Intermediates in the reduction of the antituberculosis drug PA-824, (6S)-2-nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5H-imidazo[2,1-b] [1,3]oxazine, in aqueous solution

Article abstract of DOI:10.1039/b801859f

The reduction chemistry of the new anti-tuberculosis drug PA-824, together with a more water-soluble analogue, have been investigated using pulse and steady-state radiolysis in aqueous solution. Stepwise reduction of these nitroimidazo-dihydrooxazine compounds through electron transfer from the CO ₂^- species revealed that, unlike related nitroimidazoles, 2-electron addition resulted in the reduction of the imidazole ring in preference to the nitro group. In mildly acidic solution a nitrodihydroimidazo intermediate was formed, which was reduced further to the amine product. In both alkaline and neutral solution, an intermediate produced on 2-electron reduction was resistant to further reduction and reverted to parent compound on extraction or mass spectrometric analysis of the solution. The unusual reduction chemistry of these nitroimidazole compounds, exhibiting ring over nitro group reduction, is associated with alkoxy substitution in the 2-position of a 4-nitroimidazole. The unique properties of the intermediates formed on the reduction of PA-824 need to be considered as playing a possible role in its bactericidal action. The Royal Society of Chemistry.

Full text of DOI:10.1039/b801859f

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Intermediates in the reduction of the antituberculosis drug PA-824,
(6S)-2-nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5H-
imidazo[2,1-b][1,3]oxazine, in aqueous solution†
Robert F. Anderson,*a,b Sujata S. Shinde,a,b Andrej Maroz,a Maruta Boyd,b Brian D. Palmerb and
William A. Dennyb
Received 1st February 2008, Accepted 11th March 2008
First published as an Advance Article on the web 9th April 2008
DOI: 10.1039/b801859f
The reduction chemistry of the new anti-tuberculosis drug PA-824, together with a more water-soluble
analogue, have been investigated using pulse and steady-state radiolysis in aqueous solution. Stepwise
•
−
reduction of these nitroimidazo-dihydrooxazine compounds through electron transfer from the CO2
species revealed that, unlike related nitroimidazoles, 2-electron addition resulted in the reduction of the
imidazole ring in preference to the nitro group. In mildly acidic solution a nitrodihydroimidazo intermedi-
ate was formed, which was reduced further to the amine product. In both alkaline and neutral solution, an
intermediate produced on 2-electron reduction was resistant to further reduction and reverted to parent
compound on extraction or mass spectrometric analysis of the solution. The unusual reduction chemistry
of these nitroimidazole compounds, exhibiting ring over nitro group reduction, is associated with alkoxy
substitution in the 2-position of a 4-nitroimidazole. The unique properties of the intermediates formed
on the reduction of PA-824 need to be considered as playing a possible role in its bactericidal action.
1 imparts its bactericidal effect is poorly understood, but has
Introduction
been shown to be dependent on drug activation by a specific
protein, Rv3547,5 which in turn is sensitive to F420-dependent
glucose-6-phosphate dehydrogenase (FGD) activity in the bac-
terium. Analogy between the activity of the 5-nitroimidazole,
metronidazole (6), and 1 is often drawn, simply on the basis of both
compounds being nitroimidazoles.6 The reduction chemistry of the
majority of nitroaromatics, including 6 and the 4-nitroimidazole
5, is known to be dominated by the stepwise reduction of the
nitro group, with intermediates such as the nitroso form being
cytotoxic nucleophiles. Evidence for multi-electron reduction of
1 has come from voltammetric studies, which reveal reduction of
both the nitro group7 and the imidazole ring.8 In these studies,
aprotic solvents are used to stabilize the reduced intermediates for
investigation. While 6 has been extensively studied in protic sol-
vents using electrochemical9,10 and radiolytic techniques,11,12 there
has been little work on identifying the reduction intermediates
and products formed from 1. Recently, a stable metabolite of
the related clinical candidate drug OPC-67683, a nitroimidazo-
