Synthesis and reactions of new alkynyl substituted nitroxide radicals

Article abstract of DOI:10.1055/s-1999-3503

N-Oxylpyrrolidine radicals substituted with electron-withdrawing group activated olefinic or acetylenic group 2a-2h, 4, 5, 6 were synthesised from the pyrroline nitrone 1 or from aromatic aldehydes and the alkynylmagnesium bromide of 3. The mercury-catalyzed hydration of alkynyl groups of 2b,c led to the formation of ketones which could be brominated to yield new SH- specific spin labels. The alkynylmagnesium reagent 3 also proved to be versatile in the synthesis of bi- and triradicals 7, 9, 10 and spin labelled steroid derivatives 2e, 12d, 20-22.

Full text of DOI:10.1055/s-1999-3503

PAPER
1039
Synthesis and Reactions of New Alkynyl Substituted Nitroxide Radicals
Cecília P. Sár,a József Jeköb Peter Fajer,c Kálmán Hidega*
a Institute of Organic and Medicinal Chemistry, University of Pécs, H-7643 Pécs, P.O. Box 99. Hungary
Fax + 36(72)325731; E-mail: KHIDEG@main.pote.hu
b ICN Hungary Ltd., H-4440 Tiszavasvári, P.O. Box 1. Hungary
c National High Magnetic Field Laboratory, Florida State University, 1800 E. Paul Dirac Dr., Tallahassee, Florida 32310, USA
Received 11 December 1998
ondary alcohols could be oxidized to the conjugated alde-
hyde 2f or ketones 2g, 2h with activated MnO2.
Abstract: N-Oxylpyrrolidine radicals substituted with electron-
withdrawing group activated olefinic or acetylenic group 2a–2h, 4,
5, 6 were synthesised from the pyrroline nitrone 1 or from aromatic
aldehydes and the alkynylmagnesium bromide of 3. The mercury-
catalyzed hydration of alkynyl groups of 2b,c led to the formation
of ketones which could be brominated to yield new SH-specific spin
labels. The alkynylmagnesium reagent 3 also proved to be versatile
in the synthesis of bi- and triradicals 7, 9, 10 and spin labelled ste-
roid derivatives 2e, 12d, 20–22.
A conjugated ketone reagent, InVSL proved to be an ex-
cellent specific SH-reagent of ATP-ases11,12 and muscle
proteins.13–15 Where other more conventional spin labels
(maleimide, iodoacetemide) were loosely attached to pro-
tein surface, InVSL was bound rigidly enabling studies of
protein dynamics.
Key words: nitroxide free radicals, spin labelled steroids, a,b-acet-
ylenic ketones, polyradicals
Stable nitroxide free radicals with reactive groups are ef-
fective reagents in investigating the structure and function
of biomolecules.1,2 The most useful compounds for spin
labelling of macromolecules are generally attached via
their functional groups to reactive residues of the macro-
molecule,3–6 e.g. to the thiol groups of cysteine in proteins.
We have recently reported that the reaction of pyrroline
nitrones with alkynylmagnesium bromide led to 2-alk-
ynylpyrrolidine nitroxide free radicals in high yield.7 1,3-
Dipolar cycloaddition rarely occured under the described
conditions. This paper reports the synthesis of alkynyl ni-
troxide reagents for spin labelling of biologically active
molecules.
However, as a result of putative hydrophobic interactions,
in some cases the label was bound at many orientations
with respect to the protein precluding studies of protein
orientation. The acetylenic labels described here retain the
rigid binding of InVSL with the advantage of stereospe-
cific binding. Moreover, we have found that the orienta-
tion of the nitroxide moiety with respect to the protein
(myosin head) can be manipulated by varying the R group
of the ketones 2g, 2h.
The reaction of commercially available stock solution of
ethynylmagnesium bromide in THF with 2,5,5-trimethyl-
1-pyrroline N-oxide (1) resulted in the formation of 2,5,5-
trimethyl-2-ethynylpyrrolidine-1-yloxyl radical (2a). In
an analogy to the reaction with phenylethynylmagnesium
bromide (2b) described earlier,7 reaction of nitrone 1 with
a-naphthylethynyl-magnesium bromide led to the radical
2c. Nitrone 1 reacts with 2-methylbut-1-en-3-ynmagne-
sium bromide to give the conjugated en-yne compound 2d
which is a possible synthetic intermediate for a further
Diels–Alder reaction. Several nitroxide analogs of differ-
ent drugs have been developed for use in assays and for in-
vestigating receptor site interactions.8–10 Steroid hormone
spin labelled at its ethynyl group 2e could be obtained
when 17-ethynylestradiol reacted first with an excess (3
equiv) of ethylmagnesium bromide and then with the ni-
trone 1. This reaction was extended to commercialy avail-
able acetylenic alcohols (propynol, but-3-yn-2-ol and 1-
phenylprop-2-yn-1-ol) without tetrahydropyranyl protec-
tion of the hydroxyl group unlike to our earlier work7 and
led directly to propargylic alcohols. The primary and sec-
These paramagnetic acetylenic ketones 2h, 4, 5 could be
synthesised not only from nitrone 1 but also from 1-acet-
oxy-2-ethynyl-2,5,5-trimethylpyrrolidine (3) when it was
reacted first with two equivalents of ethylmagnesium bro-
mide and then with aromatic aldehydes to form secondary
alcohols which could be oxidized readily with MnO2 to
carbonyl compounds.
