Synthesis and characterisation of 13C and 15N isotopomers of a 1-acyl-7-nitroindoline

Article abstract of DOI:10.1002/jlcr.494

Efficient methods are described for synthesis of isotopomers of the water-soluble, photolabile 1-acyl-7-nitroindoline 5 with either ¹³C in the carbonyl of the acyl substituent or ¹⁵N in the nitro group. The isotopic incorporations we

Full text of DOI:10.1002/jlcr.494

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Labelled Cpd Radiopharm 2001; 44: 619–626.
DOI: 10.1002/jlcr.494
13
Synthesis and characterisation of C and
15N isotopomers of a 1-acyl-7-nitroindoline
John E. T. Corrie1,*, Andreas Barth2 and George Papageorgiou1
1 National Institute for Medical Research, The Ridgeway, Mill Hill,
London NW7 1AA, UK
2
.
Institut fu.r Biophysik, Johann Wolfgang Goethe-Universitat,
Frankfurt D-60590, Germany
Summary
Efficient methods are described for synthesis of isotopomers of the
water-soluble, photolabile 1-acyl-7-nitroindoline 5 with either 13C in the
%
carbonyl of the acyl substituent or 15N in the nitro group. The isotopic
incorporations were verified by IR difference spectroscopy coupled with
flash photolysis.
Key Words: nitration; protecting group; infrared spectroscopy; photo-
lysis; FTIR
Introduction
We have previously reported1,2 that photolysis of 1-acyl-7-nitroindo-
lines 1 and 2 in neutral aqueous solution cleanly yields a carboxylate
%
%
anion and a 7-nitrosoindole 3 or 4 (Scheme 1). Release of the
%
%
carboxylate anion upon flash irradiation is rapid and the reagents are,
forexample, suitable forstudy of synaptic transmission by l-glutamate
in mammalian brain tissue, where sub-millisecond release of the
neurotransmitter is needed to mimic normal physiology.3,4
*Correspondence to: J. E. T. Corrie, National Institute of Medical Research, The Ridgeway,
Mill Hill,London NW7 1AA, UK.
Contract/grant sponsor: Royal Society
Copyright # 2001 John Wiley & Sons, Ltd.
Received 2 May 2001
Accepted 21May 2001
Published online \ \ \ \

620
J. E. T. CORRIE ET AL.
Scheme 1.
As part of a programme to examine the mechanism of this novel
photocleavage reaction, we planned to use time-resolved infrared
spectroscopy, as we had done previously for 1-(2-nitrophenyl)ethyl
phosphates.5,6 To facilitate band assignment in this approach, we
required particular 15N and 13C isotopomers of a 1-acyl-7-nitroindoline
with high watersolubility and chose 5, previously made in a non-
%
isotopic form, as a suitable model. We describe here the synthesis and
IR characterisation of two isotopomers carrying either 13C in the
carbonyl of the 1-acyl group or 15N in the 7-nitro group.
1
In ourprevious synthesis, the N-acyl substituent was derived from
glutaric anhydride but a different approach was necessary for the 13C
isotopomer to ensure labelling only at the required position. Scheme 2
shows the initial stages of the successful route, leading to the key alcohol
10. The p-methoxyphenyl protecting group7 was chosen in preference to
the more commonly used p-methoxybenzyl to ensure its survival under
the severe conditions necessary for hydrolysis of the nitrile 7. Blocking
%
the hydroxyl group also avoids problems of intramolecular participa-
tion and facilitates isolation and purification of the early precursors 7
%
and 8. The methods described here may be of wider use for synthesis of
%
labelled hydroxycarboxylic acids.
Forsynthesis of the [ 15N]isotopomer, our previous route was also
somewhat unsatisfactory, as the isotopic nitrogen would have been
introduced at an early stage. We therefore altered the sequence of
synthetic steps, enabling the isotope to be introduced in the final step.
Copyright # 2001 John Wiley & Sons, Ltd.
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ISOTOPOMERS OF A 1-ACYL-7-NITROINDOLINE
621
Scheme 2.
Scheme 3.