dihydrooxazole, has been identified as the desnitro analogue;13
however, there is no information on more active intermediates
which could possibly play a role in the cytotoxicity of this drug. It
is known that in aqueous solution 5-nitroimidazoles, such as 6, are
generally of higher one-electron reduction potential, E(1), than 4-
nitroimidazoles;14 however, the influence of the fused oxazine ring
on E(1) of 1 is unknown. In this study we have used both pulse
and steady-state radiolysis in aqueous solution to investigate the
reduction of 1, its more soluble analogue without the lipophilic
sidechain (2) and the related compound 2-methoxy-1-methyl-4-
nitro-1H-imidazole (4). Also, UV-vis spectral studies and mass
spectrometry analysis are used to follow both the reduction of
the compounds and identify the intermediates/products which
Multi-drug resistance has become a major setback in controlling
tuberculosis, a disease which kills approximately two million
people annually and is the greatest single infection worldwide.1
There is an urgent need for new drug leads to be identi-
fied, followed by the rational design of improved therapeu-
tics. PA-824, ((6S)-2-nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-
6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine) (1) (Scheme 1), shows
good bactericidal activity against both latent and replicating My-
cobacterium tuberculosis,2 is active in preclinical murine models2–4
and entered Phase I clinical trial in 2005. The mechanism by which
Scheme 1
aDepartment of Chemistry, The University of Auckland, Private Bag
92019, Auckland, 1142, New Zealand. E-mail: r.anderson@auckland.ac.nz;
Fax: +64 9 3737422; Tel: +64 9 3737599 extn 88315
bAuckland Cancer Society Research Centre, The University of Auckland,
Private Bag 92019, Auckland, 1142, New Zealand
† Electronic supplementary information (ESI) available: Isotropic Fermi
contact couplings for radical anion of 2. See DOI: 10.1039/b801859f
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are formed. DFT calculations are carried out to gain insight on
the nature of the radical species formed upon their one-electron
reduction.
Results and discussion
(a) Radical absorption spectra
One-electron reduction of 1 and 2 was carried out by electron
•
−
transfer from the CO2 species, which reacted with both com-
pounds with a rate constant of 2.0 × 109 M−1 s−1. The obtained
time-resolved difference spectra (between the pre-irradiated parent
compound and its radical) are the same for both compounds
over a wide range in pH (4–11), and a representative spectrum
is presented in Fig. 1. The radical species measured 20 ls and
10 ms after the pulse, I, shows a narrow increase in absorption
centred at 270 nm, a broad but weaker absorption at 440 nm
and bleaching centred at 340 nm. Spectral changes are seen with
increasing acidity below pH 4 as an increase in absorption at
290 nm and a decrease at 415 nm, leading to a proposed radical
pKa of I of 1.65 0.2 (Fig. 1, Insert B).
Fig. 2 Transient absorption spectra observed following the pulse radioly-
sis (2.5 Gy in 200 ns) of deaerated aqueous solution containing 1 (100 lM),
2-methylpropan-2-ol (0.1 M) buffered at pH 10 (pyrophosphate, 5 mM).
Measurements made at 2 ls (᭺) and 5 ms (●) following the electron pulse.
Insert: Oscilloscope trace of the change in transmittance vs. time following
pulse radiolysis (arrow).
Fig. 3 Transient absorption spectra observed following the pulse radi-
olysis (2.5 Gy in 200 ns) of N2O-saturated aqueous solution buffered at
pH 10 (pyrophosphate, 5 mM) containing (a) 2-methylpropan-2-ol (0.1 M),
measured at 1 ls (᭺), and (b) 1 (100 lM) and 2-methylpropan-2-ol (0.1 M),
at 5 ms (●). Insert: Change in absorbance vs. pH for (b).
Fig. 1 Transient absorption spectra (presented as the change in ab-
sorbance relative to unirradiated solution) observed following the pulse
radiolysis (2.5 Gy in 200 ns) of N2O-saturated aqueous solution containing
2 (100 lM), sodium formate (0.1 M) buffered at pH 7 (phosphate, 5 mM).
Identical measurements were made at 50 ls and 10 ms (᭺) following the
electron pulse. Insert A: Oscilloscope trace of the change in transmittance
vs. time following pulse radiolysis (arrow). Insert B: Changes in absorption
with pH.
