Retooling manganese(III) porphyrin-based peroxynitrite decomposition catalysts for selectivity and oral activity: A potential new strategy for treating chronic pain

Article abstract of DOI:10.1021/jm201233r

Redox-active metalloporphyrins represent the most well-characterized class of catalysts capable of attenuating oxidative stress in vivo through the direct interception and decomposition of superoxide and peroxynitrite. While many interesting pharmacologic

Full text of DOI:10.1021/jm201233r

Article
pubs.acs.org/jmc
Retooling Manganese(III) Porphyrin-Based Peroxynitrite
Decomposition Catalysts for Selectivity and Oral Activity: A Potential
New Strategy for Treating Chronic Pain
Smita Rausaria,† Mahsa M. E. Ghaffari,† Andrew Kamadulski,† Kenny Rodgers,‡ Leesa Bryant,§
Zhoumou Chen,§ Tim Doyle,§ Michael J. Shaw,‡ Daniela Salvemini,§ and William L. Neumann*,†
‡
†Department of Pharmaceutical Sciences, School of Pharmacy, and Department of Chemistry, Southern Illinois University,
Edwardsville, Illinois 62026, United States
§Department of Pharmacology and Physiology, St. Louis University, St. Louis, Missouri 63104, United States
S
* Supporting Information
ABSTRACT: Redox-active metalloporphyrins represent the
most well-characterized class of catalysts capable of attenuating
oxidative stress in vivo through the direct interception and
decomposition of superoxide and peroxynitrite. While many
interesting pharmacological probes have emerged from these
studies, few catalysts have been developed with pharmaceutical
properties in mind. Herein, we describe our efforts to identify
new Mn(III)−porphyrin systems with enhanced membrane
solubilizing properties. To this end, seven new Mn(III)-
tetracyclohexenylporphyin (TCHP) analogues, 7, 10, 12, 15,
and 16a−c, have been prepared in which the beta-fused
cyclohexenyl rings provide a means to shield the charged metal
center from the membrane during passive transport. Compounds 7, 15, and 16a−c have been shown to be orally active and
potent analgesics in a model of carrageenan-induced thermal hyperalgesia. In addition, oral administration of compound 7 (10−
100 mg/kg, n = 5) has been shown to dose dependently reverse mechano-allodynia in the CCI model of chronic neuropathic
pain.
catalysts (PNDCs).1,2,11−13 It should also be noted that
Mn(III) and Fe(III) corroles (the one carbon ring contracted
INTRODUCTION
Strategies that employ redox-active transition metal complexes
for the in vivo catalytic detoxification of tissue-damaging
reactive oxygen and reactive nitrogen species have shown great
■
derivatives of porphyrins) have also recently emerged as potent
SOD mimic/PNDCs.14−19
As a result of the pioneering work of Fridovich20−22 and
promise in animal and cell-based studies of diseases where
subsequent development of the SOD mimic approach,23 most
current metalloporphyrin-based catalyst systems have been
designed with an eye toward SOD activity first. The isomeric
Mn(III)-tetrakis-(meso-N-alkylpyridinium)porphyrins [e.g.,
Mn(III)-4-TMPyP5+ 3a and ortho-homologues 4a and
4b]1,11,12,24 are outstanding catalysts in this regard. These
compounds dismute superoxide with some of the highest rate
constants known for synthetic catalysts. The Fe(III) variants of
these systems [i.e., Fe(III)-4-TMPyP5+, 3b] also possess
excellent SOD activity.1 The key design feature for SOD
activity is the incorporation of highly electron-withdrawing N-
alkylpyridinium groups in the meso-positions of the porphyrin
macrocycle. For compounds such a Mn(III)-4-TMPyP5+ 3a and
related congeners, the metal-centered reduction potentials are
subsequently adjusted to values that are nearly ideal for the
rapid dismutation of superoxide.1,25 Thus, the electron-deficient
oxidative stress plays a role.1,2 To date, a number of catalytic
antioxidant systems have been developed, with three of the
most predominant classes represented by Mn(II) polyazama-
crocycles, such as 1 (SC-72325; M40403);3−5 Mn(III)
ethylenebis(salicylimine) (Salens), such as 2 (Euk-134);6 and
Mn(III) and Fe(III) porphyrins, such as 3a [Mn(III)-4-
TMPyP5+] and 3b [Fe(III)-4-TMPyP5+], respectively (Figure
1).1,2 These classes vary significantly regarding their selectivity
toward, and mechanisms of decomposition of, two critical
mediators of oxidative and nitroxidative stress: superoxide and
peroxynitrite (formed from the near diffusion-controlled
reaction of superoxide and nitric oxide).2 Most representatives
of the Mn(II) polyazamacrocycle class have been reported as
selective superoxide dismutase (SOD) mimics,7,8 and Mn(III)
Salens have been studied primarily as SOD/catalase mimics9
[although Mn(III) Salens do react with peroxynitrite].10 The
Mn(III) and Fe(III) porphyrins are clearly the most versatile
catalytic antioxidants from this group and have been studied
primarily as dual SOD mimic/peroxynitrite decomposition
Received: September 16, 2011
Published: November 14, 2011
© 2011 American Chemical Society
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Figure 1. Transition metal-based catalytic antioxidants.
Scheme 1. SOD Activity (A) and PNR Activity (B) of Mn(III) Porphyrins
Mn(III) complex can be readily reduced to the Mn(II) form by
superoxide, which is oxidized to molecular oxygen. The proton-
dependent reoxidation of the Mn(II) form by superoxide
reforms the Mn(III) complex and completes the dismutation
process (and catalytic cycle) by producing hydrogen peroxide
(Scheme 1A).1,12
In addition to SOD activity, compounds such as Mn(III)-4-
TMPyP5+ 3a have been shown to function as potent
peroxynitrite reductases (PNRs).1,12 The mechanism of action
for these types of catalysts has been studied thoroughly by
Groves12 and involves an overall one-electron reduction of
peroxynitrite to nitrogen dioxide with concomitant oxidation of
the Mn(III) catalyst to the Mn(IV)O species. Endogenous
coreductants such as ascorbate or glutathione have been shown
to rapidly reduce both the potentially damaging nitrogen
dioxide to nitrite and the Mn(IV)O species back to
Mn(III).11,12 This completes a reductase type catalytic cycle
and detoxifies peroxynitrite (Scheme 1B, right cycle).