The alternate synthesis of alcohol 10 (unlabelled) that facilitates this
procedure is shown in Scheme 3, together with the final two steps of the
preparation of 5 that followed a common route for either isotopomer,
%
namely phosphorylation of 10 to give the phosphotriester 12. Treatment
of [13C] 12 with non-isotopic NaNO3–TFA, ornon-isotopic 12 with
[15N]NaNO3–TFA then effected the required nitration and concurrently
cleaved the t-butyl protecting groups to release the appropriate
isotopomerof 5.
%
The isotopic incorporations were verified by difference IR spectro-
scopy of 5 before and after photolysis. Figure 1 shows the difference
%
spectrum for the non-isotopic compound, where negative bands
represent vibrations present prior to photolysis and positive bands are
from species formed by photolysis.5,6 The time frame of the spectrum
shown is averaged over the interval from 70 ms to 29 s after the light
flash and contains no time-resolved information, i.e. individual spectra
at 70 ms and at 29 s were identical, which confirms in this time frame the
absence of reactions after photolysis.
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J. E. T. CORRIE ET AL.
Figure 1. IR difference spectrum of photolysis of compound 5 in aqueous solution
%
The most prominent change in the difference spectrum for ½13Cꢀ5 was
for the strong antisymmetric carboxylate vibration of the photoreleased
%
product, that shifted as expected from 1556 to 1512 cmꢁ1, while in the
spectrum of ½15Nꢀ5, the antisymmetric and symmetric nitro stretches of
%
the starting compound shifted from 1538 to 1510 cmꢁ1 and 1375 to
1362 cmꢁ1, respectively. The magnitudes of these isotopic shifts for the
nitro bands are consistent with previous results.6 These characteristic
primary isotope shifts confirm that the isotopic substitutions are in the
expected positions. Other effects of the isotopic substitutions were more
ꢁ1
complex. Forexample, the two negative bands at 1649 and 1618 cm
,
unaffected by 15N substitution appeared as three negative bands at 1635,
1603 and 1577 cmꢁ1 upon 13C substitution. The amide band present in
the starting compound is expected near 1650 cmꢁ1 and a shift to
1603 cmꢁ1 with 13C substitution is consistent with this. Full assignment
of the remaining bands in this region and elsewhere in the spectrum,
where there is substantial overlap of positive and negative bands, would
require further experimentation. Notably there was no evidence for a
nitroso monomer band at ꢂ1500 cmꢁ1 in the photoproducts. This is
consistent with data for the pure nitrosoindole 3 [spectrum in CHCl3].1
%
Evidently, the nitroso group of this compound undergoes rapid
dimerisation. A detailed analysis of the IR spectra was beyond the
scope of the present work, particularly as the time scale of the
photocleavage was beyond the time resolution of equipment available
to us.
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 619–626

ISOTOPOMERS OF A 1-ACYL-7-NITROINDOLINE
623
Experimental
General details are as previously described.2 [13C]KCN was from
Cambridge Isotope Laboratories and [15N]NaNO3 was from Aldrich.
Details given are for non-isotopic syntheses and the isotopic materials
were prepared using identical methods.
5-(4-Methoxyphenoxy)pentanonitrile 7
%
A solution of potassium cyanide (1.43 g, 22 mmol) and 4-(4-methoxy
phenoxy)butyl bromide (5.18 g, 20 mmol), prepared as described,8 in
EtOH (50 ml) and water(10 ml) was heated underreflux for18 h. The
solvent was evaporated and the residue was dissolved in Et2O and
washed with water. The organic phase was washed with brine, dried and
evaporated to give 7 as white crystals (3.52 g, 87%), mp 36.5–37.58C
%
(EtOAc–petroleum ether) (Found: C, 67.31; H, 7.31; N, 6.49. C12H15
NO2 ꢃ 0:5 H2O requires C, 67.27; H, 7.53; N, 6.54%); dH (90 MHz) 6.81
(4 H, s, ArH), 3.84–4.04 (2 H, m, ArOCH2), 3.76 (1 H, s, OMe), 2.32–2.52
(2 H, m, CH2CN) and 1.72–1.96 (4 H, m, CH2CH2).
