Design, synthesis, and in vitro evaluation of cyclic nitrones as free radical traps for the treatment of stroke

Article abstract of DOI:10.1021/jm960243v

Analogs of the cyclic nitrone free radical trap 1 (3,3-dimethyl-3,4- dihydroisoquinoline N-oxide, a cyclic analog of phenyl-tert-butylnitrone (PBN)) were prepared in which (1) the fused phenyl ring was replaced with a naphthalene ring, an electron rich heterocycle, or a dimethylphenol, (2) the nitrone-containing ring comprised five, six, or seven atoms, and (3) the gem- dimethyl group was replaced with spirocyclic groups. The most active antioxidant, which bears a dimethylphenol fused to a 7-membered ring nitrone (compound 6h), inhibited lipid peroxidation in vitro with an IC₅₀ of 22 μM, a 75-fold improvement over that of 1. The previously observed correlation between lipophilicity and activity vs lipid peroxidation in vitro has been further substantiated and refined by this study. Moreover, certain classes of compounds (namely, dimethylphenols 6g, h and furan 6j) have now been found which are considerably more active in vitro than expected on the basis of their log k'(w) values.

Full text of DOI:10.1021/jm960243v

4988
J . Med. Chem. 1996, 39, 4988-4996
Design , Syn th esis, a n d in Vitr o Eva lu a tion of Cyclic Nitr on es a s F r ee Ra d ica l
Tr a p s for th e Tr ea tm en t of Str ok e
Thomas L. Fevig,* S. Marc Bowen, David A. J anowick,† Bryan K. J ones, H. Randall Munson,
David F. Ohlweiler, and Craig E. Thomas‡
Hoechst Marion Roussel, Inc., 2110 East Galbraith Road, Cincinnati, Ohio 45215
Received March 28, 1996X
Analogs of the cyclic nitrone free radical trap 1 (3,3-dimethyl-3,4-dihydroisoquinoline N-oxide,
a cyclic analog of phenyl-tert-butylnitrone (PBN)) were prepared in which (1) the fused phenyl
ring was replaced with a naphthalene ring, an electron rich heterocycle, or a dimethylphenol,
(2) the nitrone-containing ring comprised five, six, or seven atoms, and (3) the gem-dimethyl
group was replaced with spirocyclic groups. The most active antioxidant, which bears a
dimethylphenol fused to a 7-membered ring nitrone (compound 6h ), inhibited lipid peroxidation
in vitro with an IC50 of 22 µM, a 75-fold improvement over that of 1. The previously observed
correlation between lipophilicity and activity vs lipid peroxidation in vitro has been further
substantiated and refined by this study. Moreover, certain classes of compounds (namely,
dimethylphenols 6g,h and furan 6j) have now been found which are considerably more active
in vitro than expected on the basis of their log k′w values.
In tr od u ction
There are numerous disease states, including stroke,
in which radical-induced oxidative tissue damage is
prevalent.1 For example, the occurrence of lipid per-
oxidation2,3 with a concomitant decrease in the lipid
F igu r e 1.
soluble R-tocopherol has been reported4 following cere-
bral ischemia/reperfusion injury. In order to minimize
this type of damage, various classes of radical traps, or
X-ray crystal structures suggest that the nitrone double
bond in 1 is sterically more accessible than that in PBN.
antioxidants, are being developed. Among these are
nitrones, typified by phenyl-tert-butylnitrone (PBN,
Figure 1), which can react with short-lived oxygen- or
carbon-centered radicals at the nitrone double bond. The
result is a relatively stable nitroxide radical which
cannot propagate destructive radical chain reactions
and has a sufficient lifetime to diffuse from the site at
which it is generated, thereby preventing concentrated,
debilitating tissue damage. Indeed, PBN has shown
intriguing activity as a neuroprotectant in certain
animal models, and free radical trapping has been
proposed as the mechanism of action in these studies.5
We have recently reported the design and synthesis
of cyclic analogs of PBN,6 the simplest of these being
compound 1 (Figure 1). The original premise for the
synthesis of 1 and related analogs was that cyclic
compounds should be more reactive toward radicals both
electronically, by virtue of increased planarity resulting
in better orbital overlap between the nitrone double
bond and the aromatic ring, and sterically, because of
restricted rotation. However, subsequent molecular
modeling studies7 suggested that, in their lowest energy
conformations, PBN was actually more nearly planar
than 1. The X-ray crystal structures8 show that in both
molecules, at least in the solid state, the phenyl ring
and the nitrone double bond form a dihedral angle of
about 14°. Hence, there is no evidence to sustain the
original argument with respect to planarity and orbital
overlap. On the other hand, both the modeling and the
Moreover, high-level HOMO/LUMO calculations7 for the
two molecules show that 1 has a higher energy HOMO
orbital and a lower energy LUMO orbital than those
for PBN. Thus, 1 should be more reactive toward both
electrophilic, oxygen-centered radicals (which occur
mainly through SOMO-HOMO interactions) and toward
nucleophilic, carbon-centered radicals (which occur
mainly through SOMO-LUMO interactions).9 In fact,
when tested for its ability to inhibit radical-induced lipid
peroxidation or to trap hydroxyl radicals in vitro, 1 was
substantially more potent than PBN.7 Also, 1 possesses
outstanding solubility characteristics, being highly soluble
both in water and in nonpolar organic solvents.
Although possessing these favorable properties, 1 still
suffers from low potency (the IC50 in an in vitro lipid
peroxidation assay is 1.67 mM), and side effects (seda-
tion at doses greater than 100 mg/kg, and an iv LD50 of
320 mg/kg). The overall result is an unacceptable
therapeutic index. In this study we describe our efforts
to improve the therapeutic index by increasing potency.
The following paper in this issue addresses the issue of
side effects.