To have a closer comparison, with the known InVSL and
the acetylenic ketones, the acetylenic ketone 2h was re-
duced to an allylic type of alcohol which could be conve-
niently oxidized back to the a,b-unsaturated ketone 6
(Scheme 1).
Compound 3 also proved to be a key intermediate for the
synthesis of bi- and polyradicals. The synthesis and appli-
cation of bi- and polynitroxides in physics, chemistry, bio-
chemistry as well as in MRImaging techniques are of
current interest.16,17 The in situ generated Grignard re-
agent of 3 readily reacts with nitrone 1 or with 2,5-dime-
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C. P. Sár et al.
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Reagents and conditions: a) R–≡–MgBr/THF/1, –10°C to r.t., 2 h, then NH4Cl/MnO2/CHCl3, r.t. or reflux, 30 min (46–78%); b) ascorbic acid
(5 equiv)/dioxane/H2O (2:1), 40°C, 15 min, then CHCl3/Et3N/AcCl, 0°C to r.t., 1 h (87%); c) EtMgBr (2 equiv)/THF/3, 50°C, 30 min, then
aromatic aldehyde, –10°C to r.t., 1 h, NH4Cl, then MnO2/CHCl3, reflux, 15 min (52–71%); d) SMEAH/THF, 5 h, r.t., then 10% NaOH then
CHCl3 /MnO2, reflux, 30 min (74%)
Scheme 1
Reagents and conditions: a) EtMgBr (2 equiv)/THF/3, 50°C, 30 min, then 1, –10°C to r.t., 2 h, then NH4Cl/MnO2/CHCl3, r.t., 30 min (66%); b)
EtMgBr (2 equiv)/THF/3, 50°C, 30 min, then 2,5-dimethyl-1-pyrrolineN-oxide, 2 h, then NH4Cl/MnO2/CHCl3, r.t., 30 min (58%); c) HC∫CMg-
Br/THF/8, –10°C to r.t., 2 h, then NH4Cl/MnO2/CHCl3, r.t., 30 min (62%); d) EtMgBr (2 equiv)/THF/3, 50°C, 30 min, then 8, –10°C to r.t., 2
h, then NH4Cl/MnO2/CHCl3, 30 min (39%)
Scheme 2
thyl-1-pyrroline N-oxide to give biradical 7 or nitrone- Alkynylmagnesium halides could also be reacted with 3-
radical 8. The nitrone intermediate 8 was reacted again formyl-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrol-1-
with ethynylmagnesium bromide to yield biradical 9 or yloxyl radical (11) without any protection of the N-oxyl
with another Grignard derivative of compound 3 to trirad- function to paramagnetic acetylenic alcohols which could
ical 10 (Scheme 2).
be oxidized to acetylenic ketones 12a–d (Scheme 3).
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Synthesis and Reactions of New Alkynyl Substituted Nitroxide Radicals
1041
presence of mercury salt. The a-methylene group of para-
magnetic ketones 17a, 17b could be brominated22 to
a-bromoketones 18a,b without any protection of the ni-
troxide moiety. The bromine was then substituted with io-
dine when reacted with NaI to give the a-iodoketone 18c
which is more reactive for selective S-spin labelling of
protein thiols (Scheme 4).
In addition to previously described a-halogenated spin la-
bel ketone reagent23,24 and a,b-unsaturated ketone (In-
VSL) a comparative study with 18c and 2g, 2h, 4, 5, 6
offers a possibility in investigating the influence of the re-
active arm on the orientation in labelled protein when the
reactive arm is attached in position 2 of pyrrolidine ring.
In a model reaction 2-alkynyl substituted nitroxide com-
pound 2h activated with electron-withdrawing group was
suitable for conjugated addition of thiols such as the SH
group of N-acetyl-cysteine methyl ester (19) (Scheme 5).
Reagents and conditions: R–≡–MgBr/THF, then 11, –10°C to r.t., 2 h,
NH4Cl then MnO2/CHCl3, reflux, 30 min (41–75%)
Scheme 3
The 1,3-dipolar cycloaddition reaction of phenylacetylene
with compound 1 in toluene at 110°C led to moderate
yield of isoxazoline 13 which gets partially transformed to
benzoyl-aziridine 14 at this temperature. However, com-
pound 14 was further rearranged in a ring-expansion reac-
tion to a dihydropyrrolopyrrole compound 15 similarly as
Reagents and conditions: N-acetylcysteine methyl ester/dioxane/H2O
described before.18,19 Reductive cleavage of the N–O (2:1)/K2CO3, r.t., 2 h (62%)
bond of isoxazoline 13 with Zn/AcOH20,21 afforded an
Scheme 5
amino alcohol which was then oxidized selectively to the
paramagnetic alcohol 16 followed by oxidation of the
Spin labelled steroid derivatives were synthesized for in-
vestigation of steroid receptor sites. The preparation of
spin-labelled mestranol from commercially available
benzyl alcohol to aryl ketone 17a by MnO2. This type of
ketones could also be obtained when the alkynylaryl com-
pounds 2b, c were treated with dilute sulfuric acid in the
Reagents and conditions: a) toluene, reflux, 2 h (14: 21%, 15: 34%); b) Zn/AcOH/EtOH, 60°C, 45 min, NH4OH, pH 10; c) H2O2/MeOH/H2O
(3:1), cat. Na2WO4, 24 h (68%); d) MnO2/CHCl3, reflux, 15 min (87%); e) Hg(OAc)2/5% H2SO4/dioxane, r.t., 24 h (58–62%); f) PHT, 2-pyr-
rolidone/THF, reflux, 30 min (65–72%); g) NaI/THF, reflux, 2 h (78%)
Scheme 4
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C. P. Sár et al.