Alternatively, electron-poor complexes can also reduce
peroxynitrite via a two-electron process.26,27 This pathway
requires preliminary reduction of the Mn(III) catalyst to the
Mn(II) form by cellular reductants, a process that is again
contingent on having an electron-poor metal center. Reaction
of the Mn(II) center with peroxynitrite can produce nitrite
directly with concomitant oxidation of the Mn(II) form by two
electrons, and oxygen atom transfer, to generate the Mn(IV)O
species.2,27 Cellular reductants would again be necessary to
reduce the Mn(IV)O form and complete the reductase-like
cycle (Scheme 1B, center cycle). The Fe(III)-based systems are
also quite interesting regarding their reactivity with peroxyni-
trite in that they can function, at least partially, as isomerases by
catalytically converting peroxynitrite to nitrate ion.28
In general, the emphasis of metalloporphyrin chemistry
research programs in these areas has primarily been on
optimizing catalytic activity and further elucidating the
mechanisms of catalysis. To this end, some very impressive
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Figure 2. Membrane-soluble catalytic antioxidants.
and comprehensive studies have been reported that correlate
rates of catalysis with reduction potential of the metal center
(for SOD activity),29 rates and mechanism of catalysis with
secondary coreductant (PNR activity),11,12,30 and rates of
catalysis with number and proximity of positive charges
(electrostatic guidance for both SOD and PNR activity).1,30,31
However, thus far, only a limited number of studies have been
concerned with enhancing the druglike properties of the
catalyst beyond the functional metal center. Attempts to
increase the lipophilicity and membrane solubility of com-
pounds related to Mn(III)-4-TMPyP5+ 3a through regiochem-
ical variations and homologation of the alkylpyridinium
substituents have met with some success, yielding compounds
like Mn(III)-2-TE-PyP5+ 4a and Mn(III)-2-TnHex-2-PyP5+ 4b
(Figure 2).32−34 These compounds are extremely interesting
catalysts and have shown improved cellular and subcellular
penetration.32,35 Other intriguing studies have shown that
further modification of meso-substituents can in fact yield
complexes that are orally active. In this regard, Mn(III)-5,15-
for these studies as it accomplishes our design strategy and is
readily prepared.
Thus, we hypothesized that the Mn(III)-TCHP complex 7
would afford a monocationic metalloporphyrin system in which
the symmetric zones of lipophilicity afforded by the beta-fused
cyclohexenyl substitution would effectively “shield” the polar
region of charge on the metal center from the membrane
(during passive transport and oral absorption). This idea is
analogous to the charge-shielded membrane transport
component of ionophore activity (e.g., the high lipid solubility
of the valinomycin-potassium ion complex).55 Furthermore, it
is now recognized that in addition to lipid solubility and global
reduction of polar surface area, molecular rigidity, as estimated
by the number of rotatable bonds, is another strong contributor
to oral bioavailability.56 These factors tend to be more
important than molecular weight in predicting oral absorp-
tion.56 Thus, we proposed that rigidifying hydrophobic
functionality such as the fused cyclohexenyl groups of
Mn(III)-TCHPs may contribute effectively to oral bioavail-
ability in these types of systems. To test our hypothesis, a
series of Mn(III)-TCHPs of varying lipophilicity (compounds
7, 10, and 12) and with varying functionality (15 and 16a−c)
were prepared, characterized, and evaluated in vivo as orally
active PNR catalysts in models of inflammatory and neuro-
pathic pain.
bis(methoxycarbonyl)-10,20-bis-trifluoromethylporphyrin36
5
and Mn(III)-5,10,15, 20-tetraethylporphyrin37 6 are examples
of orally active porphyrins that have been reported as SOD
mimics with catalase activity.
Our desire to identify PNR catalyst scaffolds with oral
bioavailability was motivated by our continued interest in the
role of peroxynitrite in inflammatory and neuropathic
pain.38−40 Thus, orally active and preferably selective PNR
catalysts were needed as pharmacological probes for in vivo
studies of these chronic pain conditions and also to potentially
serve as therapeutic lead scaffolds for translational studies. In
looking through the literature, we were drawn to the significant
recent advances that have been made in the construction of
highly functionalized porphyrin systems for use in the areas of
materials science,41−43 nonlinear optics,44,43 optical imaging
and sensing,45−48 and photodynamic therapy.47,49 Because in a
number of studies porphyrin systems were designed to be
highly lipophilic for various materials applications (e.g., films50),
we thought this might be fertile territory to mine for
membrane-soluble catalyst ideas. One particular porphyrin
scaffold, used primarily as a synthetic intermediate, that stood
out from the others was the symmetric hexahydro-29H,31H-
tetrabenzo[b,g,l,q]porphine (hereafter referred to as tetracyclo-
hexenylporphyrin, TCHP).45,51,52 We have used similar cyclo-
hexyl and cyclohexenyl substitutions in previous studies of
Mn(II) polyazamacrocycles (as SOD mimics) for both
conformational control of metal chelation and to impart
membrane solubilizing character to the complex for enhanced
bioavailability.4,53,54 Other lipophilic fused-ring porphyrin
systems (of geochemical interest)52 may also be useful for
catalyst development, but we settled upon the TCHP scaffold
CHEMISTRY
■
To determine whether our metal charge-shielding structural
paradigm based upon the TCHP scaffold would provide catalysts
with enhanced druglike properties, we prepared a preliminary set
of three compounds with varying degrees of meso-substitution.
Thus, the readily available 2-ethoxycarbonyl-4,5,6,7-tetrahydro-
2H-isoindole51,52,57 8 (Scheme 2) was prepared via the Barton-
Zard reaction58 from 1-nitrocyclohexene and ethyl isocyanoace-
tate as described in the literature.51,52,57 Compound 8 was then
converted to bis(4,5,6,7-tetrahydro-2H-isoindolyl)methane 9 by
reaction with dimethoxymethane in acidic media followed by
basic hydrolysis and decarboxylation at high temperature.45
Dipyrromethane 9 was then converted to the Mn(III)-TCHP 7
by reaction with formaldehyde in refluxing acetic acid under
aerobic conditions to form the ligand followed by complexation
with MnCl2 and in situ oxidation to the Mn(III) complex under
basic aerobic conditions. The ligand system for Mn(III)-5,15-
diphenyl-TCHP 10 was prepared by the method reported by
Vinogradov and Cheprakov.45 Thus, dipyrromethane 9 was
allowed to react with benzaldehyde mediated by TFA followed
by oxidation of the protoporphyrin system with DDQ. Mn(III)
complex 10 was prepared by reacting the corresponding
porphyrin ligand with MnCl2 using the same procedure as
that used to form complex 7. Finally, the ligand system for
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a
Scheme 2. Synthesis of Mn(III)-TCHPs with Varying Levels of meso-Substitution
a
Reagents and conditions: (a) CH2(OCH3)2, p-TsOH, AcOH, 95%. (b) (CH2OH)2, KOH, 195 °C, 80%. (c) CH2O, AcOH, reflux. (d) MnCl2,
CHCl3, CH3OH, 2,6-lutidine, air, 80%. (e) PhCHO, cat. TFA, CH2Cl2. (f) DDQ, CH2Cl2, 15% for two steps (from e). (g) PhCHO, BF3·OEt2,
CH2Cl2, 50%.