Previous studies from our labs6,7,10 focused mainly on
placing various substituents on the aromatic ring, and
to a lesser extent on expanding the nitrone-containing
ring and replacing the gem-dimethyl moiety with spi-
rocyclic groups. Herein we describe the synthesis and
evaluation of compounds in which (1) the fused phenyl
ring is replaced with more extended aromatic systems
or electron rich heterocycles, (2) a second antioxidant
functionality (o,o′-dimethylphenol) is added, (3) the
nitrone-containing ring is contracted, and (4) the ni-
trone-containing ring is expanded and/or the gem-
† Current address: Abbott Laboratories, 100 Abbott Park Rd, Abbott
Park, IL 60064-3500.
‡ Current address: Eli Lilly and Company, Greenfield Laboratories,
P.O. Box 708, Greenfield, IN 46140.
X Abstract published in Advance ACS Abstracts, November 15, 1996.
S0022-2623(96)00243-9 CCC: $12.00 © 1996 American Chemical Society

Cyclic Nitrones as Free Radical Traps
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25 4989
Sch em e 1a,b
Ta ble 1. In Vitro Activity and log k′w Values for PBN and
Cyclic Nitrones
a
Reagents: (a) tetrahydropyran-4-one or cyclohexanone, (b)
b
MeMgBr, (c) NaCN, H2SO4. Letter designations correspond to
structures given in Table 1.
11g,h (Scheme 3). Carboxylic esters 7g,h 14 were treated
with MeMgBr and brominated in the 4-position to
furnish tertiary alcohols 8g,h . A double Mannich
reaction with pyrrolidine and formaldehyde then pro-
vided 9g,h . Numerous attempts to hydrogenolyze the
bromine and both pyrrolidines in one step were unsuc-
cessful, the more hindered C-N bond being inert to a
wide variety of conditions.15 Resubmission of a chro-
matographically purified, debrominated monopyrroli-
dine (not shown) to several hydrogenation conditions
also failed to give 11g. Finally, we found that the amino
groups could be displaced by thiols16 under harsh
conditions (with concomitant debromination) to afford
10g,h . These were readily desulfurized upon treatment
with RaNi to give 11g,h .
The Ritter reaction of 11g,h with NaCN gave the
cyclized imines 5g,h directly via capture of the nitrilium
ion intermediate by the electron rich phenol. This
reaction was observed in the synthesis of thiophene 6c
as well. The yields were low with the thiophene and
11h due to competing reactions of the tertiary cation
intermediates, but 11g gave imine 5g in ca. 50% yield.
Conversion of the imines into the corresponding nitrones
could be carried out as described above (NaBH4 reduc-
tion; H2O2/Na2WO4 oxidation), but because of the high
water solubility of these compounds, it was preferrable
to perform the reduction step by catalytic hydrogenation
(RaNi).
Compounds 6i-n were not accessible by the above
route. For example, in an attempted synthesis of
thiophene 6i, the Ritter reaction gave neither the
cyclized imine nor the formamide because of intermo-
lecular reactions of the tertiary cation. Likewise, a
5-membered ring could not be formed by cyclization of
the requisite formamide as described above. In order
to avoid these problems, isocyanates were chosen as
cyclization substrates since this functional group could
be generated under milder conditions via the Curtius
rearrangement.
The Curtius rearrangement substrates (carboxylic
acids 12) were readily prepared as shown in Scheme 4.
The isocyanates (13) formed smoothly upon treatment
of the acids with diphenyl phosphorazidate. For the
heterocycles, anhydrous H3PO417 or BF3‚Et2O proved to
be the best cyclization catalysts, whereas only FeCl3
allowed formation of the isoindolones (i.e., 14k , Scheme
5) to occur in reasonable yield.18 In the latter case, an
activating substituent (OMe) appropriately placed on
the aromatic ring was also required. This resulted in
the formation of regioisomers and necessitated removal
of the activator19 (Scheme 5) in order to obtain the
unsubstituted analog 6n for direct comparison to
a
b
See ref 22. Calculated from eq 1. c These values represent
single determinations. The r2 values for linear regression analyses
of the raw chromatographic data were >0.999 in each case. See
ref 21.
dimethyl group is replaced by a spirotetrahydropyran
or a spirocyclohexyl moiety.
Ch em istr y
Compounds 6a -f (Table 1) were prepared by the
previously described route,6 as summarized in Schemes
1 and 2. This involved preparation and reaction of
tertiary alcohols 2 with NaCN under Ritter11 conditions
to give formamides 3. Cyclization of the formamides
by a method developed by Larsen and co-workers at
Merck12 afforded tricyclic intermediates 4. These could
be hydrolyzed or, preferrably in some cases, pyrolyzed6b
to give the desired imines. The imines were converted
into the requisite nitrones most efficiently by prelimi-
nary reduction (NaBH4) to the corresponding amines,
followed by tungstate-catalyzed H2O2 oxidation.13
Synthesis of the o,o′-dimethylphenol analogs 6g,h
followed the same general route, but additional steps
were required to obtain the Ritter reaction substrates,

4990 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25
Fevig et al.
Sch em e 2a,b
a
b
Reagents: (a) (COCl)2; (b) FeCl3; (c) H2SO4/EtOH, ∆ or ∆; (d) NaBH4; (e) H2O2, cat. Na2WO4. Letter designations correspond to
structures given in Table 1.
Sch em e 3a
a
Reagents: (a) MeMgBr; (b) NBS; (c) 37% aqueous HCHO, pyrrolidine, ∆; (d) RSH, 180 °C, no solvent; (e) RaNi, EtOH, ∆; (f) NaCN,
H2SO4; (g) NaBH4, or H2/RaNi; (h) H2O2, cat. Na2WO4.
Sch em e 4a,b
Sch em e 5a
a
Reagents: (a) BBr3, (b) 5-chloro-1-phenyl-1H-tetrazole, K2CO3,
a
Reagents: (a) ethyl isobutyrate, LiN(TMS)2; (b) KOH; (c) MeI,
(c) H2, Pd/C, (d) BH3/THF, ∆, (e) H2O2, cat. Na2WO4.
NaH; (d) DPPA, Et3N, ∆; (e) anhydrous H3PO4, or BF3‚Et2O, or
FeCl3; (f) BH3/THF, ∆; (g) H2O2, cat. Na2WO4. b Letter designations
correspond to structures given in Table 1.