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Reagents and conditions: a) MeI/K2CO3/18-crown-6/dioxane/H2O (5:1), 2 h, r.t. (76%); b) Fe powder/AcOH, 50°C, then r.t., K2CO3 (63%); c)
SMEAH/THF, 0°C to r.t., 3 h, then 10% NaOH, then MnO2/CHCl3, r.t., 30 min (70%)
Scheme 6
mestranol was not successful, the starting material was re-
covered unreacted. Instead we used the compound 2e ob-
tained from estradiol which was selectively alkylated at
the phenolic hydroxyl group to compound 20. The reduc-
tion of N-oxyl function to basic pyrrolidine 21 could be
carried out selectively, without the involvement of other
functions of the molecule similarly as it was described
earlier.25 The dehydration which often occurs in com-
pounds containing tertiary alcohols in acetic media was
not observed. The reduction of alkynyl function of 2e to
alkene 22 makes possible the investigation of the differ-
ences in alkenes and alkynes in receptor affinity and bio-
logical activity (Scheme 6).
mode. 10 mL of the sample solution in MeOH was introduced via the
thermospray interface. The mobile phase was MeOH/H2O (1:1) so-
lution containing 0.1 M NH4OAc. The capillary tip temperature was
230°C, the electrode voltage was 180 V and the source temperature
was 210°C. ESR spectra were obtained from a 10–5 molar solution
(CHCl3) using Bruker 300-E spectrometer. Flash column chroma-
tography was performed on Merck Kieselgel 60 (0.04–0.063 mm).
The physical and spectroscopic data of compounds 2b, 2f were the
same as described.7
2,5,5-Trimethyl-2-ethynylpyrrolidines 2a–h and 3-Oxoalky-
nylpyrrolines 12a–d; General Procedure
To a stirred solution of alkynylmagnesium bromide (0.01 mol) in
THF (a stock solution of ethynylmagnesium bromide was used for
the synthesis of 2a and 12a or prepared from ethylmagnesium bro-
mide and the corresponding alkynyl compound;26 in the case of
compounds 2f,g,h, 12c the amount of ethylmagnesium bromide was
two equiv or three equiv for 2e, 12d due to the presence of hydroxyl
groups) was added dropwise a solution of 1 (0.01 mol, 1.27 g) or 11
(0.01 mol, 1.68 g) in THF (20 mL) at –10°C. After stirring the mix-
ture at r.t. for 2 h, aq NH4Cl solution (40 mL) was added. The or-
ganic phase was separated, dried (MgSO4) and evaporated. The
residue was dissolved in CHCl3 (20 mL), activated MnO2 (0.01 mol,
870 mg) was added and O2 was bubbled for 30 min or in the case of
compounds 2f,g,h and 12a–d refluxed for 30 min. The mixture was
filtered, evaporated and purified by flash chromatography using
hexane/Et2O or hexane/EtOAc to give 2a–h, 12a–d.
In summary, a facile synthesis of a,b-acetylenic ketones
useful for spin labelling of biomolecules was completed.
The present method has the advantage over the previously
reported method insofar that for obtaining hydroxy acety-
lenes via Grignard reagents no protection of the hydroxy
group is necessary. This method proved to be useful in the
synthesis of new physiologically active spin-labelled
alkynyl steroids. Alkynyl groups were converted in a mer-
cury catalyzed reaction to ketones containing active meth-
ylene groups in a-position bromination of which results in
a-bromo ketone reagents. These reagents are used as spin
labels of a wide range of biomolecules.
2-Ethynyl-2,5,5-trimethylpyrrolidin-1-yloxyl Radical (2a)
Yield: 1.19 g (78%); mp 69–71°C; Rf 0.59 (hexane/Et2O, 2:1).
IR (nujol): n = 3252 (∫CH), 2097 cm–1 (C∫C).
MS: m/z (%) = 152 (M+, 19), 138 (17), 107 (96), 56 (100).
Melting points were determined on a Boetius micro melting point
apparatus and are uncorrected. Elemental analyses (C, ± 0.2; H, ±
0.17; N, ± 0.17; S, ± 0.22) were performed on EA 1110 Elemental
Analyser apparatus or (Hal) were carried out titrimetrically by
Schöniger’s method. The IR spectra (Specord 85) were in each case
consistent with the assigned structure. Mass spectra were recorded
on a VG TRIO-2 instrument in the EI mode (70 eV, direct inlet) or
with thermospray technique. Samples were analysed in the by-pass
2-[2-(1-Naphthyl)ethynyl]-2,5,5- trimethylpyrrolidin-1-yloxyl
Radical (2c)
Yield: 1.97 g (71%); mp 110–112°C; Rf 0.33 (hexane/Et2O, 2:1).
IR (nujol): n = 1582 cm–1 (C=Carom).
