Synthesis of analogs of the radiation mitigator JP4-039 and visualization of BODIPY derivatives in mitochondria

Article abstract of DOI:10.1039/c3ob40489g

JP4-039 is a lead structure in a series of nitroxide conjugates that are capable of accumulating in mitochondria and scavenging reactive oxygen species (ROS). To explore structure-activity relationships (SAR), new analogs with variable nitroxide moieties were prepared. Furthermore, fluorophore-tagged analogs were synthesized and provided the opportunity for visualization in mitochondria. All analogs were tested for radioprotective and radiomitigative effects in 32Dcl3 cells.

Full text of DOI:10.1039/c3ob40489g

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Synthesis of analogs of the radiation mitigator JP4-039
and visualization of BODIPY derivatives in
mitochondria†
Cite this: DOI: 10.1039/c3ob40489g
Received 9th March 2013,
Accepted 15th May 2013
Marie-Céline Frantz,a Erin M. Skoda,a Joshua R. Sacher,a Michael W. Epperly,b
DOI: 10.1039/c3ob40489g
Julie P. Goff,b Joel S. Greenbergerb and Peter Wipf*a,c
www.rsc.org/obc
JP4-039 is a lead structure in a series of nitroxide conjugates that
are capable of accumulating in mitochondria and scavenging
reactive oxygen species (ROS). To explore structure–activity
relationships (SAR), new analogs with variable nitroxide moieties
were prepared. Furthermore, fluorophore-tagged analogs were
synthesized and provided the opportunity for visualization in
mitochondria. All analogs were tested for radioprotective and
radiomitigative effects in 32Dcl3 cells.
Introduction
The 4-amino-TEMPO (4-AT) derivative JP4-039 is a promising
lead among an emerging family of mitochondria-targeted nitr-
oxides whose structures were inspired by the antibiotic grami-
cidin S (GS) and its microbial membrane affinity (Fig. 1).1,2
The first generation hybrid molecule XJB-5-131 is composed of
an alkene peptide isostere that mimics the leucyl-D-phenyl-
alanine dipeptide in GS, a side chain-protected ornithylvalyl-
proline tripeptide taken directly from GS, and a nitroxide
subunit. The truncated analog JP4-039 contains a shortened
alkene dipeptide isostere moiety directly attached to the nitr-
oxide. The nitroxide fulfills several critical functions. It is
effective at catalyzing the dismutation of superoxide radical
anions and other reactive oxygen species (ROS) generated in
mitochondria into H2O2.3 Nitroxides also trap electrons and
scavenge radicals escaping from the oxidative phosphorylation
(OXPHOS) pathway. In solution, nitroxides are in a dynamic
equilibrium with hydroxylamines and nitroxonium species,
which also respond to the redox state of the environment. GS
Fig. 1 Structural correlations between gramicidin S, XJB-5-131, and JP4-039.
adopts a type II′ β-turn structure that buries several polar
amide groups inside the molecule and thus may facilitate
membrane transport.4,5 Both XJB-5-131 and JP4-039 fold into a
β-turn secondary structure in the solution and solid state,
respectively, and exhibited enrichments of 30- to 600-fold in
mitochondria.1,6 Localization of ROS trapping agents to mito-
chondria is important since ca. 90% of all ROS are generated
in these ATP-producing cell organelles.7
Our previous studies demonstrated that XJB-5-131 is
superior to 4-AT or TEMPO alone in preventing actinomycin
D-induced DNA fragmentation and upregulation of caspase 3,
thus reducing apoptosis and enhancing cell survival in mouse
embryonic cells (MECs).1a In addition to its antiapoptotic1a
and radioprotective2 activities in cells and in mice, XJB-5-131
can also prolong survival in a rat model of lethal hemorrhagic
shock.8 XJB-5-131 readily partitions into the brain and shows
promising efficacy in improving neurocognitive outcome after
traumatic brain injury (TBI) in rats by preventing cardiolipin
oxidation and reducing neuronal death.9a,b Furthermore,
aDepartment of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA.
E-mail: pwipf@pitt.edu
bDepartment of Radiation Oncology, University of Pittsburgh Cancer Institute,
Pittsburgh, PA 15232, USA
cDepartment of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh,
PA 15260, USA
†Electronic supplementary information (ESI) available: Experimental procedures
and complete spectroscopic data for new compounds and protocols for
irradiation survival curves. CCDC 928524. For ESI and crystallographic data in
CIF or other electronic format see DOI: 10.1039/c3ob40489g
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Fig. 2 Structures of stable cyclic nitroxides.