1, although HOMO/LUMO energy calculations7 suggest
that these compounds should be less reactive toward
radicals than 1 (but more reactive than PBN), from a
frontier molecular orbital standpoint. Finally, we had
observed in previous studies6,7,10 a general trend that
in vitro activity against lipid peroxidation was correlated
with lipophilicity. Accordingly, some spirocyclic and
ring-expanded analogs (6d -f) were targeted.
The cyclic nitrones prepared (6a -n ) are listed in
Table 1, along with their IC50 values for inhibition of
lipid peroxidation in vitro. In addition, the relative
lipophilicity of the compounds was determined by a well-
established chromatographic technique (log k′w val-
ues).21 Compound 1 and PBN are also included for
comparison. The in vitro assay procedure involved
measuring the ability of the test compounds to inhibit
oxidation of soybean phosphatidylcholine liposomes.22
A mixture of Fe2+/Fe3+ was used to initiate peroxidation,
as this system produces an as yet uncharacterized, weak
oxidant which reacts solely with polyunsaturated fatty
1. Compounds 6k -m were also prepared and submit-
ted for testing (see Table 1 for structures and Scheme
5 and Experimental Section for synthesis).
Resu lts a n d Discu ssion
Several approaches toward increasing the potency of
the cyclic nitrones (relative to 1) were taken in this
study. The electronic effects of increasing the degree
of conjugation of the nitrone double bond (naphthalenes
6a ,b), or placing a heteroatom in conjugation with it
(heterocycles 6c,i,j), were expected to afford more reac-
tive compounds. The dimethylphenol analogs (6g,h )20a
were obviously members of the well-known class of
antioxidants based on R-tocopherol,20b-d which are
generally substantially more potent than 1 in the lipid
peroxidation assay. Compounds in which the nitrone
is contained within a 5-membered ring (e.g. 6n ), were
predicted to be somewhat more sterically accessible than

Cyclic Nitrones as Free Radical Traps
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25 4991
F igu r e 2. Plot of log(1/IC50) vs log k′w for 1 and 6a -n . IC50
values in mM units were used. log k′w values were measured
as described in ref 21. Points labeled a-n correspond to
compounds 6a -n .
F igu r e 3. Plot of measured vs predicted IC50 values for 1 and
6a -n . Predicted values were calculated with eq 1. See text.
Points labeled a-n correspond to compounds 6a -n .
acids.23 Thus, measurement of nitrone trapping of lipid-
derived oxygen- or carbon-centered radicals is not
subject to the interference of initiating radicals.
The data described above show that increased lipo-
philicity, the presence of a phenol, and the incorporation
of an electron rich heterocycle (furan, but not thiophene)
can each have an appreciable beneficial effect on the
antioxidant activity. The remaining parameters inves-
tigated in this study, the degree of conjugation of the
nitrone (naphthalenes 6a and 6b), and the size of the
nitrone-containing ring (five, six, or seven atoms) seem
to have relatively little effect on the radical-trapping
ability of the compounds. The naphthalenes are quite
potent, but in both plots (Figures 2 and 3) these
compounds show no significant deviation from the
expected values based on log k′w. Thus, the added
aromatic ring apparently contributes nothing beyond
lipophilicity to these molecules.
In order to assess the effect of the ring size we can
compare three pairs of compounds in which ring size is
the only difference. With spirotetrahydropyrans 6d
(IC50 ) 1240 µM, log k′w ) 1.65) and 6e (IC50 ) 310
µM, log k′w ) 1.98), the increase in potency on going
from a 6-membered ring to a 7-membered ring is
essentially in line with the increase in lipophilicity.
In general, the more lipophilic compounds are more
active in vitro, as expected based on previous results.
A plot of log(1/IC50) vs log k′w (Figure 2) shows the trend,
but fails to give a statistically valid quantitative struc-
ture-activity relationship. The major outliers in the
plot seem to be spirocyclohexyl derivative 6f and the
phenolic compounds 6g,h ,m . Compound 6f is the most
lipophilic member of the series, and it is possible that
micelle formation, which impairs incorporation into the
liposomes, is confounding the data. The phenolic com-
pounds 6g,h ,m (points g, h, and m in Figure 2) are well-
behaved among themselves, showing a steady increase
in potency as the log k′w increases from 0.68 for 6m to
2.29 for 6h . We believe the relationship between the
three phenolic compounds and the remaining, nonphe-
nolic analogs reflects the expected increase in potency
resulting from the presence of a second antioxidant
moiety. If separate lines were drawn to fit these two
sets of points (excluding point f), one can see that the
lines would have approximately the same slope, but the
line for the phenols would be offset by about 1 log unit
in the direction of greater activity. Thus, while log k′w
alone gives a poor correlation with activity, if we add
an indicator variable for the presence (D ) 1) or absence
(D ) 0) of a phenol, the statistically significant eq 1 is
obtained24 which correlates the antioxidant activity of
this series with lipophilicity. The predicted IC50 values
shown in Table 1 were calculated with this equation.
Likewise, the data for phenols 6g (IC50
) 66 µM, log
k′w ) 1.82) and 6h (IC50 ) 22 µM, log k′w ) 2.29) support
the contention that there is little or no difference
(beyond lipophilicity) between 6- and 7-membered rings.
On the other hand, the 5-membered ring nitrone 6n
(IC50 ) 1360 µM, log k′w ) 1.61) is perhaps slightly more
active than its 6-membered ring counterpart 1 (IC50
)
1670 µM, log k′w ) 1.90) despite being more polar,
suggesting that a 5-membered ring nitrone might be
inherently more reactive toward free radicals. However,
the ring size clearly has no dramatic impact on the
activity of the nitrones.
log(1/IC50) ) 0.701 ((0.076)log k′w +
1.06 ((0.054)D -1.20 ((0.013) (1)
We believe that the lipophilicity correlation is to some
extent inherent in the assay, i.e. compounds having
greater residence time in the lipid phase should be more
effective at protecting the lipid from peroxidation.25
Therefore, the most interesting compounds are those for
which in vitro potency exceeds that expected on the
basis of log k′w values, indicating inherently superior
radical trapping ability. Of the compounds reported
here, only the phenolic compounds 6g,h ,m and furan
6j seem to fit this description. The effect of the phenol,
however, does not seem to be additive as hoped; simple
phenolic compounds, such as vitamin E and probucol,
lacking a nitrone moiety yield IC50 values of about 10
n ) 15, r2 ) 0.729, r ) 0.854, q2 ) 0.502,
standard error of estimate ) 0.350,
standard error of prediction cross-validated ) 0.474.