Mn(III)-5,10,15,20-tetraphenyl-TCHP 12 was prepared by
another method reported in the literature.59 Thus, compound
8 was hydrolyzed and decarboxylated in one pot to tetra-
hydroisoindole 11.45,59 This somewhat unstable compound was
then allowed to react with benzaldehyde mediated by BF3·OEt2,
followed again by oxidation of the protoporphyrin system with
DDQ. Mn(III) complex 12 was prepared exactly as described for
complexes 7 and 10 above.
The octanol−water partition coefficients (Log P values) for
these three Mn(III) TCHP complexes were measured by the
slow stir method,60 and the results indicated high lipid
solubility (Table 1). Complex 7 afforded a Log P = 3.77,
while bis-meso-phenyl analogue 10 afforded a Log P = 2.78. The
lower Log P value for compound 10 may be due to the known
deviation from planarity of deca- and doceca-substituted
porphyrin ring systems.61 To reduce steric interactions between
the cyclohexenyl groups and the meso-phenyl substituents, the
normally planar porphyrin undergoes a conformational saddling
distortion, which may provide slightly more solvent access to
the cationic Mn(III) center than in the non-meso-phenyl-
substituted analogue 7. This effect may serve to expose the
Mn(III) center in compound 10 to solvent to a greater extent
than the more planar and therefore more fully charge-shielded
complex 7. The addition of two more meso-phenyl groups
resulting in the tetraphenyl-TCHP complex 12 afforded a Log
P > 5, most likely due to the addition of lipophilicity in all beta-
and meso-positions.
Vinogradov and Cheprakov.45 The esters were reduced to the
bis-meso-benzyl alcohol derivative 14 in good yield using
LiAlH4. Complexation with MnCl2 under aerobic basic
conditions as before afforded the bis-meso-benzyl alcohol-
functionalized Mn(III)-TCHP 15. This complex then served as
the key intermediate for the postchelate synthesis of three new
reversed ester complexes 16a−c by reaction with the
appropriate anhydride in DMF (Scheme 3). We were
unsuccessful in measuring the Log P values for compounds
15 and 16a−c due to severe emulsion problems using standard
octanol/water systems. Thus, these compounds were evaluated
in terms of relative lipophilicity by determination of their
respective reversed phase high-performance liquid chromatog-
raphy (RPHPLC) retention factors (longer retention = greater
lipophilicity)62 as a mixture under isocratic conditions. This
study revealed that the bis-meso-alcohol derivative 15 is
substantially less lipophilic than parent compound 7 (measured
Log P = 3.77), while the bis-meso-acetate 16a, the bis-meso-
propionate 16b, and the bis-meso-isobutyrate 16c are
progressively more lipophilic (Figure S1 in the Supporting
Information).
RESULTS AND DISCUSSION
■
Inhibition of Aryl Boronic Acid Oxidation Assay for
Peroxynitrite Decomposition Activity. To evaluate the
effectiveness with which each complex could intercept and
decompose peroxynitrite, we needed a moderate throughput
assay in which conditions could be easily varied with replicate
runs. All complexes and known standards were therefore
assayed for their ability to inhibit aryl boronate oxidation. The
oxidation of 4-acetylphenylboronic acid to phenol, by attack of
peroxynitrite on the electrophilic boron atom followed by
Mn(III)-5,15-diphenyl-TCHP 10 was chosen for further
synthetic elaboration due to its intermediate lipophilicity and
the ease with which the two meso-phenyl groups could be
functionalized. Thus, 5,15-bis(4-methoxycarbonylphenyl)-
TCHP 13 was prepared according to the method of
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endogenous reductants are plentiful, and their reduction of
the Mn(IV)O form back to the resting Mn(III) complex is fast,
a reductase catalytic cycle is enforced.11,12 Under these
conditions, the second-order rate constant for oxidation of
Mn(III)-TCHPs is also the catalytic rate constant for the
reductase cycle. Inhibition results for all Mn(III)-TCHP
complexes, as well as the known compound Mn(III)-4-
TMPyP5+ 3a, are presented in Table 1. The known PNR
catalyst Mn(III)-4-TMPyP5+ 3a showed a 62.4% inhibition.
From this value, we calculated the apparent second-order rate
constant for the oxidation of the Mn(III) form of 3a to the
Mn(IV)O form to be k = (2.6 ± 0.2) × 106 M−1 s−1, which is in
close agreement with value measure by stopped-flow kinetic
methods.12,66 Thus, this assay provides a convenient method
for the rapid, approximate in vitro measurement of activity
toward the decomposition of peroxynitrite prior to in vivo
studies. All compounds were to some extent effective at
intercepting peroxynitrite and inhibiting boronate oxidation.
Estimated apparent second-order rate constants for the Mn(III)-
TCHPs are in the range of 105 to 106 M−1 s−1. These are very
respectable values given the monocationic nature of our
Mn(III)-TCHPs, as electrostatic guidance has been invoked as
being partially responsible for the efficiency with which
polycationic complexes such as 3a catalyze the decomposition
of the peroxynitrite anion and superoxide radical anion.1,31
Electrochemistry and SOD Activity. As mentioned
above, the removal of electron-withdrawing meso-substituents
and the addition of the weakly inductive electron-donating
cyclohexenyl groups result in Mn(III)-TCHP complexes that
are substantially more electron rich than MnTE-2-PyP5+ (and
related congeners). The electrochemistry of complex 10 was
studied as a representative example of the behavior of this series
of Mn(III)-TCHP charge-shielded catalysts in nonpolar
solvents to mimic membrane space compartmentalization.