5-(4-Methoxyphenoxy)pentanoic acid 8
%
A solution of the nitrile 7 (2.05 g, 10 mmol) in a mixture of acetone
%
(60 ml) and methanol (30 ml) was treated with 2 M aqueous NaOH
(10 ml) and H2O2 (27.5 wt% aqueous solution, 10 ml). The solution was
stirred at room temperature for 1 h and treated with further H2O2
(10 ml). After 2 h the solution was concentrated and the residue was
dissolved in water and extracted with EtOAc. The combined organic
phases were washed with brine, dried and evaporated to give 5-(4-
methoxyphenoxy)pentanamide as a white solid (2.21 g, 99%). The crude
amide was dissolved in dioxane (20 ml), treated with 0.5 M aqueous
H2SO4 (100 ml) and heated under reflux for 2.5 h. After cooling to room
temperature the solution was washed with EtOAc and the combined
organic phases were washed with brine, dried and evaporated to give 8
%
as white plates (1.57 g, 70%), mp 95–968C (EtOAc–petroleum ether)
(Found: C, 64.23; H, 7.10. C12H16O4 requires C, 64.27; H, 7.19%);
dH (400 MHz) 6.82 (4 H, s, ArH), 3.91–3.94 (2 H, m, ArOCH2),
3.76 (1 H, s, OMe), 2.42–2.46 (2 H, m, CH2CO2H) and 1.81–1.84
(4 H, m, CH2CH2).
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Methyl 1-[5-(4-methoxyphenoxy)pentanoyl]indoline-5-acetate 9
%
Methyl indoline-5-acetate (0.28 g, 1.5 mmol), prepared as previously
described,1 was dissolved in dry MeCN (30 ml), cooled under nitrogen
to 08C and treated with DMAP (0.49 g, 4 mmol) and 5-(4-methoxyphe-
noxy)pentanoic acid 8 (0.23 g, 1.2 mmol), followed by 1-(3-dimethyl-
%
aminopropyl)-3-ethylcarbodiimide hydrochloride (0.38 g, 2 mmol). The
solution was stirred at room temperature for 18 h, concentrated under
reduced pressure and a solution of the residue in EtOAc was washed
successively with 1 M aqueous HCl, saturated aqueous NaHCO3 and
brine, dried and evaporated. Flash chromatography [EtOAc–petroleum
ether(2:3)] gave 9 as white crystals (0.44 g, 95%), mp 90–918C (EtOAc–
%
petroleum ether) (Found: C, 69.59; H, 6.81; N, 3.46. C23H27NO5 requires
C, 69.50; H, 6.85; N, 3.52%); dH (600 MHz) 8.17 (1 H, d, J 8.3, 7-H),
7.07–7.11 (2 H, m, 4, 6-H), 6.82 (4 H, s, ArH), 4.05 (2 H, t, J 8.5, 2-H),
3.96 (2 H, t, J 6.0, ArOCH2), 3.76 (3 H, s, OMe), 3.68 (3 H, s, CO2Me),
3.57 (2 H, s, 4-CH2), 3.17 (2 H, t, 3-H), 2.49 (2 H, t, J 6.9, NCOCH2) and
1.86–1.94 (4 H, m, CH2CH2).
Methyl 1-(5-hydroxypentanoyl)indoline-5-acetate 10
A solution of 9 (596 mg, 1.5 mmol) in acetonitrile (240 ml) was
%
cooled to ꢁ158C and treated with an ice-cold solution of ceric
ammonium nitrate (2.47 g, 4.5 mmol) in water (60 ml). The
mixture was stirred at ꢁ158C for5 min and quenched by the
addition of saturated aqueous NaHSO3 (10 ml). The solvent was
removed under reduced pressure and a solution of the residue
in EtOAc was washed successively with saturated aqueous NaHSO3,
saturated aqueous NaHCO3 and brine, dried and evaporated. Flash
chromatography (EtOAc) gave 10 as white needles (197 mg, 45%), mp
68–698C (EtOAc–petroleum ether) (Found: C, 65.91; H, 7.23; N, 4.66.
C16H21NO4 requires C, 65.96; H, 7.26; N, 4.81%); dH (600 MHz) 8.17
(1 H, d, J 8.3, 7-H), 7.07–7.11 (2 H, m, 4,6-H), 4.05 (2 H, t, J 8.5, 2-H),
3.65–3.69 (3 H, m, CH2OH), 3.68 (3 H, s, CO2Me), 3.57 (2 H, s, 4-CH2),
3.18 (2 H, t, 3-H), 2.46 (2 H, t, J 6.9, NCOCH2) and 1.82–1.89 (2 H, m,
CH2CH2) and 1.64–1.69 (2 H, m, CH2CH2).