A plot of the measured vs calculated IC50 values gives
a different perspective on the data set (Figure 3). The
major outlier in this plot is furan 6j, which seems to be
much more potent than anticipated from lipophilicity
measurements. This effect was suspected from analysis
of the plot in Figure 2, but is more dramatically evident
here.

4992 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25
Fevig et al.
µM in this assay.10,22 Hence, the activity of the phenol
moiety may actually be diminished as a consequence of
being in conjugation with the nitrone functionality, a
situation which was prompted by consideration of
synthetic accessibility.
chromatography (FC) was carried out using EM Science silica
gel 60 (40-63 µm) according to the literature procedure.27
Melting points were determined on a Thomas-Hoover Uni-
melt capillary melting point apparatus and are uncorrected.
IR spectra were recorded on a Mattson Galaxy Series 5020
infrared spectrophotometer with samples prepared as indi-
cated and are reported in wavenumbers (cm-1). 1H NMR and
13C NMR spectra were recorded on Varian Unity (300 or 400
MHz) or Gemini (300 MHz) spectrometers with chemical shifts
(δ) reported in ppm relative to tetramethylsilane (0.00 ppm)
and chloroform-d (77.0 ppm), respectively. Mass spectra were
obtained on a Finnigan MAT Model TSQ 700 mass spectrom-
eter system using electron impact or chemical ionization with
the molecular ion designated as M+ or (M + H)+ given in
parentheses. Combustion analyses were obtained with a
Perkin-Elmer Model 2400 elemental analyzer.
Con clu sion
The significant new findings of this study can be
summarized as follows: (a) new chemistry was devel-
oped in order to prepare some compounds inaccessible
by the previously reported route, (b) the relative lipo-
philicities (log k′w) of the compounds were measured
(rather than calculated) by a rapid, convenient, and
reliable method, (c) two different classes of compounds
were found to be significantly more active than expected
on the basis of their lipophilicity, and, most importantly,
(d) an equation was derived from the data which allows
us for the first time to find such compounds. This latter
point represents a significant departure from, and
extension of, our previous studies.
More specifically, the results described above dem-
onstrate that relatively simple modifications of the
nitrone radical trap PBN can yield large increases in
potency in an in vitro assay measuring inhibition of lipid
peroxidation. Incorporation of the nitrone unit within
a ring, as shown also in previous studies,6,7,10 gives a
ca. 10-fold improvement of the IC50, presumably as a
result of favorable changes in the HOMO and LUMO
energy levels, and greater steric accessibility of the
nitrone double bond. Other desirable structural fea-
tures revealed by this work include the presence of a
phenolic hydroxyl, a furan ring in place of the fused
phenyl ring, and log k′w values of ca. 2.0-2.5. The
scatter in the data evident in Figures 2 and 3 is believed
to result from the smaller contributions of electronic or
steric effects to the activity of the compounds. Com-
pound 6h , which combines several of the best features
noted above (cyclic nitrone, phenolic hydroxyl, log k′w
of 2.29), is the most potent nitrone we have synthesized
to date. With an IC50 of 22 µM, this compound is 650-
fold more potent than PBN in the in vitro lipid peroxi-
dation assay.
Bioch em ica l Meth od s. Preparation of the liposomes and
analysis of oxidation products were carried out essentially as
described by Thomas.22 Compound 1 was included as an
internal standard during testing of new compounds. If the
value obtained for 1 varied by more than 15% from the value
given in Table 1, the experiment was excluded.
Deter m in a tion of log k′w Va lu es.21 Compounds were
dissolved in MeOH at 0.5 mg/mL, injected onto a Zorbax Rx-
C18 column (15 cm × 4.6 mm i.d.; 5 µm) from MAC-MOD
Analytical, Inc., and eluted with various percentages of MeOH
(32-62%, three concentrations/compound) in pH 7.4 phosphate
buffer. The flow rate was 1 mL/min, the column temperature
was 30 °C, and the UV detector wavelength was λ ) 230 nm.
log k values were calculated by using the equation: log k )
log((t - t0)/t0), where t is the retention time of the compound
of interest and t0 is the elution time of MeOH, which is not
retained on the column. Linear regression analysis (r2
>
0.999) was performed on the three data points for each
compound and the resulting line extrapolated to 100% aqueous
to give the log k′w values listed in Table 1.
Gen er a l P r oced u r e for Oxid a tion of Am in es to Ni-
tr on es (P r oced u r e A). The amine (1 equiv) was dissolved
in MeOH and treated sequentially with Na2WO4 (0.1 equiv)
and 30% H2O2 (3 equiv). The resulting mixture was stirred
at room temperature until TLC analysis indicated complete
reaction (ca. 4 h). The reaction mixture was poured into brine
containing Na2S2O3 (to destroy excess peroxide) and extracted
several times with EtOAc (until aqueous phase showed little
or no product by TLC). The organic phase was dried (Na2-
SO4), filtered, and evaporated. The crude product was purified
as indicated.
Gen er a l P r oced u r e for Hyd r olysis of Ester s or Nitr iles
(P r oced u r e B). The ester or nitrile (1 equiv) was added to a
solution of KOH (2.5 equiv) in 10% aqueous MeOH, and the
resulting mixture was heated at reflux until TLC showed the
absence of starting material. The mixture was cooled and most
of the MeOH evaporated. The residue was diluted with water
and extracted with Et2O (2×, discarded). The aqueous phase
was acidified by adding dilute HCl and extracted with EtOAc
(3×). The organic phase was washed with brine, dried
(MgSO4), filtered, and evaporated. The crude product was
used without further purification.