Complex 10 features a cyclic voltammetry response in CH2Cl2,
which includes a reversible (presumably porphyrin-based)
oxidation at 0.89 V vs ferrocene and a chemically reversible
Mn(II/III) couple at −0.79 V vs ferrocene. The latter features a
much broader peak separation than the former, which is
consistent with CV observations on other Mn (III)-porphyrin
complexes. The increased peak separation is consistent
with a fast follow-up reaction where the axial halide ligand
is lost from the Mn(III) atom's coordination sphere upon
reduction.
Table 1. Inhibitory Effects of Mn(III)-TCHPs on the
Peroxynitrite-Mediated Oxidation of 4-Acetylphenylboronic
Acid
The conversion of nonaqueous measurements to the NHE
scale (for comparison to polar water-soluble/membrane-
insoluble catalysts such as MnTM-4-PyP5+) is complicated by
consideration of junction potentials and other problems.67
There are multiple reports of the value of E°′ for ferrocene vs
NHE, which range from −0.4 to −0.6 V vs NHE.67 Using this
range to convert our observed value for the Mn(II/III) couple
to the NHE scale, it appears that the reduction potentials for
Mn(III)-TCHP 10 and related complexes in this series are
between −0.2 and −0.4 V. These estimates are consistent with
the reports of E°′ values for Mn(II/III) couple for a number of
5- and 6-coordinate tetraphenylporphyrin complexes of
manganese, which range from −0.43 to −0.54 V vs NHE.68
In addition, these values confirm the electron-rich nature of the
Mn(III)-TCHP complexes reported herein. Thus, the metal-
based reduction potentials of Mn(III)-TCHPs will be out of the
range useful for SOD activity (with +0.30 V optimal for high
SOD activity).1 Accordingly, our design concept for enhancing
druglike properties and facilitating membrane penetration also
a
b
Measured by the slow stir method.60 ND = not determined due to
severe emulsion formation (see the Supporting Information for the
evaluation of lipophilicity by HPLC).
rearrangement, is a clean conversion, and the second-order rate
constant for this reaction has been accurately measured to be
k = 1.6 × 106 M−1 s−1 using stopped flow methods.63 The
extent to which a given Mn(III)-TCHP complex could inhibit
this reaction would be a direct measure of the ability of the
complex to compete with the aryl boronate for peroxynitrite.
Thus, in effect, this method allows a crude estimation of the
apparent second-order rate constant of the reaction of the
complex with peroxynitrite based upon its ability to compete
with the known boronate reaction. Under conditions of low
peroxynitrite concentrations in vivo,64,65 the second-order rate
constant for the rate-limiting oxidation of a Mn(III) complex to
the Mn(IV)O form with concomitant decomposition of
peroxynitrite defines the efficiency of the process.11,12 If
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a
Scheme 3. Synthesis of Mn(III)-TCHPs with Alcohol and Ester Functionality
a
Reagents and conditions: (a) LiAlH4, THF, 80%. (b) MnCl2, CHCl3, CH3OH, 2,6-lutidine, air, 74%. (c) Ac2O, DMF, 91%; propionic anhydride,
DMF, 84%; isobutyric anhydride, DMF, 85%.
limits SOD activity. Data comparing the measured SOD activity
of complex 7 versus known SODm FeTM-4-PyP5+ using
the xanthine/xanthine oxidase-luminol assay69 are plotted in
Figure 3.
studies with SOD mimics have described a modest version of
such an effect in which less potent but more lipophilic complexes
are equally effective in vivo as their related analogues with
much greater activity but 10-fold lower lipophilicity.1,33 The Log
P of compound 7 is many orders of magnitude greater than
both Mn-TM-4-PyP5+, its homologated isomer MnTE-2-PyP5+,
and even long-chain N-alkyl analogues which still show negative
values.1,33
Carrageenan-Induced Hyperalgesia. Intraplantar injec-
tion of carrageenan in rats leads to the time-dependent
development of edema and thermal hyperalgesia.72 This
inflammatory response has been correlated with high levels of
peroxynitrite flux as determined by the detection of nitro-
tyrosine formation and the effectiveness of PNDCs and
inhibitors of peroxynitrite formation in treating the ensuing
inflammation.38−40,73,74 In addition to its potent pro-inflam-
matory and pro-apoptotic effects, the peroxynitrite-derived
nitration and modification of protein functions essential for
normal neuronal homeostasis are key disrupting factors that
contribute to the mechanisms of hyperalgesia.38−40
Figure 3. SOD activity of Mn(III)-TCHP 7. FeTMPyP5+ (▲), but
not Mn(III)-THCP 7 (●), has potent SOD activity; P < 0.001 by
linear regression analysis, n = 3 at each concentration.
As can be seen in Figure 4, wheareas Mn(III)-TE-2-PyP5+ or
Mn(III)-TCHP 7 blocked the development of carrageenan-
induced hyperalgesia when given systemically by ip injection
(10 mg/kg, n = 3), only 7 is able to block hyperalgesia when
given orally by gavage (100 mg/kg, n = 3). The 100 mg/kg
dose was used as a screening dose for oral activity before
measuring the absolute oral bioavailability of promising lead
molecules. In fact, postmortem evaluation of the animals
revealed a substantial amount of the dark red complex 7 left in
the gut (in precipitated form). Thus, a much smaller fraction of
the 100 mg/kg dose was actually absorbed and therefore
responsible for the pharmacological activity in this demanding
assay. This is consistent with the great potential of a catalytic
drug strategy. Nearly 100% inhibition is seen for 2 h with a
substantial inhibitory effect maintained out to 5 h. It is
noteworthy to mention that to achieve a similar degree of
inhibition (at the 2 h time point) with the nonselective
cyclooxygenase (COX)-1/COX-2 inhibitor ibuprofen, a dose of
300 mg/kg was required (99 ± 5% inhibition, n = 6, P < 0.05 as
compared to carrageenan alone). Under the same conditions
and at a dose of 300 mg/kg, acetaminophen or aspirin
Clearly, complex 7 shows only minimal SOD activity, while
FeTM-4-PyP5+ potently inhibits superoxide-mediated oxidation
of luminol. Peroxynitrite, however, is an extremely strong oxidant
(ONOO−, 2H+/•NO2 E°′ = 1.4 V and ONOO,2H+/NO2− E°′ =
1.2 V)70 and is reactive with most metalloporphyrins, as reflected
by the boronate oxidation assay inhibition results for 7 and all
Mn(III)-THCP complexes. Interestingly, sparing superoxide
may actually be beneficial for physiological signaling,71 and
selectivity for peroxynitrite will be of great value in sorting out
mechanistic pharmacological details regarding which reactive
oxygen/nitrogen species (superoxide vs peroxynitrite) is the
linchpin toxic species. Thus, our design concept originally aimed
at enhancing druglike properties has affordeded PNR complexes
with both enhanced lipid solubility (as reflected by their Log P
values and/or their RPHPLC retention factors) and selectivity
toward the decomposition of peroxynitrite. While complex 7
shows inhibition in the aryl boronate oxidation assay, which is
roughly one-half that of highly cationic catalysts such as Mn-TE-
2-PyP5+, it is greatly enhanced membrane solubility compensates
for this lower activity (in vivo results, next section). Previous
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Figure 4. Inhibition of carrageenan-induced hyperalgesia by Mn(III)-TE-2-PyP5+ 4a and Mn(III)-TCHP 7. Systemic (ip) injection (left panel). Oral
†
gavage (right panel). *P < 0.001 vs Veh, and P < 0.001 vs carrageenan.
