C24H25N1O4Na, predicted 414.1676, found 414.1667.
4.5 Cytoprotection in the presence of mitochondrial
dysfunction
N-(4-Hydroxyphenylethyl)-4-(3-methyl-1,4-naphthoquinone-2-
yl) butanamide (49). Naphthoquinone 49 was prepared
according to general procedure
B from 4-(3-methyl-1,4-
Cytoprotection against mitochondrial dysfunction was mea-
sured as previously described.3 Briefly, 5000 HepG2 cells per
well were preincubated with quinones (10 μM) in a 96 well
plate for 2 days prior to being challenged by the mitochon-
drial complex I inhibitor rotenone (1 μM) for 6 hours in
Hanks balanced salt solution (HBSS). After post incubation
with only quinones in HBSS for additional 24 h, cell viability
was quantified by analysing ATP content per well using a
naphthalen-2-yl)-butanoic acid (21) (235.6 mg, 0.9122 mmol)
and tyramine (119.0 mg, 0.8675 mmol) and the product
purified by flash chromatography (80% ethyl acetate/hexanes)
to give 49 as yellow solid in 33% yield (108.9 mg, 0.2885
mmol). mp 116–118 °C.
δH (400 MHz; CDCl3): 1.76 (quin, J = 7.5 Hz, 2H), 2.12 (s, 3H),
2.24 (t, J = 7.2 Hz, 2H), 2.56–2.60 (m, 2H), 2.71 (t, J = 7.0 Hz,
2H), 3.44–3.49 (m, 2H), 6.22 (t, J = 5.5 Hz, 1H), 6.75 (d, J = 8.4
Hz, 2H), 6.96 (d, J = 8.4 Hz, 2H), 7.62–7.65 (m, 2H), 7.97–8.00
(m, 2H); 13C NMR δC (100 MHz, CDCl3): 12.7, 24.4, 16.3, 34.7,
36.1, 41.1, 115.7, 126.3, 129.8 (2 x C), 129.9, 132.0, 132.1, 133.53,
133.59, 144.2, 146.2, 155.3, 173.1, 185.0, 185.2; νmax/cm−1: 3365
(N–H), 3306 (–OH), 2935, 1654 (CO), 1616, 1595, 1541, 1516,
1375, 1330, 1296, 715 cm−1; HRMS [M + Na]: for C23H23N1O4Na,
predicted 400.1519, found 400.1510.
luciferase-based
reaction
as
described
previously.3
Cytoprotection is displayed as a percentage of the untreated
(no rotenone) control. Data represent the average of 3 inde-
pendent experiments with n = 6 wells within each experi-
ment. Error bars = SD.
4.6 Acute rescue of ATP levels
Acute rescue of ATP levels in the presence of the mitochon-
drial inhibitor rotenone was measured as previously de-
scribed.2 Briefly, 15 000 HepG2 cells per well were seeded in a
96 well plate and allowed to attach overnight. After 24 h, cells
were incubated with quinones (10 μM) along with the mito-
chondrial complex I inhibitor rotenone (10 μM) for 1 hour in
glucose-free growth media before immediately measuring
ATP levels using a luciferase-based reaction as described pre-
viously.2 Acute rescue of ATP levels is displayed as a percent-
age of the untreated (no rotenone) control. Data represent
the average of 3 independent experiments with n = 6 wells
within each experiment. Error bars = SD.
N-(3,4-Dimethoxyphenylethyl)-4-(3-methyl-1,4-naphthoquin-
one-2-yl)butanamide (6r). Naphthoquinone 51 was prepared
according to general procedure
B from 4-(3-methyl-1,4-
naphthalen-2-yl)-butanoic acid (21) (187.5 mg, 0.7260 mmol)
and 3,4-dimethoxyphenylethylamine (146.6 mg, 0.8088 mmol)
and the product purified by flash chromatography (90% ethyl
acetate/hexanes) to give 51 as pale orange crystalline solid in
38% yield (117.0 mg, 0.2776 mmol). mp 105–108 °C.
