Mechanistic investigation of the oxidation of hydrazides: Implications for the activation of the TB drug isoniazid

Article abstract of DOI:10.1039/c2ob26419f

Aryl hydrazides are oxidised to acyl radicals through a mechanism involving diimide intermediates that are prone to nucleophilic acyl substitution. This oxidation occurs regardless of the oxidant involved, however there is no evidence that the acyl radical formed undergoes further oxidation to the corresponding acylium ion, even in the presence of strong oxidants. This study may provide insight into the mechanism of isoniazid resistance in Mycobacterium tuberculosis. The Royal Society of Chemistry.

Full text of DOI:10.1039/c2ob26419f

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Mechanistic investigation of the oxidation of
hydrazides: implications for the activation of the TB
Cite this: Org. Biomol. Chem., 2013, 11,
170
drug isoniazid†
Ruth I. J. Amos,a,b Brendon S. Gourlay,a Brian F. Yates,a Carl H. Schiesser,b,c
Trevor W. Lewisa and Jason A. Smith*a,b
Aryl hydrazides are oxidised to acyl radicals through a mechanism involving diimide intermediates that
are prone to nucleophilic acyl substitution. This oxidation occurs regardless of the oxidant involved,
however there is no evidence that the acyl radical formed undergoes further oxidation to the corre-
sponding acylium ion, even in the presence of strong oxidants. This study may provide insight into the
mechanism of isoniazid resistance in Mycobacterium tuberculosis.
Received 20th July 2012,
Accepted 8th November 2012
DOI: 10.1039/c2ob26419f
www.rsc.org/obc
Introduction
Isoniazid, (1, Scheme 1) the hydrazide of isonicotinic acid, is a
commonly used antibiotic against Mycobacterium tuberculosis.1
As resistance to the frontline antibiotics isoniazid, ethambu-
tol, rifamycin and pyrazinamide increases, an understanding
of the mode of action and mechanisms of resistance are key to
the development of new antibiotics for multi-drug resistant
tuberculosis (MDR-TB).2 Isoniazid acts by inhibiting the fatty
acid synthesis pathway of Mycobacterium tuberculosis, effec-
tively preventing the production of mycolic acid required for
cell capsule synthesis.3 The target enzyme is InhA, an enoyl–
acyl carrier protein.3,4 Sacchetini elucidated the structure of
the inhibitor in the active site of the enzyme InhA and found it
to be an isonicotinic acyl-NADH adduct (2, Scheme 1),5 con-
firming that a reactive intermediate from isoniazid and NAD+
(3, Scheme 1) combine to give the true inhibitor of the bacter-
ium. As a consequence, isoniazid requires activation in order
to react with NAD+ and be effective against the bacterium
(Scheme 1).5
Scheme 1 Isoniazid (1) combines with NAD+ (3) under oxidative conditions to
form the true inhibitor of InhA 2.
The activation of isoniazid was postulated to involve a free
radical and this hypothesis was supported by electron spin
resonance (ESR) spectroscopy studies.6,7 In the bacterium this
activation is performed by the catalase-peroxidase enzyme
KatG, therefore, oxidation of isoniazid plays an important role
in this process.5,6 In many drug resistant strains of the bacter-
ium, activation is inhibited as a result of a single amino acid
residue change (S315T) in the KatG enzyme that appears to
slightly contract the ligand access channel to the active site.8
Investigations into the mechanism of oxidation of isoniazid
may give insight into drug resistance and provide new leads in
the development of more effective antibiotics for MDR-TB.
Very little was known about the mechanism for the oxi-
dation of hydrazides, although it was well established that an
electrophilic species is generated and has been exploited for
nucleophilic acyl substitution reactions.9,10 The oxidation of
hydrazides to give carboxylic acids in water, and esters in the
aSchool of Chemistry, University of Tasmania, Hobart, Australia.
E-mail: Jason.Smith@utas.edu.au; Fax: +613 6226 2858; Tel: +613 6226 2182
bAustralian Research Council Centre of Excellence for Free Radical Chemistry and
Biotechnology, Australia
cSchool of Chemistry and Bio21 Molecular Science and Biotechnology Institute,
The University of Melbourne, Victoria, 3010, Australia
†Electronic supplementary information (ESI)
available. See DOI:
10.1039/c2ob26419f
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Scheme 2 Proposed intermediates in the oxidation of hydrazides.