6.9 0.1 (insert Fig. 3). However, some radical–radical reactions
between I and the alkyl radical occur under pulse radiolysis
conditions, as evidenced by the decrease in the visible absorption
band at 440 nm after 5 ms.
One-electron reduction of 1 and 2 was also carried out using
the e− in deaerated solutions containing 2-methylpropan-2-ol
aq
(0.1 M) as an•OH/H• radical scavenger. This produces what is
generally considered to be a relatively inert alkyl radical (Fig. 2).
The same spectrum as for I above is initially produced followed
over 5 ms by a shift in the absorption centred at 270 nm to a
greater absorption at 290 nm. The change in radical absorption
was investigated by studying the reaction of the alkyl radical of
2-methylpropan-2-ol (produced in N2O-saturated solution) with
(b) Conductivity measurements
Changes in conductance following pulse radiolysis were compared
to those observed following the oxidation of DMSO in N2O-
saturated solution as a standard. When the differences in radical
yield are taken into account, similar changes to that of the standard
were found for the compounds in solutions adjusted to pH 4.3–5.5
with HClO4 and pH 10.5 with NaOH (data not shown). Oxidation
2. The alkyl radical was found to react slowly (ca. 7
1.5 ×
•
106 M−1 s−1) to produce II, which has an absorption band in
the 270–290 nm region with an extinction coefficient of ca.
1500 M−1 cm−1 (Fig. 3). The absorption of II can account for most
of the spectral changes observed in Fig. 2 and possesses a pK of
of DMSO by the OH radical results in the rapid formation
of sulfinic acid (pKa 2.28) which increases the conductance of
the pre-pulse solution under acidic conditions, and decreases
the conductance under basic conditions (due to neutralization).15
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These results imply that the one-electron reduced species, I, of
both 1 and 2 at pH ≥4 are radical anions.
(e) Steady-state radiolysis, HPLC and mass analysis of products
•
−
The reduction of compounds 1, 2 and 4 by the CO2 species
upon accumulated radiation dose was monitored using UV-vis
spectrophotometry. For 1 and 2 the loss in absorption of the
parent compound, centred at 340 nm, resulted in the formation
of products which absorbed maximally at lower wavelengths, with
the maintenance of isobestic points. The pH-dependent spectral
data for 2 is given in Fig. 5. Products could be separated from
their parent compounds using HPLC but some of these, formed
by radiolysis under high pH conditions, were unstable in the
pH 3.5, ammonium formate–acetonitrile–water solvent used in the
analysis. Typical chromatograms, obtained on the radiolysis of 1
at different pH, are displayed in Fig. 6. Converting the absorption
at 340 nm into concentration and plotting its loss against radiation
(c) One-electron reduction potential, E(1)
It is widely recognized that E(1) at pH 7 is a controlling parameter
in the activation of several classes of bioreductive drugs. Redox
equilibria were established between species I of both 1 and 2 and
the redox indicator tetraquat (E(1) = −635
6 mV16) on the
20 ls timescale following their reduction by the e−aq. Equilibrium
constants, K = 81.4 14.4 and 102 10 gave similar values for
E(1) of −534 7 mV and −527 6 mV for 1 and 2 respectively.
While these potentials are relatively low, restricting the range of
proteins which could possibly carry out one-electron reduction
of 1, they are considerably higher than the E(1) of the F420 5-
deazaflavin cofactor at −650 mV.17 However, 5-deazariboflavin is
only known as a 2-electron competent cofactor in protein function,
with a two electron reduction potential of −310 mV.18 This fact
points to the possible importance of a reduction intermediate at
the 2-electron level.