δH (400 MHz; CDCl3): 1.77 (quin, J = 7.2 Hz, 2H), 2.16 (S,
3H), 2.22 (t, J = 7.2 Hz, 2H), 2.58–2.62 (m, 2H), 2.74 (t, J = 7.2
Hz, 2H), 3.46–3.51 (m, 2H), 3.79 (s, 3H), 3.81 (s, 3H), 5.94 (t, J
= 5.6 Hz, 1H), 6.68–6.76 (m, 3H), 7.63–7.66 (m, 2H), 7.98–8.02
(m, 2H); δC (100 MHz, CDCl3): 12.7, 24.3, 26.3, 35.2, 36.1,
40.7, 55.8, 55.9, 111.4, 111.9, 120.7, 126.25, 126.29, 131.4,
132.0, 132.1, 133.4, 133.5, 144.0, 146.2, 147.7, 149.0, 172.3,
184.8, 185.1; νmax/cm−1: 3377 N–H), 3296, 2935, 1656 (CO),
1595, 1516, 1462, 1329, 1294, 1261, 1236, 1157, 1141, 1028,
717 cm−1; HRMS [M + Na]: for C25H27N1O5Na, predicted
444.1781, found 444.1773.
4.7 Cyclic voltammetry
Cyclic voltammetry (CV) studies were carried out using a
Metrohm 797 VA fitted with
a glassy carbon working
electrode, a platinum auxiliary electrode and a saturated calo-
mel reference electrode (SCE). Measurements were performed
at room temperature in 20 mL of a 0.1 M NBu4ClO4 solution
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in CH3CN containing the naphthoquinones at a concentra-
tion of 1 mM. The electrochemical cell was deoxygenated by
purging with N2 for 2 minutes before scanning between
−0.850 and 0.500 V (vs. SCE) at a scan rate of 100 mV s−1. The
electrodes and measurement cell were rinsed with CH3CN be-
tween each experiment.
10 D. M. Fash, O. M. Khdour, S. J. Sahdeo, R. Goldschmidt, J.
Jaruvangsanti, S. Dey, P. M. Arce, V. C. Collin, G. A.
Cortopassi and S. M. Hecht, Bioorg. Med. Chem., 2013, 21,
2346–2354.
11 T. Palecek, M. Fikrle, E. Nemecek, L. Bauerova, P. Kuchynka,
W. E. Louch, I. Spika and R. Rysava, Curr. Pharm. Des.,
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Author contributions
13 D. R. Lynch, S. L. Perlman and T. Meier, Arch. Neurol.,
2010, 67, 941–947.
Conceived and designed the experiments: JAS, NG, KLW, MN.
Performed the experiments: KLW, MN, MNK, MC. Analysed
the data: KLW, MN. Contributed reagents/materials/analysis
tools: JAS, NG, TWL, AB. Wrote the paper: KLW, MN, NG, AB,
JAS.
14 G. M. Buyse, N. Goemans, M. van den Hauwe, D. Thijs,
I. J. M. de Groot, U. Schara, B. Ceulemans, T. Meier and L.
Mertens, Neuromuscular Disord., 2011, 21, 396–405.
15 G. M. Buyse, T. Voit, U. Schara, C. S. M. Straathof, M. G.
D'Angelo, G. N. Bernert, J.-M. Cuisset, R. S. Finkel, N.
Goemans, C. Rummey, M. Leinonen, O. H. Mayer, P.
Spagnolo, T. Meier and C. M. McDonald, Pediatr. Pulmonol.,
2016, 52, 508–515.
16 C. M. McDonald, T. Meier, T. Voit, U. Schara, C. S. M.
Straathof, M. G. D'Angelo, G. Bernert, J.-M. Cuisset, R. S.
Finkel, N. Goemans, C. Rummey, M. Leinonen, P. Spagnolo
and G. M. Buyse, Neuromuscular Disord., 2016, 26, 473–480.
17 G. M. Buyse, T. Voit, U. Schara, C. S. M. Straathof, M. G.
D'Angelo, G. Bernert, J.-M. Cuisset, R. S. Finkel, N. Goemans,
C. M. McDonald, C. Rummey and T. Meier, Lancet, 2015, 385,
1748–1757.
Conflicts of interest
Associate Professor Nuri Gueven acts as scientific consultant
to Santhera Pharmaceuticals (Pratteln, Switzerland) that de-
velops idebenone for diverse neuromuscular and mitochon-
drial indications.
Acknowledgements
We gratefully acknowledge the Royal Hobart Hospital Re-
search Foundation (RHHRF) and the University of Tasmania
School of Natural Sciences – Chemistry for financial support
and the University of Tasmania Central Science Laboratory
for providing access to NMR spectroscopy services. K. L. W.
thanks the University of Tasmania for a Tasmanian Graduate
Research Scholarship. We thank Santhera Pharmaceuticals
(Pratteln, Switzerland) for providing us with idebenone.
18 S. M. Fiebiger, H. Bros, T. Grobosch, A. Janssen, C.
Chanvillard, F. Paul, J. Dörr, J. M. Millward and C. Infante-
Duarte, J. Neuroimmunol., 2013, 262, 66–71.
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