Having learned how to maximize in vitro radical
trapping activity, we are now focusing attention on
optimizing the physicochemical properties of the cyclic
nitrones so as to obtain potent compounds with good
solubility and brain penetration and minimal side
effects (see the following paper in this issue).
Exp er im en ta l Section
Gen er a l Meth od s. Except where noted otherwise, re-
agents and starting materials were obtained from common
commercial sources and used as received. Tetrahydrofuran
(THF) was distilled from sodium benzophenone ketyl im-
mediately prior to use. Other reaction solvents, and all
chromatographic, recrystallization, and workup solvents, were
spectroscopic grade and used as received. Reactions were
carried out under an atmosphere of dry N2 in oven-dried flasks
when required.
Thin layer chromatography (TLC) was performed on glass-
backed, silica gel 60F-254 plates (EM Science). Spots were
visualized by one or more of the following methods: UV light,
I2 vapor, or staining with phosphomolybdic acid, Ce(SO4)2, or
KMnO4.26 Gas chromatography (GC) was performed on a
Hewlett-Packard 5890 Series II gas chromatograph equipped
with a Hewlett-Packard 3392A integrator. Separations were
carried out on a 15 m × 0.32 mm i.d. fused silica capillary
column (DB-5, 0.25 mm film) from J & W Scientific. Flash
Gen er a l P r oced u r e for t h e Cu r t iu s R ea r r a n gem en t
(P r oced u r e C). To a solution of the carboxylic acid (1 equiv)
in toluene at 0 °C were added Et3N (0.95 equiv) and diphenyl
phosphorazidate (0.95 equiv). The mixture was stirred at 0
°C for 30 min and then heated at reflux for 3 h. The mixture
was cooled, washed with cold NaHCO3 solution (2×) and brine
(2×), then dried (MgSO4), filtered, and evaporated. The crude
product was used without purification.
Gen er a l P r oced u r e for Red u ction of La cta m s (P r oce-
d u r e D). The lactam (1 equiv) was carefully (gas evolution)
added to BH3‚THF solution (1 M in THF, 2.5 equiv). After
gas evolution subsided, the mixture was heated at reflux
overnight. The reaction mixture was cooled, treated cautiously
with MeOH (ca. 50% by volume, gas evolution) and 1 M NaOH
solution, and heated at reflux for 7 h. The resulting mixture
was cooled and extracted with EtOAc (2×). The organic phase
was extracted with 1 M HCl (2×), and the aqueous phase was
neutralized by adding NaHCO3. The product was extracted

Cyclic Nitrones as Free Radical Traps
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25 4993
into EtOAc (2×), and the organic phase was washed with
brine, dried (MgSO4), filtered, and evaporated. The amines
were used without further purification.
3.66 (s, 2), 3.59 (s, 2), 2.68 (s, 2), 1.53 (s, 1), 1.15 (s, 6); 13C
NMR (CDCl3) 158.7, 154.1, 137.3, 130.0, 129.5, 128.9, 124.5,
123.8, 121.7, 114.2, 113.9, 113.9, 71.0, 55.2, 55.2, 45.3, 36.2,
34.9, 31.8, 29.5, 27.7; MS (CI/CH4, 70 eV) m/ z 499 (M + H)+,
481, 427, 389, 346, 327, 287, 237, 207, 175, 155, 122, 121 (base
peak), 109, 91.
3-(2-Hydr oxy-2-m eth ylpr opyl)-2,6-dim eth ylph en ol (11g).
Raney nickel (RaNi, ca. 20 g) was washed five times with water
and twice with anhydrous EtOH. A slurry of this catalyst in
EtOH was then added to a solution of bis(sulfide) 10g (3.01 g,
6.04 mmol) in EtOH (30 mL). The resulting mixture was
heated at vigorous reflux for 2 h and then cooled. The
supernatant was decanted, and the catalyst was washed
successively with MeOH and EtOAc (2×). The decanted
organic layers were combined and evaporated. The residue
was purified by FC (10:1 CH2Cl2/iPrOH) to give 11g as a pale
yellow oil (0.98 g, 84%): 1H NMR (CDCl3) 6.94 (d, 1, J ) 7.7),
6.72 (d, 1, J ) 7.7), 2.81 (s, 2), 2.24 (s, 6), 1.24 (s, 6); 13C NMR
(CDCl3) 152.4, 135.1, 127.4, 123.4, 122.9, 121.0, 71.6, 45.7, 29.4,
15.9, 12.8; MS (CI/CH4, 70 eV) m/ z 195 (M + H)+, 177 (base
peak), 175, 149, 136, 91, 79.
4-Br om o-3-(2-h yd r oxy-2-m et h ylp r op yl)p h en ol (8g).
MeMgBr solution (3 M in Et2O, 150 mL, 450 mmol) was diluted
with 150 mL of THF and cooled to -78 °C. The ester 7g14
(14.5 g, 87.3 mmol) was dissolved in THF and added dropwise
over 15 min (vigorous gas evolution). Vigorous mechanical
stirring was required to prevent solidification of the reaction
mixture. The cooling bath was removed after the addition was
complete, and the reaction mixture was allowed to stir
overnight. Excess MeMgBr was quenched by adding saturated
NH4Cl solution, and the resulting mixture was poured into
0.5 M HCl and extracted with EtOAc (2×). The organic phase
was washed with brine, dried (Na2SO4), filtered, and evapo-
rated. The resulting crude product was dissolved in warm
CH2Cl2 and diluted with hexane to produce a white, crystalline
solid: mp 91-94 °C (12.3 g, 85%); 1H NMR (CDCl3) 7.14 (t, 1,
J ) 7.7), 6.80-6.68 (m, 3), 2.70 (s, 2), 1.23 (s, 6); 13C NMR
(CDCl3) 155.9, 139.0, 129.4, 122.6, 117.3, 113.8, 71.5, 49.4, 29.0;
MS (EI, 70 eV) m/ z 166 (M+), 152, 151, 133, 115, 108 (base
peak), 107, 90, 79, 77. Anal. (C10H14O2) C, H.
3,3,5,7-Tetr a m eth yl-3,4-d ih yd r oisoqu in olin -6-ol (12g).