If enzymatic reduction of 1 does occur in the bacterium, its
radical anion would be subject to back-oxidation by O2. We have
determined this rate constant to be 1.9
0.2 × 107 M−1 s−1
from measurements of the pseudo-first-order decay rates of the
radical anion absorption at 450 nm for a range of oxygen
concentrations. The rate constant is similar to that measured for
other 4-nitroimidazoles of similar E(1).19
(d) DFT calculations for the radical anion of 2
The distribution of the HOMO on one-electron-reduced 2 was
calculated using DFT at the UB3LYP/6-31G(d,p) level. Electron
distribution is shared mainly between the nitro substituent and
over the C2–C3 region of the imidazole ring (using systematic
numbering for 2 – see Scheme 1) (Fig. 4). The HOMO population
at C3 is slightly higher than that on the N and O atoms of the nitro
group, making C3 a favourable position for protonation or other
electrophilic attack. The spin density distribution in the radical
anion indicates that radical–radical reactions can occur at several
sites on the compound; at C2 and C3 as well as at the atoms of the
nitro group (see ESI†).
Fig. 5 Changes in UV-vis absorption spectra, observed during the
c-irradiation (absorbed doses 0–150 Gy) of N2O-saturated aqueous
solutions of the compound 2 (50 lM), containing sodium formate (50 mM)
and phosphate buffer (5 mM) at different pH.
Fig. 6 Reverse-phase HPLC chromatograms (A–E) of products obtained
•
−
following the reduction of 1 (∼30 lM) by the CO2 radical, generated
in N2O-saturated solutions containing sodium formate (50 mM) and
phosphate buffer (5 mM) under different pH conditions: (A) unirradiated,
(B) pH 4.0, at the 2-electron reduction level, (C) pH 4.0, 6-electron
reduction, (D) pH 7.0, 2-electron reduction, and (E) pH 10.5, 2-electron
reduction. The chromatograms were obtained at the kmax of the products,
and each chromatogram has been scaled for maximum signal intensity.
Fig. 4 HOMO of the radical anion (one-electron-reduced species)
of the structure 2, surface isovalue = 0.05. Calculated using DFT
UB3LYP/6-31G(d,p)
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•
−
Table 1 Radiolytic yield for the loss of compounds 1, 2, 4 and formation of products following reduction by the CO2 radical in aqueous solution
Compd
Glossa/lM
kmax/nm of
products
Ion massc/
g mol−1
Glossg/lM
kmax/nm of
products
Ion massc,h
g mol−1
/
(Ion mass+)
pH
4.0
Gy−1
Gy−1
1 (360.6)
1
0.30 0.01
0.23 0.01
262 V
IVb
293 III
293 III
362.6d
346.6
0.13 0.01
250 VI
330.5d
7.0
(360.6)e,f
(360.6)
1
10.5
0.31 0.01
0i
—
(360.6)f
170.4d
2 (200.4)
2
4.0
5.8
0.33 0.01
0.19 0.01
262 V
262 V
IVb
202.4d
202.4
0.14 0.01
250 VI
186.4e
2
7.0
0.29 0.01
IVb
186.4
293 III
293 III
293 III
(200.4)e,f
(200.4)f
(200.4)f
2
2
8.0
10.5
0.29 0.01
0.33 0.01
0i
—
(200.4)f
214.4
4 (158.4)
4
4.0
7
0.34 0.01
0.33 0.01
262
262
290
290
160.4d
0.16 0.01
258
(158.4)f
4
10.5
0.62 0.02
(158.4)f
0i
(158.4)f
a Loss of compounds measured at 332 nm. b Hydroxylamine is assumed to absorb only weakly. c Positive ion. d Only mass peak. e Predominant peak in
mass spectrum. f Product reverts to parent compound upon mass spectrometry. g Loss of initially formed product V measured at 262 nm (pH 4). h Product
arising from secondary reduction. i No loss in absorption at 290 nm on further irradiation.
dose enabled G values (radiation yield in lM Gy−1) for the loss of
1 and 2 to be calculated (Table 1). Comparing these values with
formed at pH 7). The hydroxylamine is expected to have a UV-
vis absorption spectrum of much lower intensity than that of the
parent nitroimidazole12 but this could not be determined from our
experiments. Thus a range of different intermediates and products
are formed via multiple 1-electron reduction events of 1 and 2,
which are dependent on the pH of the solution (Scheme 2). Similar
data was obtained for 4 in regard to product formation at the 2-
electron reduction level. However, products produced on further
reduction of 4 must fragment and undergo radical–radical events.
since the major product is of increased mass. A product with the
same increased mass was obtained upon the 6-electron reduction
of 5, which underlines the instability of these reduced small-sized
nitroimidazoles in the mass spectrometer.