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Synthesis and Reactions of New Alkynyl Substituted Nitroxide Radicals
1043
MS: m/z (%) = 278 (M+, 3), 264 (13), 248 (34), 152 (100).
Et2O) giving the title compound as off-white crystals; yield: 5.09 g
(87%); mp 56–58°C; Rf 0.43 (hexane/Et2O, 2:1).
2-(3-Methylbut-3-en-1-yn-1-yl)-2,5,5-trimethylpyrrolidin-1-
yloxyl Radical (2d)
Yield: 1.29 g (67%); oil; Rf 0.50 (hexane/Et2O, 2:1).
IR (film): n = 1600 cm–1 (C=C).
IR (nujol): n = 3180 (∫CH), 2080 (C∫C), 1768 cm–1 (C=O)
MS: m/z (%) = 195 (M+, 2), 180 (2), 153 (24), 138 (100).
MS: m/z (%) = 192 (M+, 8), 178 (55), 150 (21), 124 (100).
Alkynyl Aryl Ketones 2h, 4, 5; General Procedure
To a solution of EtMgBr (8.0 mmol) in anhyd THF (25 mL) was
added compound 3 (780 mg, 4.0 mmol) dropwise. After stirring the
mixture at 50°C for 30 min, the appropriate aromatic aldehyde (4.0
mmol) was added in THF at –10°C. After stirring for additional 1 h
at r.t. aq satd NH4Cl solution (30 mL) was added. The organic phase
was separated, dried (MgSO4) and evaporated to dryness. The resi-
due was dissolved in CHCl3 (20 mL), activated MnO2 (2 g) was
added and the mixture was refluxed for 15 min, filtered and evapo-
rated. The crude product was flash chromatographed with hexane/
Et2O or hexane/EtOAc to give 2h, 4, 5.
17α-[2-(2,5,5-Trimethyl-1-oxylpyrrolidin-2-yl)ethyn-1-yl]es-
tra-1,3,5(10)-triene-3,17β-diol Radical (2e)
Yield: 1.94 g (46%); mp 111–113°C; Rf 0.35 (CHCl3/Et2O, 2:1).
IR (film): n = 3200 (OH), 1606 cm–1 (C=Carom).
TSP: m/z = 423 (M+H)+.
2,5,5-Trimethyl-2-(3-oxobut-1-yn-1-yl)pyrrolidin-1-yloxyl
Radical (2g)
Yield: 1.20 g (62%); oil; Rf 0.48 (hexane/EtOAc, 2:1).
IR (film): n = 2210 (C∫C), 1674 cm–1 (C=O).
MS: m/z (%) = 194 (M+, 2), 180 (15), 164 (8), 41 (100).
(2h); yield: 0.73 g (71%).
2,5,5-Trimethyl-2-[3-oxo-3-(2-pyridyl)prop-1-yn-1-yl]pyrroli-
din-1-yloxyl Radical (4)
Yield: 0.54 g (52%); thick orange oil; Rf 0.19 (hexane/EtOAc, 2:1).
IR (nujol): n = 2200 (C∫C), 1646 cm–1 (C=O).
TSP: m/z = 258 (M+H)+.
2,5,5-Trimethyl-2-[3-oxo-3-(2-thienyl)prop-1-yn-1-yl]pyrroli-
din-1-yloxyl Radical (5)
2,5,5-Trimethyl-2-(3-oxo-3-phenylprop-1-yn-1-yl)pyrrolidin-1-
yloxyl Radical (2h)
Yield: 1.77 g (69%); thick orange oil; Rf 0.45 (hexane/EtOAc, 2:1).
IR (film): n = 2210 (C∫C), 1640 (C=O), 1592 cm–1 (C=Carom).
TSP: m/z = 257 (M+H)+.
2,5-Dihydro-2,2,5,5-tetramethyl-3-(1-oxoprop-2-yn-1-yl)-1H-
pyrrol-1-yloxyl Radical (12a)
Yield: 1.44 g (75%); mp 163–165°C; Rf 0.43 (hexane/EtOAc, 2:1).
Yield: 0.57 g (54%); orange oil; Rf 0.21 (hexane/Et2O, 2:1).
IR (nujol): n = 2200 (C∫C), 1660 cm–1 (C=O).
TSP: m/z = 263 (M+H)+.
IR (nujol): n = 3234 (∫CH), 2088 (C∫C), 1680 (C=O), 1604
cm–1 (C=C).
MS: m/z (%) = 192 (M+, 22), 178 (11), 147 (75), 41(100).
2,5,5-Trimethyl-2-(3-oxo-3-phenyleth-1-en-1-yl)pyrrolidin-1-
yloxyl Radical (6)
2,5-Dihydro-2,2,5,5-tetramethyl-3-(1-oxo-3-phenylprop-2-yn-
1-yl)-1H-pyrrol-1-yloxyl Radical (12b)
Yield: 1.61 g (60%); mp 75–76°C; Rf 0.65 (hexane/EtOAc, 2:1).
IR (film): n = 2200 (C∫C), 1640 (C=O), 1610 cm–1 (C=C).
MS: m/z (%) = 268 (M+, 4), 254 (8), 238 (19), 129 (100).