Powdered NaCN (3.79 g, 77.3 mmol) was placed in a flask
cooled in an ice bath. A mixture of concentrated H2SO4 in an
equal volume of HOAc (70 mL total) was prepared, cooled to
room temperature, and then added to the NaCN through a
dropping funnel (CAUTION! HCN generated). The mixture
was stirred vigorously during the addition. Tertiary alcohol
11g (12.5 g, 64.4 mmol) in 30 mL of HOAc was added via pipet
to the acid/cyanide mixture (at room temperature) over a 2.5
h period, and the resulting red reaction mixture was stirred
overnight at room temperature. Because of the high water
solubility of the product, the aqueous phase was saturated with
NaCl and extracted six times with EtOAc to obtain an
acceptable recovery of material. The organic phase was dried
(Na2SO4), filtered, and concentrated. The residue was filtered
through silica gel with 10:1 CH2Cl2/MeOH, and the appropriate
fractions were combined and diluted with hexane to produce
5g as yellow crystals, mp 220-234 °C (dec.). A lower Rf
product was also isolated. This material displayed the same
type of TLC behavior (blue fluorescence) and a 1H NMR
spectrum very similar to that of 5g, and is thought to be a
symmetrical, macrocyclic dimer.28 Upon being left to stand
in solution, this byproduct was slowly converted into 5g, which
then precipitated. The solid was collected, and two more crops
of the yellow crystals were subsequently obtained from the
mother liquor, for a total yield of 6.8 g (52%): 1H NMR (CD3-
OD, 3.30 ppm) 7.76 (s, 1), 7.10 (s, 1), 4.93 (s, 1), 2.82 (s, 2),
2.10 (s, 3), 2.07 (s, 3), 1.33 (s, 6); 13C NMR (CD3OD, 49.05 ppm)
158.2, 136.2, 134.9, 127.6, 126.9, 54.3, 38.2, 27.3, 17.0, 11.4;
MS (CI/CH4, 70 eV) m/ z 204 [(M + H)+, base peak], 188, 177,
122.
This material (12.3 g, 74.2 mmol) was dissolved in DMF and
cooled to 0 °C. Solid N-bromosuccinimide (14.77 g, 83.0 mmol)
was added in small portions (the yellow color was allowed to
dissipate between additions) over 1.5 h. After the addition
was complete, stirring at 0 °C was continued for 30 min. The
mixture was then poured into water and extracted with EtOAc
(3×). The organic phase was washed with water (1×) and
brine (1×), dried (MgSO4), filtered, and evaporated. The
residue was dissolved in EtOAc/CH2Cl2 and diluted with
hexane to afford 8g as white crystals, mp 139-141 °C (12.7 g,
70%). Concentration of the mother liquor gave two more crops
(1.2 and 1.0 g) of crystals, bringing the total to 14.9 g (82%):
1H NMR (acetone-d6, 2.05 ppm) 8.43 (s, 1), 7.34 (d, 1, J ) 8.7),
7.01 (d, 1, J ) 2.7), 6.64 (dd, 1, J ) 8.7, 2.8), 2.89 (s, 2), 1.21
(s, 6); 13C NMR (acetone-d6, 20.83 ppm) 147.9, 131.0, 124.6,
111.3, 107.1, 106.2, 62.4, 39.6, 20.8; MS (EI, 70 eV) m/ z 246/
244 (M+), 231/229, 201, 188/186, 163, 150, 131, 108, 107, 91,
77, 63, 59 (base peak). Anal. (C10H13BrO2) C, H.
4-Br om o-3-(2-h yd r oxy-2-m eth ylp r op yl)-2,6-bis(p yr r o-
lid in -1-ylm eth yl)p h en ol (9g). The bromophenol 8g from the
previous step (5.7 g, 23.3 mmol) and pyrrolidine (4.8 mL, 58.2
mmol) were placed in a flask with a reflux condenser. Aqueous
formaldehyde (37%, 4.7 mL, 58.2 mmol) was added to the
mixture, causing a vigorous exothermic reaction. The yellow
mixture was stirred and heated at ca. 85 °C for 6 h, with an
additional 2 equiv of pyrrolidine and formaldehyde being added
after 3 h. The reaction mixture was cooled, poured into water,
and extracted with EtOAc (3×). The organic phase was
washed with brine, dried (Na2SO4), filtered, and evaporated.
The residue was taken up in CH2Cl2 and diluted with hexane
to afford 9g as white crystals, mp 111-113 °C. Two more
crops of crystals were obtained upon concentration of the
mother liquor. The total yield of 9g was therefore 8.0 g
(83%): 1H NMR (CDCl3) 8.50 (br s, 1), 7.19 (s, 1), 3.75 (v br s,
8), 3.16 (v br s, 2), 2.62 (v br s, 8), 1.84 (br s, 4), 1.78 (br s, 4),
1.38 (v br s, 6); 13C NMR (CDCl3) 156.3, 139.2, 131.0, 125.8,
121.8, 115.8, 68.7, 58.1, 53.3, 52.3, 49.1, 46.8, 34-28 (v br, gem-
dimethyl), 23.7, 23.2; MS (CI/CH4, 70 eV) m/ z 413/411 (M +
H)+, 397/395, 395/393, 370/368, 342/340 (base peak), 324/322,
290, 283, 262, 211, 183, 145, 100. Anal. (C20H31BrN2O2) C,
H, N.