The above results point to the importance of the oxazine ether
ring in the reduction chemistry of 1 and 2. Voltammetric studies
in protic media on the reduction of the 2-methylhydroxy analogue
of 521 showed it to exhibit typical nitroimidazole behaviour, i.e.
proceeding through the formation of its radical anion, followed
by further reduction and disproportionation reactions to form the
nitroso, hydroxylamine and amine derivatives. We also find that
the reduction of 5 under our experimental conditions did not lead
to the formation of either III or V (data not shown).
•
the G value of the radiolytically-produced CO2 − species (0.66 lM
Gy−1)20 indicates that that 2 electrons are required to convert all
of the parent compound into products for solutions at pH ≥7.
The predominant product at these pH values, III, identified by
its strong absorption band at 293 nm, is assumed to be unstable
to mass spectrometry analysis, as the same molecular mass as
the parent compound was obtained on analysis. Similar spectral
changes (but with a maximum absorption of the product at 295 nm
at greater intensity) have been reported upon treatment of 1 with
strong alkali (pH ≥12),7 which points to it, and possibly III, being
a product which can also be formed by hydrolysis. We have found
that this product, formed by the action of alkali on 1, only partially
reforms the 332 nm band of 1 upon decreasing the pH of the
solution to ≤7. Also III was found to be unstable over time at pH 7
(half-life ca. 1 h). No reduction of III was observed to occur upon
•
−
further irradiation, i.e. radiolytic reduction by the CO2 species
was arrested at the 2-electron-reduced level. This is markedly
different to observations made with other nitroimidazoles where
•
−
reduction by the CO2 species in steady-state radiolysis is to
the 4-electron-reduced level.12 Under acidic conditions, pH 4,
a single product is formed with slightly greater than 2-electron
stoichiometry and absorbs maximally at 262 nm. Mass analysis
of this product, V, shows it to have gained 2 mass units over
both 1 and 2. Further reduction of V with 4 electrons leads again
to the formation of VI, which absorbs maximally at 250 nm.
This process most likely proceeds via intermediates IIb, V, and
reduction of the nitro group to the hydroxylamine, followed by
water elimination to form the amine, VI. The molecular mass
and overall 6-electron stoichiometry identifies this final product
as the amine derivative of the compounds. Reduction of 1 and
2 at pH 5.8 leads to the formation of a different predominant
product, IV, with ca. 4-electron stoichiometry. Mass analysis of
IV is consistent with the formation of the hydroxylamine derivative
of the compounds. (A minor amount of IV, relative to III is also
(f) NMR studies on products formed by steady-state radiolysis
NMR analysis was attempted on the single product V produced
•
upon the reduction of 2 (1.5 mM) by the CO2 − species under low
pH conditions. The product formed proved to be much more polar
than 2 and resisted extraction from the aqueous solution into non-
protic solvents. The solution was concentrated to dryness and the
residue purified by reverse phase semi-preparative HPLC, using
a buffer-free eluant. A single peak almost eluting with the void
volume of the column was collected, concentrated to dryness, and
1
the residue was subjected to detailed H and 13C NMR analysis
(1D and 2D). Despite eluting as a single peak on HPLC, the
NMR analysis indicated the presence of two major products.
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is proposed to result from the new proton now at the C8a position.
The resonance at d 5.5 displayed a 3-bond coupling correlation
with the carbon resonance at d 165.0, indicating them to be in the
same molecule. In the second component of the mixture an ABX
1
coupling system in the H NMR spectrum was consistent with
the –NCH2CHNO2–system which would result from reduction of
the C2–C3 bond. Overall, the NMR spectra are consistent with
V containing the products resulting from reduction of each of the
double bonds of the imidazole ring. This is also consistent with
the observed mass of this product, being two units higher than
the starting material 2. Another possibility for this product, that
it results from cleavage of the C8a–O bond of the oxazine ring,
could be discounted, since an authentic sample of the alcohol
which would be produced was available for comparison.