Scheme 3 Reagents; (i) benzothiazole, AcOH, 5 M H2SO4, KMnO4; (ii) Ph2Se2,
presence of alcohols is well known. However, there would
appear to be some inconsistencies surrounding the mechan-
ism of this transformation.9–12 Some studies invoke the pres-
ence of diimide intermediates (4) during the oxidation of
hydrazides (5)11,12 and the possibility of nucleophilic acyl sub-
stitution of these intermediates (Scheme 2). However, it seems
generally accepted in the literature that an acylium ion (6), or
acyl cation, could be formed by oxidation, leading to carboxylic
acids when the oxidation is carried out in water, and ester for-
mation in the presence of alcohols.9,10 Recently it has been
shown unequivocally that the oxidation of benzhydrazide
forms acyl radicals (7, R = H).13 Preliminary investigations sup-
ported this observation and showed that isoniazid also forms
an acyl radical14 but the involvement of an acylium ion in the
oxidation of hydrazides is ambiguous.
CH3CN/H2O, KMnO4; (iii) TEMPO, CH3CN/H2O, KMnO4.
Fig. 1 TEMPO adduct from isoniazid.
Oxidation of 8 with potassium permanganate in aqueous
acetonitrile with TEMPO afforded the TEMPO adduct 11
(Scheme 3) in 41% yield. Importantly the analogous oxidation
of isoniazid (1) gave the corresponding acyl radical trapped
derivative 12 (Fig. 1) in 15% yield. The other products formed
in these reactions were the corresponding carboxylic acids,
benzoic and isonicotinic acid.
Given the conflicting mechanisms previously proposed, an
investigation into the oxidation of hydrazides was initiated to
answer these questions. The results of this mechanistic study,
involving both laboratory-based and computational tech-
niques, provides some insight into possible mechanisms of
drug resistance in S315T TB.
Oxidations in the presence of nucleophiles
For further investigations, the manganese catalyst [MnIV–
MnIV(μ-O)3L2](PF6)2 (L
= 1,4,7-trimethyl-1,4,7-triazacyclono-
nane) was utilised, as it was hypothesized to be a mimic for
oxidation by the KatG enzyme, due to its catalase activity.17
This catalyst forms a MnVvO species as the active oxidant.18
Results
Trapping of acyl radicals
Initial experiments on the oxidation of the phenyl derivative of Under a nitrogen atmosphere in acetonitrile : pyridine (9 : 1)
isoniazid, benzhydrazide (8, Scheme 3), used the benzothiazo- with periodic acid as a co-oxidant, only a modest yield of 11
lium ion reported by Minisci as a diagnostic probe for the was observed (9–20%). However, when the solution was purged
presence of acyl radicals in the permanganate oxidation of with nitrogen throughout the reaction, the yield was dramati-
alcohols to carboxylic acids.15 When the hydrazide 8 was oxi- cally increased to 58% (see Table 1).
dised with potassium permanganate in the presence of the
When oxidations were performed on a range of hydrazides
benzothiazolium ion (formed by adding benzothiazole to 5 M in methanol, instead of aqueous acetonitrile, in the presence
H2SO4), 2-benzoyl benzothiazole 9 was obtained in 16% yield of TEMPO, methyl esters (14, Scheme 4, and methyl isonicoti-
(Scheme 3).
nate) were formed in significant quantities. The reactions were
Diphenyldiselenide has also been used as a trap for acyl then repeated in acetonitrile with only three equivalents of
radicals16 and oxidation of benzhydrazide with potassium per- methanol to observe the effect of limiting the amount of
manganate in the presence of diphenyldiselenide afforded nucleophile (Table 2, Fig. 2). The ratios of ester (14) to TEMPO
phenyl selenobenzoate 10 in 34% yield (Scheme 3). The seleno adduct (15) resulting from the oxidation of various hydrazides
ester was identified by the CvO stretch in the IR spectrum at in methanol were compared by 1H NMR spectroscopy of the
1683 cm−1
.
crude reaction mixtures (Table 2).19
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Table 1 Oxidation of benzhydrazide (8) and isoniazid (1) in the presence of
TEMPO
Oxidant
Yield (%) 11
Yield (%) 12
Mn(OAc)3
MnO2
K2MnO4
KMnO4
20
81
67
41
25
35
21
84
9
8
10
15
Scheme 5 Six-centred cyclic intermediate formed during the nucleophilic acyl
substitution of the diimide 4.