attenuated hyperalgesia by 20 ± 4 and 50 ± 6%, respectively
(n = 6, P < 0.05 as compared to carrageenan alone at the 2 h
time point). Finally, ip injection of Mn(III)-TE-2-PyP5+ at a
dose 2-fold higher than the one required to block hyperalgesia
by some 90−100% by the ip route led to severe toxicities
(lethargy and piloerection); 100% mortality was seen with a
3-fold higher dose. In contrast, no observable acute toxicities
were noted with doses of complex 7 at least 6−8-fold higher
than the one needed to block hyperalgesia to the same extent.
While compounds 10 and 12 were also active at inhibiting
hyperalgesia in this model, we have discontinued work on
compounds from the dodecasubstituted series (e.g., 12) due
to insolubility. The evaluation of chronic toxicity for further
optimized analogues within this class will be the subject of
future studies.
point), and paw withdrawal latencies were increased to
approximately 90% of precarrageenan values.
Following our encouraging results with the Mn(III)-TCHP
parent system 7, we chose compound 10 for further structure−
activity relationship (SAR) work, as it affords two trans-meso-
phenyl substitutents, which are easily analogued (as described
above in the Chemistry section). To this end, we prepared bis-
meso-benzyl alcohol compound 15 originally for greater
solubility and 16a−c to modulate the in vivo performance of
15 through a prodrug type approach. Thus, bis-acetate 16a was
designed to potentially hydrolyze in vivo to 15 rapidly, while
16b and 16c would be increasingly resistant to esterase-
mediated hydrolysis, respectively.75 Results in the carrageenan
assay for this series of compounds are presented in Figure 6. All
In addition to the prophylactic treatment and thus
prevention of carrageenan-induced hyperalgesia, complex 7 is
able to reverse established hyperalgesia when dosed therapeuti-
cally. Thus, as shown in Figure 5, complex 7 (100 mg/kg) was
Figure 6. Inhibition of carrageenan-induced hyperalgesia by Mn(III)-
TCHPs 15 and 16a−c. *P < 0.05, **P < 0.01, and ***P < 0.001 vs
carrageenan.
Figure 5. Reversal of carrageenan-induced hyperalgesia by oral
administration of Mn(III)-TCHP 7. *P < 0.001 vs Veh, and †P <
0.001 vs carrageenan.
four compounds are effective in reducing hyperalgesia when
dosed orally (100 mg/kg, n = 2). On the basis of known rates
of hydrolysis for α-substituted esters (acetate > propionate >
isobutyrate)75 and previous experience with lipophilicity
requirements of metal-based catalytic antioxidants for high
dosed orally by gavage at the time of maximal hyperalgesia (3 h
postcarrageenan treatment). Within 1 h (4 h time point), pain
was substantially reduced and at 2 h after dosing 7 (5 h time
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activity in the carrageenan assay, we anticipated that bis-acetate
16a would hydrolyze and clear more rapily than 16b and 16c
(by producing the diol 15, which could be easily metabolically
conjugated or oxidized). From the data presented in Figure 6,
bis-acetate 16a produced a strong antihyperalgesic effect out to
2 h postcarrageenan, but the effect was lost at 3 h. Bis-
propionate 16b had a strong antihyperalgesic effect out to 3 h
with a decline in this effect occurring in the 4−5 h time period.
Finally, compound 16c, the bis-isobutyrate, showed potent
antihyperalgesic action out to the 5 h time point. If loss of
activity in this assay can be explained by the clearance of these
complexes from the sites of neuroinflammatory pain due to
ester hydrolysis (affording increased sites of polarity/
bioconjugation), then the results presented in Figure 6 are
consistent with the anticipated rates of hydrolysis for this series
of bis-ester complexes.
The real surprise came with compound 15, the bis-meso-
benzyl alcohol derivative (Figure 6). This compound
performed the best in the series with excellent potency out
to the 5 h time point. Thus, it is unlikely that the double
hydrolyses of the bis-esters 16a−c to the diol 15 are
responsible for the observed clearance profile of these
compounds. Partial hydrolysis of 16a−c to afford the
corresponding half alcohol-half ester derivative may be
responsible for the observed clearance trend and would still
depend upon the known hydrolysis rates for α-substituted
esters. It is possible that the diol functionality increases the
solubility of 15 enough to enhance oral absorption and
bioaccumulation in the sites of inflammation, and this is
responsible for the unexpected excellent in vivo performance of
this molecule. Determination of the absolute bioavailability of
selected members of this new family of Mn(III)-TCHP
catalysts will be the subject of future studies.