The reaction with 2 of the alkyl radical formed upon reaction of
the •OH radical with 2-methylpropan-2-ol was further studied by
isolating the product formed for NMR analysis. The steady-state
radiolysis of a N2O-saturated solution containing 2 (400 lM), 2-
methylpropan-2-ol (0.2 M), buffered at pH 7 (5 mM phosphate)
was followed spectroscopically to the point when all of 2 was
lost from solution, as evidenced by the loss of the 332 nm
absorption band. A scale-up preparation of 1.5 mM 2 in 1 M
2-methylpropan-2-ol was irradiated for product analysis (see Ex-
perimental). Thin layer chromatography revealed only one major
product of marginally greater polarity than the starting material,
and this product was recovered from the irradiation solution
and purified by chromatography on silica gel. Low resolution
mass spectrometry indicated the mass of the product to be 271,
corresponding to the addition of 2-methylpropan-2-ol to 2, and
this was confirmed by high resolution mass spectrometry, which
gave an accurate mass consistent with a formula of C11H17N3O5
1
for this material. The NMR spectrum of the purified product
showed loss of the H-3 proton of the imidazole ring, although
the characteristic resonances of the 6-membered oxazine ring and
methyl ether group were still present. A new exchangeable singlet
suggested the presence of an isolated hydroxyl group, and a new
2-proton AB quartet centred at d 3.1 (J = 14.1 Hz) indicated the
presence of an isolated methylene group, in an environment which
was moderately electron-deficient. Finally, two new three-proton
singlets at ∼d 1.1 indicated the presence of two new methyl groups.
Taken together, these features clearly identified the product as the
2-nitroimidazole, VII (Scheme 3), formed by incorporation of 2-
methyl-2-propanol at the 3-position of the imidazole ring.
Conclusions
The reduction chemistry of PA-824 exhibits atypical behaviour
to that of structurally related nitroimidazole compounds. Voltam-
metric studies8 have shown that the imidazole ring of 1 is subject
to reduction as well as the nitro group, and the present study gives
new information on the sequential reduction of these moieties.
Our results, obtained upon the step-wise reduction of 1, clearly
show that imidazole ring reduction occurs in preference to nitro
group reduction to the 2-electron-reduced level, over a range of pH
values. This behaviour can be related to the 2-alkoxy substitution
of the 4-nitroimidazole ring, as parallel results on reduction are
obtained with the 2-methoxy analogue, 4. While the lipophilic
side chain of 1 is most likely necessary to direct and bind the
compound to a specific protein, the subsequent activation of
Scheme 2
Resonances associated with the 6-membered oxazine ring were
generally maintained in both compounds, indicating this ring to
be intact. One component of the mixture contained a singlet at
d 8.25 ppm in the H spectrum, which correlated to a signal at d
165.0 ppm in the 13C spectrum. This is proposed to result from
the C3 olefinic proton of the product resulting from reduction
1
1
of the N1–C8a double bond. A singlet at d 5.5 in the H NMR
spectrum (correlated to a signal at d 80.0 ppm in the 13C spectrum)
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were determined on an Electrothermal 2300 Melting Point
apparatus. 1NMR spectra were obtained on a Bruker Avance
400 spectrometer at 400 MHz. Spectra are referenced to Me4Si.
Chemical shifts and coupling constants were recorded in units
of ppm and Hz, respectively. High resolution mass spectra were
determined on a VG-70SE mass spectrometer using an ionizing
potential of 70 eV at a nominal resolution of 1000. Atmospheric
pressure chemical ionisation mass spectra (APCI-MS) were de-
termined for methanol elutions on a ThermoFinnigan Surveyor
MSQ spectrometer. Solutions in organic solvents were dried
with anhydrous Na2SO4. Solvents were evaporated under reduced
pressure on a rotary evaporator. Thin-layer chromatography was
carried out on aluminium-backed silica gel plates (Merck 60 F254),
with visualization of components by UV light (254 nm) and
exposure to I2. Column chromatography was carried out on silica
gel (Merck 230–400 mesh). PA-824 (1)24 and 1-methyl-4-nitro-1H-
imidazole (5)25 were synthesised as described.