(NH4)2Ce(NO3)6
[Mn2(μ-O)3L2](PF6)2/H2O2
a
14
58b
20
a
[Mn2(μ-O)3L2](PF6)2/H5IO6
K3[Fe(CN)6]
a L = 1,4,7-trimethylTACN. b Nitrogen continuously bubbled through
the reaction mixture.
Scheme 6 (i) Anisole (10 equiv.), ceric ammonium nitrate (4 equiv.), aceto-
nitrile, N2 bubbled through reaction mixture.
Scheme 7 Isodesmic scheme used for calculating Bond Dissociation Energies
(BDE’s) for acyl radical formation.
Attempted trapping of acylium ion
Scheme 4 Reagents; (i) TEMPO, [Mn2(μ-O)3L2(PF6)2/H5IO6], solvent: MeOH or
MeCN with 3 equiv. MeOH.
To investigate further the possibility of cation formation benzy-
hydrazide (8) and isoniazid (1) were oxidised in a non-nucleo-
philic solvent in the presence of anisole as a nucleophile.
These reactions failed to yield any of the expected Friedel–
Crafts product 16 (Scheme 6).
Table 2 Ratio of non-radical to radical products (14 : 15)
Hydrazide
MeOH as solvent
3 equiv. of MeOH
Computational analysis of acyl radical stability
Isoniazid
Benzhydrazide
p-Methoxybenzhydrazide
p-Methylbenzhydrazide
p-Chlorobenzhydrazide
m-Bromobenzhydrazide
m-Nitrobenzhydrazide
6 : 1a
1 : 1
4 : 1
2 : 1
2 : 1
4 : 1
3 : 1
1 : 9a
1 : 8
1 : 9
1 : 8
1 : 4
1 : 2
1 : 1
Computational chemistry was utilised to investigate the bond
dissociation enthalpy (BDE) of the various aryl radicals as a
measure of radical stability. The calculations used the isodes-
mic reaction mechanism shown in Scheme 7 and were per-
formed using the Gaussian0320 software package. The level of
theory chosen after benchmarking was MPWB1K/6-311 + G
(2d,2p)//MPWB1K/6-31G(d) (see ESI† for full details). The cal-
culations were performed for 15 different aromatic groups
including those in Table 2. The resulting BDEs were close in
value, within 10 kJ mol−1. The highest BDE value was 65.2 kJ
mol−1 for p-methylbenzaldehyde and the lowest 55.7 kJ mol−1
for the m-nitrobenzaldehyde (full tables of BDE values and
benchmarking available in ESI†).
a The ratio of methyl isonicotinate to TEMPO adduct 12 was measured.
Discussion
Acyl radical trapping
The formation of 2-benzoyl benzothiazole (9) from the oxi-
dation of benzhydrazide (8) and trapping with the benzothia-
zolium ion (Scheme 3) supports the hypothesis that acyl
Fig. 2 Hammett plot for ratio of products 15 : 14 vs. Hammett constant for oxi-
dation in the presence of 3 equiv. of MeOH.
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radicals are formed from oxidation of hydrazides. The ketone Competing nucleophilic acyl substitution
would not be formed by nucleophilic acyl substitution. The
When oxidations were performed in methanol, instead of
aqueous acetonitrile, methyl esters (14 R = H, Scheme 4 and
methyl isonicotinate) were significant products of reaction. It
has been suggested in the literature that esters are formed
from the trapping of an acylium ion (6, Scheme 2) by metha-
nol.9,10 However, our preliminary research indicated that ester
formation occurs predominantly by nucleophilic acyl substi-
tution of the diimide 4 (Scheme 2), the precursor to the acyl
radical.14
The substitution of the diimide has been investigated
recently using computational techniques and was found to
proceed through a six centred cyclic intermediate involving at
least two methanol molecules (Scheme 5). The transition state
is substantially stabilised by the presence of additional
solvent25 and is therefore a likely reaction pathway to ester
formation.
While nucleophilic acyl substitution is clearly the dominant
pathway in methanol, the amount of available nucleophile was
reduced in repeat experiments to hinder this process in an
attempt to favour acyl radical formation. As expected, acyl
radical formation dominated with the reduced availability of
methanol, and the TEMPO adducts 15 and 12 were then the
major product. The apparent increase of radical formation
when less nucleophile is available for reaction supports the
hypothesis that esters are formed from a precursor to the acyl
radical and not from further oxidation of the radical to an
acylium ion.