3,3,5,7-Tetr a m eth yl-3,4-d ih yd r oisoqu in olin -6-ol N-Ox-
id e (6g). Imine 5g (1.00 g, 4.93 mmol) was hydrogenated (50
psi of H2) over RaNi (spatula scoop, washed three times with
water and three times with EtOH) in EtOH (20 mL) for 2 h at
room temperature. Filtration of the reaction mixture through
filter aid and evaporation of the solvent gave the amine as a
light yellow solid which was used without further purifica-
1
tion: yield 0.90 g (89%); H NMR (CDCl3) 6.68 (s, 1), 3.95 (s,
2), 3.62 (br s, 2), 2.45 (s, 2), 2.21 (s, 3), 2.08 (s, 3), 1.20 (s, 6);
13C NMR (CDCl3) 150.4, 131.4, 125.9, 125.1, 122.2, 121.2, 48.9,
43.9, 39.0, 27.9, 16.0, 11.0. The amine from above (0.90 g, 4.39
mmol) was oxidized according to general procedure A. The
nitrone 6g was obtained (0.66 g, 69%) as light yellow crystals:
3-(2-Hyd r oxy-2-m eth ylp r op yl)-2,6-bis[[(4-m eth oxyben -
zyl)su lfa n yl]m et h yl]p h en ol (10g). The bis(pyrrolidine)
compound 9g (5.00 g, 12.17 mmol) and 4-methoxybenzyl
mercaptan (11.24 g, 73.0 mmol) were combined in a flask
equipped with a reflux condenser. The mixture was stirred
and heated at 180 °C (sand bath in a heating mantle) for 3 h,
then cooled, diluted with CH2Cl2, and applied to a pad of silica
gel. The nonpolar impurites were eluted with CH2Cl2, and
then the crude product was eluted with 10:1 CH2Cl2/iPrOH.
The material was further purified by FC (4:1, CH2Cl2/CH3CN)
to give 10g as a pale yellow oil (4.60 g, 76%): 1H NMR (CDCl3)
7.26 (d, 2, J ) 8.5), 7.18 (d, 2, J ) 8.6), 7.00-6.80 (m, 6), 6.71
(d, 1, J ) 7.8), 3.85 (s, 2), 3.80 (s, 3), 3.79 (s, 3), 3.72 (s, 2),
1
mp 225-240 °C; H NMR (CDCl3) 7.60 (s, 1), 6.80 (s, 1), 2.97
(s, 2), 2.23 (s, 3), 2.17 (s, 3), 1.44 (s, 6); 13C NMR (DMSO-d6,
39.43 ppm) 154.1, 130.8, 127.3, 124.7, 122.7, 122.4, 120.4, 64.9,
38.1, 24.4, 16.5, 11.6; MS (EI, 70 eV) m/ z 219 (M+, base peak),
202, 187, 172, 160, 115, 91, 77. Anal. (C13H17NO2) C, H, N.
3-Th iop h en e-2-yl-2,2-d im eth ylp r op ion ic Acid (12i). To
a solution of LiN(TMS)2 (1 M solution in THF, 122 mL, 122
mmol) cooled at -78 °C was added ethyl isobutyrate (10.9 mL,
81.7 mmol). Stirring was continued at -78 °C for 1 h and then
1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU, 3
mL) was added, followed by 2-(chloromethyl)thiophene (10.8

4994 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25
Fevig et al.
g, 81.7 mmol).29 The cooling bath was removed, and the
reaction mixture was stirred overnight. The reaction mixture
was poured into cold 1 M HCl and extracted with EtOAc (3×).
The organic phase was washed with water and brine, then
dried (MgSO4), filtered, and evaporated. The residue was
purified by FC (CH2Cl2) to give the product as a yellow liquid
(16.4 g, 95%): 1H NMR (CDCl3) 7.15-7.10 (m, 1), 6.95-6.90
(m, 1), 6.77 (d, 1, J ) 3.6), 4.15 (t, 2, J ) 7.0), 3.07 (s, 2), 1.26
(t, 3, J ) 7.0), 1.21 (s, 6); 13C NMR (CDCl3) 177.0, 139.8, 126.7,
126.4, 123.9, 60.6, 43.7, 40.3, 25.1, 14.2; MS (CI/CH4, 70 eV)
m/ z 213 [(M + H)+, base peak], 193, 179, 167, 140, 139, 125,
98, 97. The ester from above (16.4 g, 77.2 mmol) was
hydrolyzed according to general procedure B to give 12i as a
milky liquid (11.0 g, 77%): 1H NMR (CDCl3) 7.15 (d, 1, J )
5.1), 6.95-6.90 (m, 1), 6.83 (d, 1, J ) 3.4), 3.10 (s, 2), 1.26 (s,
6); 13C NMR (CDCl3) 184.0, 139.4, 127.1, 126.7, 124.2, 43.6,
39.9, 24.7; MS (EI, 70 eV) m/ z 184 (M+), 139, 123, 97 (base
peak), 77.
6,6-Dim et h yl-6,7-d ih yd r o-5H -t h ien o[3,2-c]p yr id in -4-
on e (14i). Carboxylic acid 12i from the previous reaction (11.0
g, 60.0 mmol) was subjected to the Curtius rearrangement
according to general procedure C to give the isocyanate 13i
as a pale yellow liquid (9.94 g, 92%): 1H NMR (CDCl3) 7.22
(d, 1, J ) 5.1), 6.99 (m, 1), 6.89 (d, 1, J ) 3.5), 3.00 (s, 2), 1.38
(s, 6); 13C NMR (CDCl3) 138.2, 127.6, 126.7, 124.8, 58.1, 43.8,
29.9; IR (CHCl3) 2982, 2259, 1265, 1167, 704; MS (EI, 70 eV)
m/ z 181 (M+), 149, 138, 127, 123, 99, 97 (base peak), 84, 77,
71. To a mixture of 1,2-dichloroethane (DCE, 60 mL) and
anhydrous H3PO4 (35 mL, prepared from 85% H3PO4 and P2O5)
was added a solution of isocyanate 13i (5.12 g, 28.3 mmol) in
DCE (20 mL). The resulting mixture was stirred vigorously
at room temperature for 2 h and then at reflux for 4 h. The
reaction mixture was allowed to cool and separate into two
layers. The upper, organic layer was decanted, diluted with
EtOAc and Na2CO3 solution, and extracted with EtOAc (2×).