XJB-5-131 proved to be an effective therapeutic in a mouse
model of Huntington’s disease (HD) by enhancing neuronal
survival and suppressing oxidative damage to mitochondrial
DNA as well as motor decline.9c JP4-039, in addition to having
a lower molecular weight and greater solubility, retains many
of the desired physiological effects of XJB-5-131, including
radiation damage prevention and mitigation.2,10,11
Scheme 1 Synthesis of TMIO analog 12.
Preparation of 3-amino-ABNO (6) was envisioned from the
known ketone 13, which was easily accessible in one step from
acetonedicarboxylic acid and glutaraldehyde (Scheme 2).19
Conversion of 13 to oxime 14, followed by reduction to the
amine with nickel boride and N-Boc protection provided carb-
amate 15 in high yield. Removal of the benzyl group by cataly-
tic hydrogenolysis and oxidation of the resulting secondary
amine in the presence of urea–hydrogen peroxide complex
(UHP) furnished the Boc-protected nitroxide 16.20 However,
once the Boc group was removed, the resulting 3-amino-ABNO
(6) was found to be unstable,21,22 thus preventing its use in the
coupling step with 11.
To avoid the problem of the instability of amino nitroxide
6, the synthetic route to obtain this ABNO analog was modified
so that the nitroxide could be formed in the last step after
N-acylation. Reduction of oxime 14 and EDCI coupling of
the resulting primary amine with acid 11 were readily
accomplished and gave amide 18 in 57% yield over 3 steps
Aside from some early, closely related analogs of XJB-5-131
that established the function of the peptide isostere, the SAR
of these promising compounds has not been thoroughly
studied. The recent development of a large-scale JP4-039 syn-
thesis allowed for the preparation of several cyclopropane and
fluorinated analogs,12 but we had yet to modify the radical/
electron scavenging nitroxide moiety of these molecules.
Alternatives to 4-AT, namely the more stable isoindoline-based
TMIO (1),13 as well as the more reactive azabicyclic ABNO (5)14
and the azaadamantane 1-Me-AZADO (7)15 were selected for
this purpose (Fig. 2). ABNO and 1-Me-AZADO were first syn-
thesized by Dupeyre and Rassat,14a,15a and then further develo-
ped as oxidation catalysts by Iwabuchi et al.14d,15b,c They
oxidize alcohol substrates more quickly and more efficiently
than TEMPO or TEMPOL (3) due to their decreased steric hin-
drance at the nitroxide/nitroxonium reaction center. The
different ring sizes and electron-withdrawing effects of the
R-substituents in nitroxides 1–8 may also contribute to their
modified redox properties.13a,16
For the detection of GS-nitroxides in cell tissue samples, we
had previously utilized spectroscopic methods such as EPR
and MS.1,2,6 In order to more thoroughly investigate the ability
of these nitroxides to partition in tissue and cells and localize
in mitochondria, we required a fluorescence-based visualiza-
tion tool. Many fluorophores are charged, and since charge
can affect mitochondrial uptake, an overall net charge of zero
should allow for unaffected mitochondrial uptake studies. We
selected BODIPY® fluorophores for this purpose.17,18
Results and discussion
Synthesis of new nitroxide analogs
Starting with commercially available alcohol 9, homoallylic
alcohol 10 was synthesized in 5 steps according to known pro-
cedures.12 Nitroxide analogs of JP4-039 were synthesized in
two steps by Jones oxidation of 10 to the acid and EDCI-
mediated coupling with amine-functionalized building blocks.
In this fashion, TMIO analog 12 could be readily obtained
from 10 and 5-amino-TMIO (2)13c–e in 67% yield (Scheme 1).
Scheme 2 Synthesis of unstable nitroxide 6 (3-amino-ABNO).
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Scheme 5 Synthesis of 1-Me-AZADO analog 25: (A) failed coupling of 8 with
11, and (B) successful coupling of 26 with 11.
Scheme 3 Synthesis of ABNO analog 17.