The ratio of ester 14 to radical product 15 from the reduced
methanol experiments increases with increasing electron with-
drawing ability on the aryl ring. When the ratio is plotted
against the corresponding Hammett constant the result is con-
sistent with a nucleophilic acyl substitution mechanism for
ester formation.28 Electron donation has little effect on the
product ratio, with the methyl and methoxy benzhydrazides
essentially having the same ratio of products as the parent
benzhydrazide.
hypothesis that the isoniazid-NADH adduct 2 is formed by
radical trapping with NAD+ 3,21 is also supported by the for-
mation of 9 as both the benzothiazolium ion and NAD+ are
electron deficient heterocycles. However, no isonicotinoyl ben-
zothiazole was observed in a similar oxidation of isoniazid.
The formation of the phenyl selenobenzoate 10 upon oxidation
of 8 in the presence of diphenyldiselenide is also consistent
with formation of an acyl radical.16,22 The corresponding
seleno ester was not observed upon oxidation of isoniazid.
Therefore, the common radical trap TEMPO, which reacts with
carbon centred radicals at near diffusion control with a rate
constant of 1 × 109 M−1 s−1 at a temperature of 298 K,23 was
employed.
Oxidation of 8 with potassium permanganate and TEMPO
afforded the TEMPO adduct 11 (Scheme 3). Importantly the
oxidation of isoniazid gave the corresponding acyl radical
trapped derivative 12. These products are the consequence of
acyl radical generation (7, Scheme 2) and trapping, as sup-
ported by Braslau.13 It was noted that the yields appeared to
vary depending on the strength of the oxidant (Table 1,
8–84%). Significantly, these results suggest that acyl radicals
are formed when either typical single electron oxidants (such
as potassium ferricyanide) or potential two electron oxidants
were used.24
The combination of the different radical trapping
results support the hypothesis that an acyl radical is formed
upon oxidation of isoniazid by the catalase peroxidise enzyme
KatG.
The corresponding carboxylic acids, benzoic and isonicoti-
nic acid, formed in these trapping reactions were previously
believed to result from acylium ion formation (6, Scheme 2)
followed by reaction with water. The small yields of 12 (com-
pared to the corresponding benzoyl derivative 11) (Table 1)
could indicate that the acyl radical from isoniazid is much less
stable than that derived from benzhydrazide and oxidised
further to the cation (6). However, it is possible that the car-
boxylic acids could also be formed by the trapping of an acyl
radical with O2.11,25
The Hammett constant for the pyridyl group predicts a 1 : 1
ratio similar to the nitro derivative but when methanol is
limited, isoniazid (1) is less susceptible to nucleophilic acyl
substitution than predicted. However, when isoniazid (1) is
oxidised with methanol as the solvent, the methyl ester is the
major product (Table 2). The contrasting experimental results
are supported by calculations that predict that bulk solvent is
required for the stabilisation of the transition state during
nucleophilic acyl substitution of the diimide more for isonia-
zid than for other aryl hydrazides.27
The manganese catalyst [MnIV–MnIV(μ-O)3L2](PF6)2 (L =
1,4,7-trimethyl-1,4,7-triazacyclononane) was utilised for the
majority of our experiments and is a mimic for the oxygen
evolving centre of photosystem II.17 Thus it could generate a
low concentration of oxygen in the presence of periodic
acid.18,26 When the reaction solution was purged with nitrogen
throughout the reaction, the yield of the radical product 12
was dramatically increased from 9–20% to 58% (See Table 1).
One reason for this increased yield may be that purging the
reaction mixture with nitrogen gas removes traces of oxygen.
Evidence against the formation of the acyl cation
As a consequence this reduces the competing trapping of an Oxidation of benzyhydrazide or isoniazid in the presence of
acyl radical with O2 to give the carboxylic acid (Scheme 4).25
anisole as a nucleophile failed to yield any Friedel–Crafts
A
such, 12 was now observed as the major product. These exper- product 16 that would be expected if an acylium ion were
iments demonstrate that, under the conditions described formed (Scheme 6). The acyl radical would have been formed
above, the major oxidation pathway most likely involves for- under these conditions, as oxidation with ceric ammonium
mation of the acyl radical (7, Scheme 2).
nitrate (Table 1) showed the presence of the acyl radical by the
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formation of the TEMPO adduct 11. The lack of addition esters by the oxidation of hydrazides in the presence of alco-
product on the anisole ring is further supporting evidence that hols in this and previous studies.
oxidation of the acyl radical to a cation is unlikely under these
conditions.