To a solution of compound 2h (1.28 g, 5.0 mmol) in anhyd THF (15
mL) was added dropwise with stirring a 70% toluene solution of so-
dium bis(2-methoxyethoxy)aluminum hydride (SMEAH, 4 mL) in
THF (10 mL). The resulting mixture was stirred under Ar for 5 h at
r.t. and then cautiously decomposed by the dropwise addition of
10% NaOH (10 mL). The organic phase was washed with brine (15
mL), dried (MgSO4) and evaporated. The residue was dissolved in
CHCl3 (15 mL), MnO2 (1.74 g, 20.0 mmol) was added and the mix-
ture was refluxed for 30 min then filtered and evaporated. The prod-
uct was purified by flash column chromatography with hexane/Et2O
giving the title compound; yield: 956 mg (74%); mp 57–58°C; Rf
0.17 (hexane/Et2O, 2:1).
2,5-Dihydro-3-(4-hydroxy-4-methyl-1-oxopent-2-yn-1-yl)-
2,2,5,5-tetramethyl-1H-pyrrol-1-yloxyl Radical (12c)
Yield: 1.43 g (57%); mp 138–139°C; Rf 0.57 (CHCl3/MeOH, 9:1).
IR (film): n = 3550–3300 (OH), 2090 (C∫C), 1672 (C=O), 1620
cm–1 (C=C).
MS: m/z (%) = 250 (M+, 10), 236 (14), 187 (18), 43 (100).
17α-[3-(2,5-Dihydro-2,2,5,5-tetramethyl-1-oxyl-1H-pyrrol-3-
yl)-3-oxoprop-1-yn-1-yl]estra-1,3,5(10)-triene-3,17β-diol Radi-
cal (12d)
IR (film): n = 1660 (C=O), 1610, 1600 cm–1 (C=C).
TSP: m/z = 259 (M+H)+.
Yield: 1.90 g (41%); mp 113–115°C; Rf 0.31 (CHCl3/Et2O, 2:1).
Biradical 7 and Nitrone Radical 8; General Procedure
IR (nujol): n = 3300–3200 (OH), 2200 (C∫C), 1645 (C=O), 1600
cm–1 (C=Carom).
TSP: m/z = 463 (M+H)+.
To a organomagnesium derivative of compound 3 (5.0 mmol) pre-
pared as above was added a solution of nitrone 1 or 2,5-dimethyl-1-
pyrroline N-oxide (5.0 mmol) in THF at –10°C. After stirring for
additional 2 h at r.t., aq satd NH4Cl solution (30 mL) was added.
The organic phase was separated, dried (MgSO4) and evaporated to
dryness. The residue was dissolved in CHCl3 (30 mL), activated
MnO2 (100 mg) was added and a stream of O2 was bubbled for 30
min, filtered and evaporated. The product was flash chromato-
graphed on silica gel with hexane/EtOAc or CHCl3/MeOH.
1-Acetoxy-2-ethynyl-2,5,5-trimethylpyrrolidine (3)
To a solution of radical 2a (4.57 g, 0.03 mol) in dioxane (50 mL)
was added a solution of ascorbic acid (26.4 g, 0.15 mol) in H2O (25
mL) and the mixture was stirred at 40°C for 15 min. The colourless
solution was extracted with CHCl3 (3 × 30 mL) and dried (MgSO4)
under N2 atmosphere. Then Et3N (3.33 g, 0.033 mol) and AcCl
(2.59 g, 0.033 mol) were added at 0°C. The stirring was continued
for 1 h at r.t. then the mixture was filtered and evaporated to dry-
ness. The residue was dissolved in brine (50 mL) and extracted with
Et2O (3 × 30 mL). The organic layer was dried (MgSO4) and evap-
orated. The product was purified by flash chromatography (hexane/
1,2-Bis(2,5,5-trimethyl-1-oxylpyrrolidin-2-yl)ethyne Biradical
(7)
Yield: 0.92 g (66%); oil; Rf 0.13 (hexane/Et2O, 2:1).
MS: m/z (%) = 278 (M+, 17), 216 (7), 138 (36), 41 (100).
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2-Benzoylmethyl-2,5,5-trimethylpyrrolidin-1-yloxyl Radical
1-(2,5-Dimethyl-1-oxide-1-pyrroline-5-yl)-2-(2,5,5-trimethyl-1-
oxylpyrrolidin-2-yl)ethyne Radical (8)
(17a) from 16
To a solution of 16 (496 mg, 2.0 mmol) in CHCl3 (15 mL) was add-
ed activated MnO2 (435 mg, 5.0 mmol) and the mixture was re-
fluxed for 15 min, then filtered and evaporated. The residue was
purified by flash column chromatography with hexane/Et2O as elu-
ent; yield: 428 mg (87%); mp 72–73°C; Rf 0.28 (hexane/Et2O, 2:1).
IR (film): n = 1660 (C=O), 1580 cm–1 (C=Carom).
MS: m/z (%) = 246 (M+, 8), 214 (3), 128 (9), 105 (100).
Yield: 0.76 g (58%); oil; Rf 0.32 (CHCl3/MeOH, 9:1).
MS: m/z (%) = 263 (M+, 2), 249 (5), 113 (48), 41 (100).