(Scheme 3). Benzyl amine cleavage with ceric ammonium
nitrate (CAN), followed by UHP-assisted oxidation to the nitr-
oxide then provided the desired ABNO analog 17 in 69% yield.20
The preparation of the azaadamantyl-based nitroxide 8
(6-amino-1-Me-AZADO) was achieved in 10 steps from adaman-
tane carboxylic acid (Scheme 4).23 Selective dibromination of
the adamantane ring in neat bromine at reflux required the
presence of 2 equiv. of AlBr3 and was further accelerated by
addition of catalytic BBr3.24 Curtius rearrangement of dibromo
acid 19 provided the amine function for the attachment of the
alkene peptide isostere targeting sequence. Grob fragmenta-
tion25 of 20 in an autoclave at 160 °C followed by N-Boc protec-
tion led to the keto-alkene 21 in 42% yield over 4 steps.
According to Iwabuchi’s procedure,15b,c oxime formation led to
22, and reduction with NaBH4/MoO3 followed by iodoamina-
tion of the alkene furnished the azaadamantane core 23. After
removal of the iodine atom by catalytic hydrogenation, oxi-
dation of the crude secondary amine with sodium tungstate
Fig. 3 X-ray structure of 25 (CCDC 928524).
and hydrogen peroxide led to the Boc-protected nitroxide 24 in
58% yield over 2 steps. Deprotection of 24 with HCl in dioxane
completed the synthesis of 8.
Unfortunately, similar to the difficulties encountered with
6, coupling of 8 with acid 11 failed to furnish the desired
analog 25, possibly due to an in situ reduction of the nitroxide
to the hydroxylamine followed by double N- and O-acylations
(Scheme 5). Accordingly, an alternative synthetic route was
used to overcome the high reactivity of the AZADO nitroxide.
Diamine 26 was generated from precursor 23 in 2 steps. Selec-
tive coupling of the primary amine with acid 11, and sub-
sequent oxidation of the secondary amine allowed the
isolation of the desired AZADO analog 25 in acceptable yield.
The structure of 25 was confirmed by X-ray analysis (Fig. 3).
Synthesis of fluorophore-tagged analogs
Our first fluorophore labeled derivative contained a BODIPY®-
FL substituent in place of the N-Boc group (Scheme 6). The
direct synthesis of the labeled derivative from JP4-039 failed,
since, after the Boc group of JP4-039 was successfully removed
with HCl, treatment of the free amine with the N-hydroxy-
succinimide activated ester of BODIPY®-FL provided an
17 : 83 mixture of the desired compound and the undesired
doubly-coupled side product. According to an LC/MS/UV ana-
lysis of the crude reaction mixture, an additional acylation had
occurred at the nitroxide oxygen. In an alternative strategy, we
Scheme 4 Synthesis of 8 (6-amino-1-Me-AZADO).
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Scheme 7 Synthesis of BODIPY®-R6G-JP4-039.
Scheme 6 Synthesis of BODIPY®-FL-JP4-039 (30).
attached a BODIPY®-FL label to the alkene peptide isostere
prior to coupling to the 4-AT group. Accordingly, carboxylic
acid 11 was protected as the methyl ester to give 27. Removal
of the Boc-protecting group with TFA followed by coupling to
BODIPY®-FL-NHS (28) provided 29 in 82% yield. Saponifica-
tion of the methyl ester under standard basic conditions
resulted in extensive decomposition; therefore, pig liver ester-
ase (PLE)26 in acetone/pH 7 phosphate buffer was used to
effect a chemoselective transformation. Coupling of the result-
ing acid to 4-AT provided the desired BODIPY®-FL-labeled
compound 30 in 56% yield over 2 steps.
We expected that the formation of the biscoupling side
product could be avoided in the fluorophore conjugation
by carefully monitoring the equivalents of the BODIPY®
N-hydroxysuccinimides. Thus, to streamline the synthesis of
BODIPY®-R6G-JP4-039, we first reduced the nitroxide to the
hydroxylamine with ascorbic acid (Scheme 7). This step was
introduced since nitroxides can undergo acid-catalyzed dispro-
portionations27 and also display broadened NMR spectra
leading to challenging characterization problems.28 Removal
of the Boc group with TFA and subsequent coupling to the
NHS-derivative of BODIPY®-R6G provided 32. Oxidation to the
nitroxide provided BODIPY®-R6G-JP4-039 (33).