Chronic Neuropathic Pain. To extend the therapeutic
utility of novel and orally bioavailable “SO-sparing” Mn(III)-
TCHPs in chronic pain states of different etiologies, we
investigated the pharmacological effects of catalyst 7 in a well-
characterized model of murine neuropathic pain caused by
traumatic nerve injury, namely, chronic constriction injury
(CCI) of the sciatic nerve.76 The CCI model is a standard
model used to screen novel non-narcotic agents in chronic
neuropathic pain. As tested 7 days after surgery (time of
maximal mechano-allodynia), constriction of the sciatic nerve in
mice led to significant (P < 0.05) reduction in mechanical mean
absolute paw-withdrawal thresholds (grams, g) at forces that
failed to elicit withdrawal responses before constriction
(baseline) in ipsilateral, but not contralateral, paws, indicative
of the development of mechano-allodynia. Oral administration
of 7 (10−100 mg/kg, n = 4; Figure 7) at the time of peak
mechano-allodynia led to a rapid, dose-dependent, and near-to-
maximal reversal of neuropathic pain within 1 h postdosing
(Figure 7). Catalyst 7 had no effect on the withdrawal
responses taken from contralateral paws. The ED50 of 7 as
calculated at the time of peak reversal of mechano-allodynia was
28 mg/kg (n = 4, 95% CI = 15.64−51.67 mg/kg/day).
Figure 7. Mn(III)-TCHP 7 reverses neuropathic pain in a dose-
dependent manner. When compared to vehicle (○; n = 6), oral
Mn(III)-TCHP 7 (10, ■; 30, ▼; or 100 mg/kg, ▲; n = 4, arrow)
reversed the tactile allodynia observed on D7 after CCI in a dose- and
time-dependent manner. Mean ± SEM; n rats; ANOVA with
Bonferroni corrections. *P < 0.01, and **P < 0.001 vs vehicle.
of arthritis pain, their well-known gastrointestinal side effects
are of serious concern for chronic therapy. Despite the promise
of COX-2 inhibitors as effective analgesics with reduced
gastrointestinal side effects, the potential for severe cardiovas-
cular side effects has prompted a critical review of these
drugs.78,79 These approaches suffer from being downstream
from a key proinflammatory and neurotoxic biochemical path-
way, which generates the hyperexcitatory conditions involved in
transitioning acute to chronic pain and the maintenance of that
inflammatory pain state. The linchpin molecule is peroxyni-
trite.38−40 Peroxynitrite is a pro-inflammatory and pro-
apoptotic species that also affects the nitration and modification
of protein functions critical to neuronal homeostasis.39,40 In
that the overproduction of peroxynitrite and its subsequent
destructive reactivity manifold lies at the heart of pain of
various etiologies, molecules that can accomplish the direct
scavenging or reduction of PN will provide a novel and
analgesic and anti-inflammatory strategy.38−40,74,80 Herein, we
have shown that by employing a metal charge-shielding design
concept, Mn(III)TCHP systems have been developed as orally
active and selective PNR catalysts. These Mn(III)TCHP
complexes have been shown to rapidly destroy peroxynitrite
with minimal activity toward superoxide. The prototype
compound 7 has been shown to be quite potent in models of
inflammatory and neuropathic pain when dosed orally. Another
promising aspect of this catalytic antioxidant strategy is that
since the manganese atom is the center of activity in destroying
peroxynitrite, the periphery of the porphyrin ligand system can
be manipulated synthetically for optimization of in vivo
performance. Thus, we have shown via a bis-meso-benzylalco-
hol/ester series (15 and 16a−c) that high in vivo activity can
be maintained while tailoring clearance properties through
functionalization of the ligand periphery.
EXPERIMENTAL SECTION
■
General Methods. Analytical thin-layer chromatography (TLC)
was performed on Analtech 0.15 mm silica gel 60-GF254 plates.
Visualization was accomplished with exposure to UV light or exposure
to iodine. Solvents for extraction were HPLC or ACS grade.
Chromatography was performed by the method of Still with Merck
silica gel 60 (230−400 mesh) with the indicated solvent system. NMR
Conclusions. The traditional multifaceted drug regimens
for controlling chronic pain are marginally effective and
produce highly variable results depending upon the conditions
of the contributing pathology.77 Both chronic inflammatory and
neuropathic pain states are therefore often difficult to treat in
the clinic due to insufficient understanding of the nociceptive
pathways involved.77 While nonsteroidal anti-inflammatory
drugs (NSAIDs) are a first-choice therapy for the management
1
spectra were collected on a JEOL ECS 400. H NMR spectra were
reported in ppm from tetramethylsilane on the δ scale. Data are
reported as follows: chemical shift, multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet, b = broadened, and
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obs = obscured), coupling constants (Hz), and assignments or relative
integration. 13C NMR spectra were reported in ppm from the central
deuterated solvent peak. Data are reported as follows: chemical shift,
multiplicity, coupling information, and integration. Grouped shifts are
provided where an ambiguity has not been resolved. LC-MS were run
on a Waters Alliance−SQ 3100 system using Agilent Eclipse (XDB-
C18, 4.6 mm × 150 mm, 5-Micron) column. All compounds tested in
animals were deemed to be of >95% purity by LC-MS methods. UV−
vis spectra were recorded using an Ocean Optics Red Tide UV−vis
Spectrometer. The general procedures and apparatus for the
electrochemistry experiments have been previously reported.81
Mn(III)-TCHP Chloride (7). A mixture of TCHP (100 mg, 0.19
mmol) in CHCl3/MeOH (3:1, 60 mL) was treated with MnCl2 (362
mg, 2.88 mmol) and 2,6-lutidine (1 mL). The mixture was stirred,
open to the air, at room temperature for 1 h. The mixture was diluted
with dichloromethane (150 mL), transferred to a separatory funnel,
and washed with brine (150 mL). The layers were separated, and the
organic solution was dried (Na2SO4), filtered, and concentrated. The
residue was purified by alumina flash column chromatography, using
dichloromethane as the eluent, and the desired complex 7 was
collected as a brown solid (104 mg, 89% yield). LC-MS (85−95%
acetonitrile in 0.05% TFA over 10 min) retention time = 3.23 min,
(M − Cl)+ = 579. HRMS (Q-TOF) m/z = 579.2314 (579.2315 calcd
for C36H36MnN4 [M − Cl]+). UV/vis (1.0 × 10−5 M, CH3OH) λmax
(log ε) 363 (4.95), 389 (sh), 457 (4.67), 543 (3.83), 574 (3.65).
Mn(III)-5,15-diphenyl-TCHP Chloride (10). Compound 10 was
prepared from 5,15-bis-phenyl-TCHP (100 mg, 0.14 mmol) following
the above method. Purification by flash chromatography [neutral
alumina, CH2Cl2/MeOH (99:1)] afforded complex 10 as a brown
solid (104 mg, 97% yield). LC-MS (85−95% acetonitrile in 0.05%
TFA over 10 min) retention time = 5.73 min, C48H44MnN4, (M −
Cl)+ = 731. HRMS (Q-TOF) m/z = 731.2934 (731.2941 calcd for
C48H44MnN4 [M − Cl]+). UV/vis (1.0 × 10−5 M, CH3OH) λmax
(log ε) 373 (4.85), 396 (sh), 465 (4.73), 554 (3.85), 586 (sh).