Bi- and Triradicals 9, 10; General Procedure
To an organomagnesium derivative of compound 3 (5.0 mmol) pre-
pared as above or ethynylmagnesium bromide (5.0 mmol) was add-
ed a solution of nitrone 8 (1.32 g, 5.0 mmol) in THF at –10°C. After
stirring for additional 2 h at r.t., aq satd NH4Cl solution (30 mL) was
added. The organic phase was separated, dried (MgSO4) and evap-
orated to dryness. The residue was dissolved in CHCl3, MnO2 (100
mg) was added and a stream of O2 was bubbled for 30 min, filtered
and evaporated. The product was flash chromatographed on silica
gel with hexane/EtOAc.
Preparation of Ketones 17a, 17b from Alkynes 2b, 2c; General
Procedure
To a solution of 2b or 2c (3.0 mmol) in dioxane (15 mL) were added
5% H2SO4 (5 mL) and a catalytic amount of Hg(OAc)2 (~ 10 mg).
The mixture was allowed to stand at r.t. for 24 h. After the reaction
was complete, brine (15 mL) was added and extracted with CHCl3
(3 × 15 mL). The organic phase was dried (MgSO4) and evaporated
in vacuo. The residue was purified by flash chromatography.
1-(2-Ethynyl-2,5-dimethyl-1-oxylpyrrolidine-5-yl)-2-(2,5,5-tri-
methyl-1-oxylpyrrolidin-2-yl)ethyne Biradical (9)
Yield: 0.89 g (62%); oil; Rf 0.44 (hexane/EtOAc, 2:1).
2,5,5-Trimethyl-2-(2-oxo-2-phenylethyl)pyrrolidin-1-yloxyl
Radical (17a)
Yield: 429 mg (58%).
IR (nujol): n = 3220 (∫CH), 2090 cm–1 (C∫C).
MS: m/z (%) = 288 (M+, 9), 258 (8), 219 (40), 41 (100).
2,5-Dimethyl-2,5-bis(2,5,5-trimethyl-1-oxylpyrrolidin-2-ethyn-
2-yl)-1-oxylpyrrolidine Triradical (10)
yield: 0.81 g (39%); oil; Rf 0.40 (hexane/EtOAc, 2:1).
TSP: m/z = 416 (M+H)+.
2,5,5-Trimethyl-2-[(2-(1-naphthyl)-2-oxoethyl]pyrrolidin-1-
yloxyl Radical (17b)
Yield: 551 mg (62%); oil; Rf 0.22 (hexane/Et2O, 2:1).
IR (film): n = 1664 (C=O), 1590 cm–1 (C=Carom).
MS: m/z (%) = 296 (M+, 19), 170 (56), 155 (100), 127 (98).
Rearrangement of 4-Isoxazoline 13 to Acylaziridine 14 and Pyr-
role 15
A solution of 4-isoxazoline (13) (458 mg, 2.0 mmol) in toluene (15
mL) was refluxed for 2 h, then the solvent was evaporated in vacuo
and purified by flash column chromatography.
a-Bromoketones 18a, 18b; General Procedure
To a solution of ketone 17a, 17b (4.0 mmol) in anhyd THF (30 mL)
were added 2-pyrrolidone (426 mg, 5.0 mmol) and pyrrolidone hy-
drotribromide (PHT, 2.48 g, 5.0 mmol). The mixture was refluxed
for 30 min and allowed to cool to r.t., washed with brine (30 mL),
dried (MgSO4) and the solvent was removed in vacuo. The crude
product was purified by flash chromatography with hexane/Et2O.
2-Benzoyl-3,6,6-trimethyl-1-azabicyclo[1.3.0]hexane (14)
Yield: 96 mg (21%); oil; Rf 0.18 (hexane/Et2O, 2:1).
IR (film): n = 1682 (C=O), 1597 cm–1 (C=Carom).
MS: m/z (%) = 229 (M+, 8), 214 (83), 77 (55), 68 (100).
2-(1-Bromo-2-oxo-2-phenylethyl)-2,5,5-trimethylpyrrolidin-1-
yloxyl Radical (18a)
Yield: 846 mg (65%); oil; Rf 0.79 (hexane/EtOAc, 2:1).
8,8-Dimethyl-3-phenyl-1-azabicyclo[3.3.0]octa-2,4-diene (15)
Yield: 144 mg (34%); oil; Rf 0.78 (hexane/Et2O, 2:1).
IR (film): n = 1682 (C=O), 1590 cm–1 (C=CArom).
MS: m/z (%) = 326/324 (M+, 1/1), 215 (3), 157 (22), 105 (100).
IR (film): n = 1596 cm–1 (C=Carom).
MS: m/z (%) = 211 (M+, 92), 196 (68), 156 (100), 77 (24).
2-[1-Bromo-2-oxo-2-(1-naphthyl)ethyl]-2,5,5-trimethylpyrroli-
din-1-yloxyl Radical (18b)
Yield: 1.08 g (72%); mp 128–129°C; Rf 0.30 (hexane/Et2O, 2:1).