Fig. 4 Visualization of BODIPY®-JP4-039 (30) alone, BODIPY® alone, Mito-
Tracker alone, and combinations of 30 at 1 μM, 2.5 μM, and 5 μM concentrations
with MitoTracker in FancD20F adherent cells. Yellow-orange coloring demon-
strates MitoTracker/30 colocalization in mitochondria. At higher concentrations
of the labeled nitroxide, some cytoplasmic BODIPY®-JP4-039 is also visible
(green). White arrows point toward mitochondria and show colocalization.
colocalize in mitochondria of KM101 (human bone marrow
stroma) cell lines (see ESI, p. S31†).
The BODIPY®-labeled agents were used to visualize GS-nitr-
oxide distribution in mitochondria as a complement to the
previously used EPR and MS methods. Compound 30 was
applied for imaging in FancD20F (human Fanconi anemia)
Radioprotective and radiomitigative effects
The study of small molecules that ameliorate radiation
adherent cells.11a In comparison to Mitotracker and BODIPY® damage is pressing due to the lack of selective and potent
alone, significant colocalization of the labeled nitroxide in
mitochondria was clearly visible (Fig. 4). Compound 33, which
development candidates as well as the multiple therapeutic
applications that such small molecules could find. Radiation
minimizes spectral overlap with GFP, has also been shown to protectors, administered prior to irradiation to prevent normal
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tissue damage, can be applied in radiation oncology. Clinical occurred, the nitroxide will have no effect on the D0. If the nitr-
applications of radiation protectors also include treatments oxide is given at the time that the sublethal damage is occur-
that decrease tissue damage and prevent accidental death in ring, then more sublethal events may be necessary to kill the
workers and patients exposed to high energy beams, such as cell, thus increasing the ñ or shoulder on the curve. However,
α-, β-, and γ-radiation, cosmic and particle radiation, as well as a simple correlation between the mitigative effect of the test
UV radiation. Alternatively, compounds that are effective at compounds and the chemical reactivity of the nitroxide sub-
mitigating radiation injury even when administered 24 to 48 h units could not be established. Further studies will be necess-
after the exposure event could be applied as a countermeasure ary to determine the SAR of nitroxides in radiation mitigation.
in a radiation disaster situation such as a nuclear accident.
The fluorophore labeled compounds 30 and 33 maintained
These agents have to meet multiple safety requirements, such similar activity, indicating that they should be suitable tools
as selectivity for protection of normal tissue versus tumor with which to study the GS-nitroxides in cells. It is important
tissue, ease of administration, and minimal toxicity. Only the to note that TEMPOL (3) alone does not have a significant pro-
phosphorothioate prodrug amifostine is currently in use for tective or mitigative effect at a concentration of 10 μM, display-
this purpose.29
ing similar protection and mitigation as the negative control.
Irradiation survival assays were used to test the ability of The conjugation of the GS-derived peptide isostere sequence
selected nitroxide-conjugates to protect against, or mitigate, to the nitroxide is crucial for the observed irradiation survival
irradiation-induced damage of 32Dcl3 murine hematopoietic properties.
cells. The results for TEMPO- (JP4-039), TMIO- (12), ABNO- (17)
and 1-Me-AZADO- (25) type nitroxides demonstrate that all
structural classes are able to protect cells from multiple killing
events if given 1 h before irradiation, as shown by their
Conclusion
increased D0 (Table 1).30 If the nitroxide is given before
irradiation, it may react with the free radicals produced by
X-rays or γ-rays reacting with water in the cell, resulting in
more photons having to be delivered to result in one inactivat-
ing event, and thus increasing the dose. Notably, the protective
activity of the nitroxides used herein can be classified as
follows: 12 (TMIO) < JP4-039 (TEMPO) ≤ 25 (1-Me-AZADO) < 17
(ABNO). This order closely reflects their expected reactivity
based on steric hindrance of the nitroxide species.