Cyclic voltammetry was utilised to investigate the oxidation
of the radical to the cation, however the results were
inconclusive.
Experimental
See ESI† for general experimental details.
Computational investigations into the stability of acyl radicals
To investigate the possibility that ester formation is related to
radical stability, we turned to computational chemistry. Provid-
ing that the radicals are similar in nature, bond dissociation
enthalpies (BDEs) can be used as a measure of radical stability,
Oxidation of benzhydrazide in the presence of protonated
benzothiazole
A 3 : 2 ratio of glacial acetic acid and 5 M sulphuric acid
(2.5 mL) at 0 °C was degassed by bubbling N2(g) through the
as they indicate the amount of energy required to homolyse
the bond leading to the radicals in question.29 The calculated
solution for 15 min. To this was added 0.7 mmol benzothia-
zole (0.7 mmol) and benzhydrazide (4.5 mmol). Potassium per-
manganate (4.5 mmol) was added slowly and the solution
stirred for 15 min. The reaction was quenched by the addition
of water and sodium hydrogen carbonate and treated with pot-
assium hydrogen sulphate, and sodium sulfite to reduce
unreacted permanganate and manganese dioxide. The result-
ing solution was neutralized by the careful addition of sodium
hydrogen carbonate and extracted with dichloromethane (3 ×
BDEs for 15 different aromatic groups were close in value,
within 10 kJ mol−1, indicating that the stability of the radical
is not affected by the substitution on the aromatic ring. As
metioned in the results, the highest BDE value was 65.2 kJ
mol−1 for p-methylbenzaldehyde and the lowest 55.7 kJ mol−1
for the m-nitrobenzaldehyde (see ESI†). The formation of the
ester is favoured by electron withdrawing group substitution
on the aromatic ring (vide supra). Therefore, the similarity of
radical stabilities supports the hypothesis that interception of
under reduced pressure to leave a yellow solid which was puri-
a precursor to the radical is a more likely mechanism for ester
formation than generation of an acylium ion.
15 mL). The combined extracts were dried and evaporated
fied by column chromatography (silica gel; eluent 15% ethyl
acetate in hexanes) to give a mixture of 2-benzoylbenzothiazole
Implications in understanding resistance
(9) and benzoic anhydride. This mixture was dissolved in
ethanol and stirred overnight with 2 M sodium hydroxide. The
resulting solution was extracted with dichloromethane (3 ×
15 mL) and the combined extracts dried and evaporated under
reduced pressure to give 9 (16% yield) as a white solid. The
spectral data were consistent with that reported.30
The determination of the mechanism of oxidation of isoniazid
could assist in understanding why a single base pair mutation
in the KatG enzyme in Mycobacterium tuberculosis confers
resistance to isoniazid.8 The secondary alcohol moiety in the
threonine side chain may act as a hydrogen donor to the acyl
radical hindering the reaction with NAD+. In addition, resist-
ance may be caused by the mutation favouring the nucleophi-
lic acyl substitution pathway in preference to radical
formation. This competition is particularly relevant for isonia-
zid activation in an aqueous medium, where nucleophilic acyl
substitution could inhibit the formation of the acyl radical.