2-(2-Hydroxy-2-phenylethyl)-2,5,5-trimethylpyrrolidine-1-
yloxyl Radical (16)
To a solution of the isoxazoline 13 (1.15 g, 5.0 mmol) in EtOH (10
mL) were added a 10 M solution of AcOH (30 mL) and ethylenedi-
amine tetraacetic acid disodium salt (~ 8 g). The mixture was
warmed up to 60°C, stirred and Zn powder (3.25 g, 50 mmol) was
added. After maintaining the temperature at 60°C for 45 min, the
mixture was cooled to r.t., adjusted to pH 10 by adding 30% NH3
solution and extracted with CHCl3 (3 × 10 mL). The organic phase
was dried (MgSO4) and evaporated. The residue was dissolved in
MeOH (15 mL) then a catalytic amount of Na2WO4 (200 mg) and
H2O2 solution (30%, 5 mL) were added and the mixture was al-
lowed to stand at r.t. overnight. The solvent was evaporated to 1/4
of its volume, extracted with CHCl3, dried (MgSO4) and evaporat-
ed. The product was purified by flash chromatography using hex-
ane/EtOAc to give 16; yield: 844 mg (68%); mp 51–52°C; Rf 0.48
(hexane/EtOAc, 2:1).
IR (film): n = 1668 cm–1 (C=O).
MS: m/z (%) = 374/376 (M+, 3/3), 265 (6), 155 (100), 127 (49).
2-(1-Iodo-2-oxo-2-phenylethyl)-2,5,5-trimethylpyrrolidin-1-
yloxyl Radical (18c)
To a solution of 18a (325 mg, 1.0 mmol) in anhyd THF (10 mL) was
added NaI (300 mg, 2 mmol) and the mixture was refluxed for 2 h.
The solvent was evaporated, the residue dissolved in brine (15 mL)
and extracted with Et2O (3 × 15 mL). The organic layer was dried
(MgSO4), evaporated and purified by flash chromatography (hex-
ane/Et2O); yield: 290 mg (78%); oil; Rf 0.80 (hexane/EtOAc, 2:1).
IR (film): n = 1690 (C=O), 1595 cm–1 (C=CArom).
MS: m/z (%) = 372 (M+, not detectable), 245 (M – I, 1), 157 (10),
105 (100).
IR (film): n = 3330 (OH), 1595 cm–1 (C=Carom).
MS: m/z (%) = 248 (M+, 2), 218 (2), 128 (100), 107 (60).
S-Spin Labelled N-Acetylcysteine Methyl Ester (19)
To a solution of 2h (512 mg, 2.0 mmol) in dioxane/H2O (2:1, 15
mL) were added N-acetylcysteine methyl ester (354 mg, 2.0 mmol)
and K2CO3 (276 mg, 2.0 mmol) and the mixture was stirred for 2 h
Synthesis 1999, No. 6, 1039–1045 ISSN 0039-7881 © Thieme Stuttgart · New York

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Synthesis and Reactions of New Alkynyl Substituted Nitroxide Radicals
1045
at r.t. Then H2O (10 mL) was added and extracted with CHCl3 (3 ×
10 mL). The organic phase was dried (MgSO4), evaporated in vacuo
and purified by flash chromatography with hexane/EtOAc; yield:
538 mg (62%); mp 67–69°C; Rf 0.16 (CHCl3/Et2O, 2:1).
References
(1) Likhtenstein, G.I. Biophysical Labeling Methods in Molecular
Biology; Cambridge University Press: New York, 1993.
(2) Kocherginsky, N.; Swartz, H.M. Nitroxide Spin Labels -
Reactions in Biology and Chemistry; CRC Press: Boca Raton,
1995.
(3) Hideg, K.; Hankovszky, H.O. In Biological Magnetic
Resonance; Vol. 8.; Berliner, L.J.; Reuben, J., Eds.; Plenum:
New York, 1989, p 427.
(4) Farahbakhs, T.Z.; Hideg, K.; Hubbell, W.L. Science 1993,
262, 1416.
(5) Cornish, V.W.; Benson, D.R.; Altenbach, C.A.; Hideg, K.;
Hubbell, W.L.; Schultz, P.G. Proc. Natl. Acad. Sci., USA
1994, 91, 2910.
(6) Gettins, P.G.W.; Crews, B.; Beth, A.H.; Hideg, K. FEBS
Letters 1995, 367, 137
(7) Bárácz, M.N.; Hankovszky, H.O.; Sár, C.P.; Jerkovich, Gy.;
Hideg, K. Synthesis 1996, 205.
(8) Hideg, K.; Csekö J.; Hankovszky, H.O.; Sohár, P. Can. J.
Chem. 1986, 64, 1482.
IR (film): n = 3400–3200 (NH), 1740 (C=O), 1658, 1640 (C=O),
1600, 1590 cm–1 (C=C).
TSP: m/z = 434 (M+H)+.
17a-3-Methoxy-[2-(2,5,5-trimethyl-1-oxylpyrrolidin-2-
yl)ethyn-1-yl]estra-1,3,5(10)-triene-17b-ol Radical (20)
To a solution of 2e (423 mg, 1.0 mmol) in dioxane/H2O (5:1, 20
mL) were added K2CO3 (276 mg, 2.0 mmol), MeI (284 mg, 2.0
mmol) and 18-Crown-6 (20 mg) and the mixture was stirred at r.t.
for 2h. Then H2O (20 mL) was added and extracted with CHCl3 (3
× 20 mL). The organic layer was dried (MgSO4), evaporated and
purified by flash chromatography with CHCl3/Et2O as eluent; yield:
332 mg (76%); mp 65–67°C; Rf 0.44 (CHCl3/Et2O, 2:1).
IR (film): n = 3400–3300 (OH), 1606 cm–1 (C=Carom).
MS: m/z (%) = 436 (M+, 7), 406 (38), 227 (82), 55 (100).