When given shortly after irradiation, all agents were also
able to mitigate cell damage, according to the increase
observed in ñ.30 The ñ is the low dose range of the survival
curve where cell death results from an accumulation of events,
none of which are lethal by themselves. These may be events
which occur subsequently to the irradiation, such as pro-
duction of free radicals due to damage to the mitochondria or
other organelles and damage due to production of cellular
cytokines from the initial injury to the cell. By treating with
nitroxide after irradiation, if an inactivating event has already
We have designed a new series of mitochondria-targeted nitr-
oxide conjugates as therapeutic lead structures for diseases
where oxidative stress overcomes the natural biological
defense mechanisms against superoxide accumulation, elec-
tron leakage, and radical escape. Aging, ischemia, neurodegen-
eration, inflammatory- and radiation-induced ailments are
among the results of cellular damage that could be countered
by appropriate electronically tuned and subcellular-targeted
nitroxides.
Although the relative instability of ABNO and 1-Me-AZADO
derivatives required optimizations of the synthetic processes,
these new types of nitroxides were successfully tethered to the
mitochondria-targeting sequence used for JP4-039. The result-
ing stable conjugates demonstrate comparable or improved
biological activities in a cellular assay of radioprotection and
radiation damage mitigation. The activity of these nitroxides
depends mainly on the steric and electronic environment in
the immediate vicinity of the nitroxide center.
Table 1 Irradiation survival curve parameters in 32Dcl3 cellsa
Before irradiationb
After irradiationc
Compound
D0 (Gy)
ñ
D0 (Gy)
ñ
Controld
TEMPOL (3)
JP4-039
30 (BODIPY®-FL-JP4-039)
33 (BODIPY®-R6G-JP4-039)
12 (TMIO)
17 (ABNO)
25 (1-Me-AZADO)
1.3 0.1
1.3 0.1
1.0 0.1
1.0 0.1
1.0 0.1
1.0 0.1
N.D.
1.0 0.1
1.0 0.1
1.0 0.1
1.6 0.2
1.4 0.1
1.2 0.1
1.5 0.2
1.3 0.1
1.4 0.1
1.3 0.1
1.3 0.1
1.1 0.1
1.6 0.3
2.3 0.3 (p = 0.043)
2.6 0.5 (p = 0.048)
N.D.
1.8 0.1 (p = 0.029)
3.1 0.6 (p = 0.048)
2.6 04 (p = 0.041)
3.5 0.2 (p = 0.0001)
4.5 1.1 (p = 0.0026)
4.8 1.0 (p = 0.020)
2.5 0.3 (p = 0.007)
2.7 0.7 (p = 0.088)
2.5 0.3 (p = 0.0065)
a 32Dcl3 cells were placed in methylcellulose and incubated for 7 d at 37 °C in a CO2 incubator. Colonies of >50 cells were counted and data were
analyzed using linear quadratic or single-hit, multi-target models. For a detailed description, see ESI (pp. S29–S31). b Cells were incubated in the
presence of 10 μM nitroxides for 1 h and then irradiated to doses ranging from 0 to 8 Gy. c Cells were irradiated and then placed in media
containing 10 μM nitroxides. d Control = untreated 32Dcl3 cells. N.D. = not determined.
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Overall, the new nitroxides described in this work represent
new tool compounds for testing hypotheses for mechanism of
action and developing more effective counter measures against
redox stress and mitochondrial decay.31 Further synthetic and
biological studies on these lead structures are ongoing in our
laboratories and will be reported in due course.
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Acknowledgements
This project was supported by
a
BARDA contract
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nitroxide, the compounds were analyzed as the hydroxyl-
amine. To avoid oxidation during analysis, the hydroxyl-
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(∼10 mg mL−1) was added to the NMR tube, shaken, and
the layers were allowed to separate before the analysis.
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20 The NMR spectra of the hydroxylamines show two isomers,
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death results from an accumulation of events that individu-
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added together. D0 is the irradiation dose that reduces the
fraction of surviving cells to 37% of its previous value. The
slope of the survival curve is broken down into two parts.
The first is D1 which is the initial slope of the portion of
the curve which reduces the survival to 37% of the starting
cells and is the dose that results in this decrease. The D0 is
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0.01 to 0.0037. D0 is also said to be the dose that is
required to deliver one inactivating event per cell leading to
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21 The relative instability and difficult isolation of ABNO and
other sterically relatively unshielded nitroxides were
observed previously.14b–d Recovery of the hydroxylamine,
alone or as a mixture with the nitroxide, has also been
noted.14b,d
.
22 The isolated yellow oil decomposed into a microcrystalline,
colorless solid upon standing. The same observation was
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.
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