Oxidation of benzhydrazide in the presence of
diphenyldiselenide
A mixture of acetonitrile and water (4 : 1 ratio, 5 mL) was
degassed by bubbling N2(g) through it for 10 min. Benzhydra-
zide (2.1 mmol) and diphenyl diselenide (0.3 mmol) were
added followed by the slow addition of solid potassium per-
manganate (2.1 mmol). The resulting mixture was stirred for
15 min and the reaction quenched by the addition of water,
Conclusions
The oxidation of hydrazides under different conditions has led acidification with saturated potassium hydrogen sulfate solu-
to a greater understanding of the intermediates involved and tion and addition of solid sodium sulfite until a clear solution
their reactivity. We have demonstrated evidence for the for- was obtained. The resulting solution was neutralised by
mation of the acyl radical from the oxidation of hydrazides, in the careful addition of sodium hydrogen carbonate, extracted
particular isoniazid, and that the diimide intermediate is sus- with dichloromethane (3 × 15 mL) and the combined extracts
ceptible to nucleophilic acyl substitution in polar aprotic dried and evaporated under reduced pressure to yield a yellow
media such as methanol. Isoniazid is particularly prone to this solid which was purified by column chromatography
substitution pathway. These results are consistent with exper- (silica gel; eluent 10% ethyl acetate in hexanes) to give a
imental details in the existing literature,9–12 however we find mixture of phenylseleno ester 10 and diphenyl diselenide in a
no evidence for the further oxidation of the acyl radical to the 4 : 1 ratio by 1H NMR. This data was extrapolated to give a 34%
acylium ion. On the balance of evidence provided by this work, yield of 10.
we suggest that nucleophilic acyl substitution is likely to be
Spectral data were consistent with that previously
the major mechanism operating during the facile formation of reported.31
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General procedure for the oxidation of hydrazides in the
presence of TEMPO
General procedure for competitive trapping – methanol as
solvent
To degassed (N2(g)) methanol (5 mL) was added TEMPO
(62.5 mg, 0.4 mmol), isoniazid (54.9 mg, 0.4 mmol) and Mn
catalyst ([MnIV–MnIV(μ-O)3L2](PF6)2 (L = 1,4,7-trimethyl-1,4,7-
triazacyclononane)) (5 mg, 6 × 10−3 mmol, 1.6 mol%). Periodic
acid (180 mg, 0.8 mmol) was added slowly and the reaction
mixture stirred for a further 15 min. The solvent was removed
and the reaction worked up as previously described. The
resulting ratio of methyl ester 14 to TEMPO ester 15 was deter-
mined by 1H NMR integration.
TEMPO (62.5 mg, 0.4 mmol) was added to a solution of isonia-
zid (1) (164.5 mg, 1.2 mmol) in degassed (with N2(g)) aqueous
acetonitrile (4 : 1 MeCN : H2O, 5 mL) at 0 °C. Potassium per-
manganate (190 mg, 1.2 mmol) was added in small portions
and the solution stirred for 5 min before being warmed
to room temperature. The reaction was quenched by 0.5 M
potassium hydrogen sulphate (1 mL) and saturated
sodium sulfite solution (1 mL). The mixture was made alkaline
with sodium carbonate and extracted with dichloromethane
(3 × 20 mL). The organic extract was dried and evaporated
to give a solid which was passed through a short plug of
silica gel using 20% ethyl acetate in hexanes to give 2,2,6,6-tet-
ramethylpiperidino isonicotinate (12) as a clear solid in 41%
yield. mp. 60–61 °C. νMAX/cm−1 1755 (CO); δH(acetone-D6) 1.10
(6H, s), 1.27 (6H, s), 1.42–1.77 (6H, m), 7.90 (2H, m), 8.80 (2H,
m); δC(acetone-D6) 17.0, 21.0, 32.1, 39.2, 60.9, 123.7, 138.1,
149.7, 164.7; m/z (EI) 262.1682 (M+ C15H22N2O2 requires
262.1681) 262 (M+, 1%), 247 (84), 156 (32), 139 (13), 25 (65),
123 (100), 106 (50), 98 (27), 83 (76), 77 (37), 69 (60), 55 (76), 51
(31), 41 (50).
General procedure for competitive trapping – acetonitrile as
solvent
To a solution of Mn catalyst ([MnIV–MnIV(μ-O)3L2](PF6)2 (L =
1,4,7-trimethyl-1,4,7-triazacyclononane)) (5 mg, 6 × 10−3 mmol,
1.6 mol%) in degassed (N2(g)) acetonitrile (4.5 mL) and pyri-
dine (0.5 mL), was added TEMPO (62.5 mg, 0.4 mmol), metha-
nol (50 μL, 1.2 mmol) and isoniazid (54.9 mg, 0.4 mmol). To
this was added slowly periodic acid (180 mg, 0.8 mmol, in
1 mL 25% pyridine in acetonitrile) and the reaction mixture
stirred for a further 15 min. The solvent was removed and the
reaction worked up as previously described. The resulting ratio
of methyl ester to TEMPO ester was found by 1H NMR
integration.