(9) Horváth, L.I.; Arias, H.R.; Hankovszky, H.O.; Hideg, K.;
Barrantes, F.J.; Marsh, D. Biochemistry 1990, 29, 8707.
(10) Tian, X.; Wang, Y.; Yang, M.; Chen, Y. Life Sci. 1997, 60,
511.
(11) Esmann, M.; Hankovszky, H.O.; Hideg, K.; Pedersen, J.A.;
Marsh, D. Anal. Biochem. 1990, 189, 274.
(12) Horváth, L.I.; Török, M.; Hideg, K.; Dux, L. J. Mol. Struct.
1997, 408/409, 177.
(13) Roopnarine, O.; Hideg, K.; Thomas, D.D. Biophys J. 1993,
64, 1896.
(14) Li, H-C.; Hideg, K.; Fajer, P.G. J. Mol. Recogn. 1997, 10, 194.
(15) Adhikari, B.; Hideg, K.; Fajer, P.G. Proc. Natl. Acad. Sci.,
USA 1997, 94, 9643.
17a-[2-(2,5,5-Trimethylpyrrolidin-2-yl)ethyn-1-yl]estra-
1,3,5(10)-triene-3,17b-diol (21)
To a solution of 2e (423 mg, 1.0 mmol) in AcOH (30 mL) was add-
ed Fe powder (280 mg, 5.0 mmol) and the mixture was kept at 50°C
for 3 h. Then the mixture was allowed to cool to r.t., H2O (10 mL)
was added and the aqueous phase was decanted and basified adding
solid K2CO3. The aqueous phase was extracted with CHCl3 (3 × 20
mL), dried (MgSO4) and evaporated. The residue was purified on
silica gel with CHCl3/MeOH; yield: 257 mg (63%); mp 199–200°C;
Rf 0.35 (CHCl3/MeOH, 9:1).
IR (nujol): n = 3500–3200 (OH, NH), 1606 cm–1 (C=Carom).
MS: m/z (%) = 407 (M+, 2), 392 (19), 374 (2), 43 (100).
(16) Torii, S.; Hase, T.; Kuroboshi, M.; Amatore, C.; Jutand, A.;
Kawafuchi, H. Tetrahedron Lett. 1997, 38, 7391.
(17) Brik, M-E. Heterocycles 1995, 41, 2827, and references cited
therein.
17a-[2-(2,5,5-Trimethyl-1-oxylpyrrolidin-2-yl)ethen-1-yl]estra-
1,3,5(10)-triene-3,17b-diol Radical (22)
(18) Padwa, A.; Wong, G.S.K. J. Org. Chem. 1986, 51, 3125.
(19) Freeman, J.P. Chem. Rev. 1983, 83, 241.
(20) Ali, Sk.A. J. Chem. Res. (S) 1994, 32.
(21) Carruthers, W.; Coggins, P.; Weston, J.B. J. Chem. Soc.,
Perkin. I. 1990, 2323.
(22) Hankovszky, H.O.; Hideg, K.; Sár, P.C.; Lovas, M.J.;
Jerkovich, Gy. Synthesis 1990, 59.
(23) Raucher, D.; Sár, C.P.; Hideg, K.; Fajer, G.P. Biochemistry
1994, 33, 14317.
(24) Raucher, D.; Fajer, E.A.; Sár, C.P.; Hideg, K.; Zhao, Y.;
Kawai, M.; Fajer, P.G. Biophys. J. 1995, 68, 128s.
(25) Sár, P.C.; Kálai, T.; Bárácz, M.N.; Jerkovich, Gy.; Hideg, K.
Synth. Comm. 1995, 25, 2929.
(26) Brandsma, L. Preparative Acetylenic Chemistry; Elsevier:
Amsterdam, 1988.
To a solution of 2e (423 mg, 1.0 mmol) in anhyd THF (20 mL) was
added dropwise SMEAH (70% in toluene, 1 mL) at 0°C. After com-
pletion of the addition, the cooling bath was removed and the mix-
ture was stirred at r.t. for 3 h. Then 10% NaOH (10 mL) was added,
the organic phase was separated and the aqueous layer was extract-
ed with CHCl3 (3 × 15 mL). The combined organic extracts were
dried (MgSO4), and a catalytic amount of MnO2 (100 mg) was add-
ed and a stream of O2 was bubbled for 30 min. The mixture was fil-
tered and the solvent was removed under vacuum. The residue was
purified by flash chromatography (CHCl3/Et2O) leading to the ole-
finic compound 26; yield: 300 mg (70%); mp 113–115°C; Rf 0.17
(hexane/EtOAc, 2:1).
IR (nujol): n = 3300 (OH), 1606 cm–1 (C=C).
MS: m/z (%)= 424 (M+, 1), 394 (7), 55 (66), 43 (100).
Acknowledgement
Article Identifier:
1437-210X,E;1999,0,06,1039,1045,ftx,en;P06698SS.pdf
The authors wish to express their thanks to J. Molnár for technical
assistance and for B. Rozsnyai for microanalyses and M. Szabó
(ICN Hungary Ltd.) for mass spectral measurements.This work was
supported by grants from the Hungarian Academy of Sciences (97-
13 2,4/23) and Ministry of Welfare (80-4/1998).
Synthesis 1999, No. 6, 1039–1045 ISSN 0039-7881 © Thieme Stuttgart · New York