(6S)-6-Methoxy-2-nitro-6,7-dihydro-5H -imidazo[2,1-b][1,3]-
oxazine (2). Sodium hydride (0.10 g of a 60% dispersion in
mineral oil, 2.57 mmol) was added in one portion to a solution of
the alcohol (3)24 (0.24 g, 1.29 mmol) and iodomethane (0.20 ml,
2.83 mmol) in dry DMF (5.0 ml), which had been cooled to
5 ◦C. After 2 min the cooling bath was removed and the solution
was stirred at room temperature for 1 h. Water was added and the
solution was extracted with ethyl acetate. The extract was washed
five times with brine, and then worked up to give an oil which
was chromatographed on silica. Elution with ethyl acetate gave
2 (0.19 g, 74%) as an off-white solid, following trituration with
diethyl ether, mp 127–129 C. H NMR (400 MHz, DMSO-D6)
d ppm 8.01 (s, 1H), 4.60 (dt, J = 12.0, 2.3 Hz, 1H), 4.42 (dt, J =
12.0, 0.4 Hz, 1H), 4.24–4.15 (m, 2H), 4.03–4.00 (m, 1H), 3.34 (s,
3H). APCI-MS Found: [M + H]+ = 200. Found: C, 42.46; H, 4.76;
N, 21.16. C7H9N3O4 requires C, 42.21; H, 4.55; N, 21.10%.
Scheme 3
the 4-nitroimidazole moiety could be a pathway leading to the
bactericidal effects of 1 on M. tuberculosis.
It is presently unknown if the reduction intermediates charac-
terised in this study play a role in the bactericidal action of 1. Of
particular interest are the intermediates formed at the 2-electron-
reduction level, since these are produced for 4-nitroimidazoles
substituted with a 2-alkoxy group. The ability of the presumed
hydroxylated intermediate, III, to undergo facile reversion to
its parent compound, 1, raises the possibility that it might act
as a hydrating agent for an unsaturated species. By analogy
with fatty acid biosynthesis in E. coli,22 it is thought that the
biosynthesis of cell wall mycolic acids in M. tuberculosis proceeds
through the dehydration of hydroxymycolate by a dehydratase to
produce ketomycolate.23 It has been found that treatment with 1
interferes with this process, resulting in the accumulation of the
hydroxymycolate.2 While this could arise from the inhibition of
the uncharacterised dehydratase or from depletion of a cofactor,
rehydration of the ketomycolate by a hydrated intermediate of 1,
such as III, can be considered as an alternate possibility.
◦
1
2-Methoxy-1-methyl-4-nitro-1H-imidazole
(4). Methanol
(1.0 ml, 0.026 mol) was added in portions under nitrogen to a
stirred suspension of sodium hydride (0.27 g of a 60% dispersion
in mineral oil, 6.97 mmol) in dry tetrahydrofuran (5.0 ml). After
5 min a solution of 2,4-dinitro-1-methylimidazole26 (0.30 g,
1.74 mmol) in dry tetrahydrofuran (3.0 ml) was added and the
mixture was stirred at room temperature for 2 h. Water was added
and the solvents were removed in vacuo to leave a small volume
of aqueous residue. This was extracted with ethyl acetate and
the extract was worked up and chromatographed on silica. Ethyl
acetate–petroleum ether 1 : 9) eluted fore-fractions, while ethyl
acetate gave the product 4 (0.25 g, 91%), which crystallised from
ethyl acetate–petroleum ether as white plates, mp 130–131 ◦C. 1H
NMR (400 MHz, CDCl3) d ppm 7.45 (s, 1H), 4.12 (s, 3H), 3.52
(s, 3H). APCI-MS Found: [M + H]+ = 158. Found: C, 38.43; H,
4.50; N, 26.63. C5H7N3O3 requires C, 38.22; H, 4.49; N, 26.74%.