2,2,6,6-TETRAMETHYLPIPERIDINO BENZOATE (15, R = H). Spectral
data were consistent with that previously reported.16
2,2,6,6-TETRAMETHYLPIPERIDINO p-METHYLBENZOATE (15, R = 4-
CH3). νMAX/cm−1 1742 (CO); δH(CDCl3) 1.1–1.8 (18H, m),
7.3 (2H, d, J 7), 8.0 (2H, d, J 7); δC(CDCl3) 17.2, 21.1, 21.9,
32.2, 39.3, 60.6, 127.1, 129.4, 129.8, 143.7, 166.7; m/z (EI)
275.1883 (M+ C17H25NO2 requires 275.1885) 275 (M+, 1%), 260
(15), 136 (5), 120 (12), 119 (100), 91 (17), 83 (5), 69 (5), 55 (7),
41 (4).
2,2,6,6-TETRAMETHYLPIPERIDINO p-CHLOROBENZOATE (15, R = 4-
Cl). νMAX/cm−1 1752 (CO); δH(CDCl3) 1.1–1.8 (18H, m), 7.40
(2H, m), 8.00 (2H, m); δC(CDCl3) 17.2, 21, 32.2, 39.3, 60.7,
128.3, 129.0, 131.2, 139.5, 165.8; m/z (EI) 295.1331 (M+
C16H2235ClNO4 requires 295.1339) 295 (M+, 4%), 280 (50),
245 (6), 199 (4), 156 (12), 139 (100), 125 (12), 111 (15), 89 (5),
75 (8), 69 (8), 55 (10), 41 (5).
Procedure for attempted acyl cation trapping
A solution of anisole (500 mg, 4.6 mmol) in acetonitrile
(4.5 mL) was degassed by bubbling N2(g) through the solution
for 10 min. Light was excluded from the reaction and benzhy-
drazide (0.46 mmol) and ceric ammonium nitrate (1.62 mmol)
were added and the mixture was stirred for 3.5 h. Water was
added and the solution made alkaline by addition of solid
sodium hydrogen carbonate followed by extraction with
dichloromethane (3 × 20 mL). The extracts were dried and
solvent removed under reduced pressure to give a red residue.
GCMS analysis showed no ketone formation, only nitroanisole.
2,2,6,6-TETRAMETHYLPIPERIDINO m-BROMOBENZOATE (15,
R = 3-
Br). νMAX/cm−1 1749 (CO); δH(CDCl3) 1.1–1.8 (18H, m), 7.30
(2H, m), 7.70 (1H, m), 8.00 (1H, m), 8.2 (1H, m); δC(CDCl3)
17.2, 21.1, 32.2, 39.3, 60.8, 122.8, 128.4, 130.3, 131.9, 132.7,
136.1, 165.4; m/z (EI) 339.0823 (M+ C16H2279BrNO4 requires
339.0834) 341 (M+, 1%), 324 (58), 183 (100), 156 (19), 124 (10),
76 (11), 69 (20), 55 (38), 41 (17).
Acknowledgements
Support of the Australian Research Council through the
Centres of Excellence Program is gratefully acknowledged. JAS
thanks the University of Tasmania for support through the
IRGS program. RIJA thanks the University and the Australian
Government for an APA and the ARC Centre of Excellence for
Free Radical Chemistry and Biotechnology for a scholarship
top-up and research support. We thank NCI and TPAC for com-
puter time.
2,2,6,6-TETRAMETHYLPIPERIDINO m-NITROBENZOATE (15,
R = 3-
NO2). νMAX/cm−1 1752 (CO), 1534, 1351; δH(CDCl3) 1.1–1.8
(18H, m), 7.70 (1H, t, J 7), 8.40 (2H, m), 8.80 (1H, s); δC(CDCl3)
16.9, 20.9, 32.0, 39.1, 60.7, 124.4, 127.4, 129.8, 131.5, 135.3,
148.3, 164.4; m/z (EI) 306.1578 (M+ C16H25N2O4 requires
306.1759) 306 (M+, 1%), 291 (100), 167 (12), 156 (37), 150 (36),
123 (20), 104 (7), 69 (24), 55 (42), 41 (50).
Notes and references
2,2,6,6-TETRAMETHYLPIPERIDINO p-METHOXYBENZOATE (15, R = 4-
OCH3). Spectral data were consistent with that previously
reported.32
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