Mn(III)-5,10,15,25-tetrakis-phenyl-TCHP Chloride (12). Com-
pound 12 was prepared from 5,10,15,25-tetrakis-phenyl-TCHP (100
mg, 0.12 mmol) following the above method. Purification by flash
chromatography [neutral alumina, CH2Cl2/MeOH (98:2)] afforded
complex 12 as a brown solid (96.0 mg, 87%). LC-MS (95−100%
acetonitrile in 0.05% TFA over 10 min) retention time = 6.48 min,
C60H52MnN4, (M − Cl)+ = 883. HRMS (Q-TOF) m/z = 883.3562
(883.3567 calcd for C60H52MnN4 [M − Cl]+). UV/vis (1.0 × 10−5 M,
CH3OH) λmax (log ε) 386 (4.54), 408 (sh), 478 (4.72), 570 (3.69),
606 (sh).
purple solid complex 15 (0.33 g, 74% yield). LC-MS (50−95%
acetonitrile in 0.05% TFA over 6 min) retention time = 6.55 min,
C50H48MnN4O2, (M − Cl)+ = 791. HRMS (Q-TOF) m/z = 791.3186
(791.3152 calcd for C50H48MnN4O2 (M − Cl)+). UV/vis (1.0 × 10−5
M, CH3OH) λmax (log ε) 372 (4.52), 395 (sh), 464 (4.42), 551 (3.31),
582 (sh).
Mn(III)-5,15-bis(4-acetoxymethyl)phenyl-TCHP (16a). To a
mixture of 6 (0.100 g, 0.121 mmol) and DMF (15 mL) was added
acetic anhydride (3 mL, 31.7 mmol). The reaction was stirred at 60−
70 °C for 3 h. The solvent was evaporated, and the residue was
partitioned with CH2Cl2 (100 mL) and brine (50 mL). The layers
were separated, and the CH2Cl2 solution was dried over Na2SO4,
filtered, and concentrated to afford complex 16a as a dark red solid
(0.10 g, 91%). LC-MS (75−95% acetonitrile in 0.05% TFA over 6
min) retention time = 6.33 min, C54H52MnN4O2, [M − Cl]+ = 875.
HRMS (Q-TOF) m/z
= 875.3397 (875.3364 calcd for
C54H52MnN4O2, [M − Cl]+). UV/vis (1.0 × 10−5 M, CH3OH) λmax
(log ε) 372 (4.33), 394 (sh), 464 (4.25), 551 (3.30), 580 (sh).
Mn(III)-5,15-bis(4-propionyloxymethyl)phenyl-TCHP (16b). To
a mixture of 6 (0.1 g, 0.120 mmol) and DMF (15 mL) was added
propionic anhydride (3.00 mL, 23.4 mmol, 195 equiv). The reaction
was stirred at 60−70 °C for 4 h. The solvent was evaporated, and the
residue was partitioned with CH2Cl2 (100 mL) and brine (50 mL).
The layers were separated, and the CH2Cl2 solution was dried over
Na2SO4, filtered, and concentrated to afford complex 16b as a dark red
solid (95 mg, 84%). LC-MS (75−95% acetonitrile in 0.05% TFA over
6 min) retention time = 7.27 min, C56H56MnN4O4, [M − Cl]+ = 903.
HRMS (Q-TOF) m/z
= 903.3705 (903.3676 calcd for
C56H56MnN4O4, [M − Cl]+). UV/vis (1.0 × 10−5 M, CH3OH) λmax
(log ε) 373 (4.96), 394 (sh), 464 (4.86), 551 (3.96), 581 (sh).
Mn(III)-5,15-bis(4-isobutyryloxymethyl)phenyl-TCHP (16c). To
a mixture of 6 (0.100 g, 0.120 mmol) and DMF (15 mL) were added
isobutyric anhydride (3 mL, 18.0 mmol, 150 equiv) and DMAP (50
mg, 0.409 mmol). The reaction was stirred at 70 °C for 4 h. The
solvent was evaporated, and the residue was partitioned with CH2Cl2
(100 mL) and brine (50 mL). The layers were separated, and the
CH2Cl2 solution was dried over Na2SO4, filtered, and concentrated
to afford complex 16c as a dark red solid (0.10 g, 85%). LC-MS
(50−95% acetonitrile in 0.05% TFA over 10 min) retention time =
8.47 min, C58H60MnN4O4, [M − Cl]+ = 931. HRMS (Q-TOF) m/z =
931.4011 (931.3990 calcd for C58H60MnN4O4, [M − Cl]+). UV/vis
(1.0 × 10−5 M, CH3OH) λmax (log ε) 373 (4.76), 394 (sh), 464 (4.66),
551 (3.68), 581 (sh).
Inhibition of Aryl Boronate Oxidation Assay. Stock solutions
of 4-acetylphenylboronic acid and the PNR catalyst were prepared in
DMSO (in the 5−50 mM range). Peroxynitrite in 0.1 N NaOH
solution was kept frozen at −80 °C until needed (see the Supporting
Information). Small aliquots of the PN solution were thawed and
kept on ice, and the concentration was measured by UV spectroscopy
(λmax = 302 nm; ε = 1670 M−1 cm−1) just before measurements
were made. Peroxynitrite concentrations ranged from 58 to 77 mM
for these studies. In a typical procedure, 9.5 × 10−7 mol of
4-acetoxyphenylboronic acid (24.0 μL of stock) was dispensed into
a small vial equipped with a magnetic stir bar. A 1.00 mL amount of
250 mM phosphate buffer (pH = 7.2), which contained 0.7% sodium
dodecyl sulfate and 100 μM DTPA, was added followed by 9.5 × 10−7
mol of the PNR catalyst (aliquot from DMSO stock). To this rapidly
stirred mixture was added 9.5 × 10−7 mol of peroxynitrite by rapid
injection. The mixture was stirred for 1 min and analyzed by LC-MS
[Waters Alliance-MS3100 system; 15% acetonitrile/H2O to 95%
acetonitrile (0.05% TFA) over 10 min; Agilent Eclipse XD8-C18
column, 5 μM, 4.6 mm × 150 mm, UV detection 280 nm for 4-
hydroxyacetophenone oxidation product]. Reactions were run in
triplicate and compared to controls (also run in triplicate), which
contained everything except the PNR catalyst (amounts of DMSO that
were equivalent to those from aliquoted PNR catalyst solutions were
added to the controls to compensate for the very small effect of
DMSO). The peak areas for the phenol oxidation product were
compared for catalyst versus control runs to determine percent
inhibition.