Experimental
Synthesis
All reagents used were of analytical grade. Sodium formate,
sodium hydroxide, perchloric acid and phosphate buffers were
obtained from Merck and potassium thiocyanate from Riedel-de
Haen. All other reagents were obtained from Aldrich Chemical
Company. All solutions were prepared in water purified by the
Millipore “Milli-Q” system. Solution pH values were adjusted
using the phosphate salts (5 mM) and either NaOH or HClO4
when necessary. Analyses were carried out in the Microchemical
Laboratory, University of Otago, Dunedin, NZ. Melting points
1-[(6S)-6-Methoxy-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]-
oxazin-3-yl]-2-methyl-2-propanol (VII). A solution of the 4-
nitroimidazole (2) (6.0 mg, 0.030 mmol) in 1 M tert-butanol–
water (20 ml) was c-irradiated to 5.38 kGy. The solvents were
removed in vacuo and the residue was partitioned between ethyl
acetate and water. The extract was worked up to give an oil which
was chromatographed on silica. Ethyl acetate eluted fore-fractions,
while methanol–ethyl acetate (5 : 95) eluted VII) as a yellow oil
1978 | Org. Biomol. Chem., 2008, 6, 1973–1980
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1
(3.4 mg, 47%). H NMR (400 MHz, DMSO-D6) d ppm 4.69 (s,
by converting the changes in absorption due to the radical yields
•
−
−
1H, exch. with D2O), 4.55 (ddd, J = 11.8, 2.6, 2.6 Hz,1H), 4.41
(ddd, J = 13.7, 2.2, 2.2 Hz, 1H), 4.36 (dd, J = 11.8, 0.8 Hz, 1H),
4.07 (dd, J = 13.7, 3.3 Hz, 1H), 4.00 (m, 1H), 3.34 (s, 3H), 3.16 (d,
J = 14.1 Hz, 1H), 3.04 (d, J = 14.1 Hz, 1H), 1.19 (s, 3H), 1.16 (s,
3H). APCI-MS Found: [M + H]+ = 272. HREI-MS Found M+ =
271.1165. C11H17N3O5 requires 271.1168.
of the eaq (0.28 lM Gy−1)30 and CO2 (0.66 lM Gy−1).20
One-electron reduction potentials, E(1). The one-electron re-
duction potentials of 1 and 2, E(A/A•−), vs. NHE, were de-
termined at pH 7.0 (5 mM phosphate buffer) by establishing
redox equilibria between three mixtures of the one-electron-
reduced compounds and the reference compound tetraquat
•
(E(TeQ2+/TeQ+ ) = −635 7 mV)16 and calculating DE values
Methods
from the equilibrium constants, Ke, using the Nernst equation
Pulse radiolysis experiments, at room temperature (22
1
◦C),
and allowing for ionic strength effects.
were carried out using the University of Auckland’s 4 MeV
linear accelerator of pulse length 200 ns to deliver a typical
absorbed dose of 2.5 Gy for spectral studies and 2.5–10 Gy for
kinetic studies. The optical and conductivity detection systems
and method of dosimetry have been described previously.27,28
Steady-state radiolysis experiments were performed using a 60Co
c-source delivering a dose rate of 7.6 Gy min−1. Aqueous samples
were evacuated and purged with appropriate O2-free gasses in
glass tubes for three cycles. The tubes were fitted with a side
arm incorporating a supracil spectrophotometer cell for UV-vis
measurements. The G(loss) values obtained by the spectrophoto-
metric method, in which isobestic spectral points were maintained.
Optical spectra were recorded on an Ocean Optics HR4000GC-
UV-NIR spectrometer. Product analysis was by HPLC (Agilent
1100 liquid chromatograph coupled to an Agilent 1100 diode
array detector) utilizing a reverse-phase Alltima C18 column (5 l,
150 mm × 3.2 mm i.d.) and a gradient of aqueous (0.045 M
ammonium formate, pH 3.5) and organic (80% acetonitrile–
MilliQ water) solvent phases at a flow rate of 0.5 mL min−1.
DFT calculations were carried out using the Gaussian 03 software
package.29
Acknowledgements
This work was supported by Grant 07/243 from the Health
Research Council of New Zealand, and by the Global Alliance
for TB Drug Development, New York. We thank Sisira Kumara
for assistance with the HPLC assay.
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