In an alternate synthesis, not suitable for preparation of the
[13C]labelled isotopomer, the acid 11 (1.22 g, 4 mmol), prepared as
previously described,1 was dissolved in dry THF (80 ml), cooled to
ꢁ108C and treated dropwise with a solution of 1 M borane in THF
Copyright # 2001 John Wiley & Sons, Ltd.
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ISOTOPOMERS OF A 1-ACYL-7-NITROINDOLINE
625
(8 ml). The mixture was stirred at ꢁ108C for2.5 h and quenched with
water. The solution was saturated with NaHCO3 and extracted
thoroughly with EtOAc. The organic extract was washed with brine,
dried and evaporated and the reside was flash chromatographed
(EtOAc) to give 10 as white needless (1.06 g, 91%), identical to the
material described above.
Methyl 1-{5-[di(tert-butoxy)phosphoryloxy]pentanoyl}indoline-
5-acetate 12
A solution of 10 (437 mg, 1.5 mmol) in dry THF (20 ml) was treated
under nitrogen with 1H-tetrazole (631 mg, 9 mmol) and di-t-butyl-N,
N-diethylphosphoramidite (93% purity; 804 mg, 3 mmol) and the
mixture was stirred at room temperature for 3 h. The solution was
cooled to 08C and treated dropwise with a solution of m-chloroper-
benzoic acid (55% peracid; 1.29 g, 4.5 mmol) in CH2Cl2 (5 ml). The
solution was stirred at 48C for1 h then diluted with ether(20 ml)
and washed with 10% aqueous Na2S2O5. The organic phase was
separated and the aqueous phase was re-extracted with ether. The
combined organic phases were washed successively with 10%
aqueous Na2S2O5, saturated aqueous NaHCO3 and brine, dried
and
evaporated.
Flash
chromatography
[EtOAc–petroleum
ether (4:1)] followed by trituration with ether gave 12 as white plates
(701 g, 97%), mp 55–568C (EtOAc–petroleum ether) (Found:
C, 59.58; H, 7.76; N, 2.85. C24H38NO7P requires C, 59.62; H, 7.92;
N, 2.90%); dH (400 MHz) 8.16 (1 H, d, J 8.2, 7-H), 7.07–7.11
(2 H, m, 4,6-H), 4.05 (2 H, t, J 8.3, 2-H), 4.00 (2 H, dt, JH,P 6.3, J
7.0, CH2OP), 3.68 (3 H, s, CO2Me), 3.57 (2 H, s, 4-CH2), 3.18 (2 H, t, 3-
H), 2.46 (2 H, t, J 6.9, NCOCH2) and 1.74–1.90 (4 H, m, CH2CH2) and
1.48 (18 H, s, 2 ꢄ CMe3).
1-[5-(Dihydroxyphosphoryloxy)pentanoyl]indoline-5-acetate
disodium salt 5
%
A solution of 12 (242 mg, 0.5 mmol) in TFA (5 ml) was treated with
NaNO3 (47 mg, 0.55 mmol) and the mixture was stirred at room
temperature for 4 h. The solvent was removed under reduced pressure
and the residue was dissolved in water (30 ml) and adjusted to pH 6.4
with NaOH. The solution was washed with etherand lyophilised, then
redissolved in a small volume of 25 mM Na phosphate, pH 6.0 and
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purified by preparative HPLC as previously described.1 The recovered
material (0.36 mmol, 72%) was concentrated and stored in frozen
aqueous solution (98 mM). Analytical reverse-phase HPLC, UV and
NMR spectroscopy showed that the product was identical to that
1
previously described.
IR spectroscopy
Details of the FT-IR spectrometer and flash lamp have been described
previously.5 Solutions of 5 (100 mM) were prepared in 200 mM Na
%
MOPS buffer, pH 7.0 in D2O-based solvent and difference spectra were
recorded at 208C. The path length of the sample cell was 15 mm.
Acknowledgements
We are grateful to the Royal Society for support under the ESEP
Programme.
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Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 619–626