5,15-Bis(4-hydroxymethyl)phenyl-TCHP (14). Into a dry 250
mL round-bottom flask fitted with a magnetic stirrer and an injection
port with a rubber syringe cap under argon was added the diester
porphyrin 13 (0.700 g, 0.880 mmol) and dry tetrahydrofuran (THF)
(60 mL). To this stirred solution, maintained at 0 °C in an ice bath,
was added LiAlH4 (3.00 mL of a 1.0 M solution, 3.00 mmol). The
mixture was allowed to warm to room temperature and then heated to
50 °C and stirred for 2 h. During this time, the progress of the reaction
was followed by LC-MS. When the reaction was complete, the mixture
was cooled and carefully quenched with water. After the mixture was
partitioned with CH2Cl2 (200 mL) and water (200 mL), the desired
purple product precipitated. The precipitate was collected by filtration
and dried under vacuum to give compound 14 as a dark purple solid
1
(0.59 g, 90% yield). H NMR (CDCl3): δ −2.39 (s, 4H), 1.68 (br m,
8H), 2.25 (br m, 8H), 2.45 (s, 4H), 2.64 (br m, 8H), 3.88 (s, 8H),
5.52 (br s, 2H), 7.90 (br m, 4H), 8.27 (br m, 4H), 10.56 (s, 2H). 13C
NMR (CDCl3): δ 19.10, 22.29, 23.32, 23.75, 25.44, 56.56, 63.12,
67.50, 98.19, 119.08, 126.96, 135.51, 136.74, 137.57, 139.21, 140.79,
142.86, 145.43. LC-MS (50−95% acetonitrile in 0.05% TFA over 6
min) retention time = 4.03 min, C50H50N4O2, (M + H)+ = 739.
Mn(III)-5,15-bis(4-hydroxymethyl)phenyl-TCHP (15). A solu-
tion of 14 (0.400 g, 0.541 mmol) in CHCl3/MeOH (2:1, 45 mL) was
treated with MnCl2 (1.08 g, 8.66 mmol, 16 equiv) and 2,6-lutidine
(1 mL). The mixture was stirred, open to the air, at 50 °C for 24 h.
Removal of the solvent yielded a dark purple residue, which upon silica
gel chromatography [eluent: CHCl3/ MeOH (10:1)] afforded a dark
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CCI Model of Neuropathic Pain (CCI). CCI to the sciatic nerve
of the left hind leg in mice was performed under general anesthesia
using the Bennett model.76 Briefly, mice (weighing 25−30 g at the
time of surgery) were anesthetized with 3% isoflurane/100% O2
inhalation and maintained on 2% isoflurane/100% O2 for the duration
of surgery. The left thigh was shaved and scrubbed with
chlorohexidine, and a small incision (1−1.5 cm in length) was made
in the middle of the lateral aspect of the left thigh to expose the sciatic
nerve. The nerve was loosely ligated at two distinct sites (spaced at a 2
mm interval) around the entire diameter of the nerve using silk sutures
(6.0). The surgical site was closed with a single muscle suture and a
skin clip. In this model, maximal tactile allodynia is established
between day 5−7 and lasts for at least 1−2 months after surgery. On
day 0 (baseline, BL), mechanical withdrawal thresholds were assessed
by the von Frey test before surgery and subsequently on day 8
postsurgery. To examine changes in baseline nociceptive responses to
tactile stimulation, animals were placed in elevated cages with a wire
mesh floor and allowed to acclimate for 15 min. The plantar aspect of
hindpaws was probed with calibrated von Frey filaments (0.07, 0.16,
0.40, 1.00, and 2.00 g) purchased from Stoelting (Wood Dale, IL)
according to the up and down method.82 The mechanical threshold
was assessed three times at each time point to yield a mean value,
which is reported as mean absolute threshold (grams, g). The
development of mechano-allodynia is evidenced by a significant (P <
0.05) reduction in mechanical paw-withdrawal thresholds (grams, g) at
forces that fail to elicit withdrawal responses before CCI baseline.
Carrageenan-Induced Thermal Hyperalgesia. Lightly anes-
thetized rats [CO2 (80%)/O2 (20%)] received a subplantar injection
of carrageenan (50 μL of a 1% solution in saline) or its vehicle (50 μL
saline) into the right hindpaw. Drugs or their vehicle were given by
gavage or by intraperitoneal injection (0.2 mL) 30 min before
carrageenan or its vehicle. Hyperalgesic responses to heat were
determined by the Hargreaves' Method using a Basile Plantar Test
(Ugo Basile; Comeria, Italy)72 with a cutoff latency of 20 s employed
to prevent tissue damage. Rats were individually confined to Plexiglas
chambers. A mobile infrared generator was positioned to deliver a
thermal stimulus directly to an individual hindpaw from beneath the
chamber. The withdrawal latency period of injected paws was
determined with an electronic clock circuit and thermocouple. Results
are expressed as Paw-Withdrawal Latency Change (s). Experiments
were conducted with the experimenters blinded to treatment
conditions.
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ASSOCIATED CONTENT
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* Supporting Information
Information for the synthesis and characterization of
compounds 7-ligand, 8-, 9-, and 10-ligand, and 12-ligand;
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and nitrogen species, rescue lipoproteins from oxidative damage, and
attenuate atherosclerosis in mice. Angew. Chem., Int. Ed. Engl. 2008, 47,
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AUTHOR INFORMATION
■
Corresponding Author
*Tel: 618-650-5088. Fax: 618-650-5145. E-mail: wneuman@
siue.edu.
ACKNOWLEDGMENTS
■
Support of this research by the NIH (NIAMS, RC1AR058231)
and NSF (CHE-0911537) is gratefully acknowledged. We
thank the Donald Danforth Plant Science Center Proteomics
and Mass Spectrometry Group and the Washington University
Resource for Biomedical and Biological Mass Spectrometry for
HRMS analysis.
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