The organic extract was washed with brine (2×) and dried
(MgSO4), filtered, and evaporated. The residue was purified
by FC (6:4 CH2Cl2/CH3CN) to give a yellow solid (2.10 g, 41%):
mp 153-154 °C; 1H NMR (CDCl3) 7.43 (d, 1, J ) 5.2), 7.10 (d,
1, J ) 5.2), 6.82 (s, 1), 2.99 (s, 2), 1.38 (s, 6); 13C NMR (CDCl3)
162.6, 145.0, 130.9, 125.7, 123.0, 54.0, 37.3, 29.1; MS (EI, 70
eV) m/ z 181 (M+), 166, 151, 148, 125, 124 (base peak), 96, 83,
70. Anal. (C9H11NOS) C, H, N.
6,6-Dim eth yl-6,7-d ih yd r oth ien o[3,2-c]p yr id in e N-Ox-
id e (6i). Lactam 14i (2.76 g, 15.2 mmol) was reduced
according to general procedure D to give a dark liquid (1.82 g,
71%): 1H NMR (CDCl3) 7.07 (d, 1, J ) 5.1), 6.75 (d, 1, J )
5.1), 3.93 (s, 2), 2.66 (s, 2), 1.64 (br s, 1), 1.21 (s, 6). The crude
amine (1.82 g, 10.9 mmol) was oxidized according to general
procedure A to give the nitrone 6i as a yellow solid (660 mg,
33%, mp 130-131 °C) after recrystallization from 4:1
hexane/CH2Cl2: 1H NMR (CDCl3) 7.72 (s, 1), 7.17 (d, 1, J )
5.1), 6.89 (d, 1, J ) 5.1), 3.15 (s, 2), 1.50 (s, 6); 13C NMR (CDCl3)
131.2, 130.3, 128.6, 124.7, 123.5, 67.9, 37.41, 25.0; MS (EI, 70
eV) m/ z 181 (M+, base peak), 166, 149, 138, 134, 110, 96, 91,
77. Anal. (C9H11NOS) C, H, N.
2-(3-Meth oxyp h en yl)-2-m eth ylp r op ion ic Acid (12k ).
To an ice cold slurry of NaH (17.69 g, 60% dispersion in
mineral oil, 440 mmol) in THF (500 mL) was added a solution
of 3-methoxyphenylacetonitrile (25.0 g, 170 mmol) in THF (25
mL) over 30 min. The mixture was stirred for 30 min and
then a solution of CH3I (55.5 g, 390 mmol) in THF (25 mL)
was added over 30 min. The reaction mixture was allowed to
reach room temperature, and stirring was continued until GC
analysis indicated complete reaction (25 min). The reaction
mixture was poured into cold water/EtOAc, the layers were
separated, and the aqueous phase was extracted again with
EtOAc. The organic phase was washed with brine, dried
(MgSO4), filtered, and evaporated to give the dimethylated
nitrile30 as a dark liquid, 31.0 g (104%), which was used
without purification: 1H NMR (CDCl3) 7.31 (t, 1, J ) 8.1),
7.05-7.00 (m, 2), 6.90-6.85 (m, 1), 3.83 (s, 3), 1.72 (s, 6). The
crude nitrile (23.12 g, 132.1 mmol) was hydrolyzed according
to general procedure B to afford the carboxylic acid 12k as a
pale yellow solid (20.76 g, 81%): mp 46-47 °C; 1H NMR
(CDCl3) 7.24 (t, 1, J ) 8.0), 7.00-6.95 (m, 2), 6.85-6.80 (m,
1), 3.81 (s, 3), 1.58 (s, 6); 13C NMR (CDCl3) 182.9, 159.6, 145.4,
129.4, 118.3, 112.4, 111.7, 55.2, 46.2, 26.1; MS (CI/CH4, 70 eV)
m/ z 195 (M + H)+, 194, 177, 150, 149 (base peak), 137, 121,
109.
1-(1-Isocya n a t o-1-m e t h yle t h yl)-3-m e t h oxyb e n ze n e
(13k ). Carboxylic acid 12k (23.12 g, 132.1 mmol) was submit-
ted to the Curtius rearrangement according to general proce-
dure C to give isocyanate 13k as a yellow liquid. The crude
product (16.84 g, 96%) was used in the next step without
purification: 1H NMR (CDCl3) 7.26 (t, 1, J ) 8.2), 7.00 (m, 2),
6.85-6.80 (m, 1), 3.80 (s, 3), 1.69 (s, 6); 13C NMR (CDCl3) 159.6,
147.6, 129.5, 116.7, 112.0, 111.1, 60.7, 55.3, 33.0.
5-Meth oxy-3,3-dim eth yl-2,3-dih ydr oisoin dol-1-on e (14k)
a n d 7-Met h oxy-3,3-d im et h yl-2,3-d ih yd r oisoin d ol-1-on e
(14l). To an ice cold slurry of FeCl3 (35.69 g, 220 mmol) in
dry DCE (800 mL) was added a solution of isocyanate 13k
(19.12 g, 100.0 mmol) in the same solvent (100 mL) over 45
min. After completion of the addition, GC analysis of an
aliquot indicated complete reaction. Water (600 mL) was
added, and the resulting mixture was stirred vigorously. The
layers were separated, and the organic phase was washed with
1 M tartaric acid solution (2 × 1 L) and once with brine. The
solution was dried (MgSO4), filtered, and evaporated to a dark
liquid. This was purified by FC (1:4 hexane/EtOAc, then
EtOAc) to provide the 5-methoxyisoindolone 14k as a pale
yellow solid, mp 146-147 °C, 7.38 g (39%), and the regioiso-
meric 7-methoxyisoindolone 14l as a yellow solid, mp 155-
158 °C, 2.85 g (15%). For 14k : 1H NMR (CDCl3) 7.74 (d, 1, J
) 8.5), 7.00-6.95 (m, 1), 6.85 (d, 1, J ) 2.2), 3.89 (s, 3), 1.54
(s, 6); 13C NMR (CDCl3) 169.6, 163.2, 155.4, 125.3, 123.1, 114.2,
105.9, 58.6, 55.6, 27.8; MS (EI, 70 eV) m/ z 191 (M+), 176 (base
peak), 161, 133, 118, 88, 77.
For 14l: 1H NMR (CDCl3) 7.51 (t, 1, J ) 8.0), 6.95 (d, 1, J
) 8.0), 6.88 (d, 1, J ) 8.0), 6.28 (br s, 1), 3.98 (s, 3), 1.51 (s, 6);
13C NMR (CDCl3) 168.6, 157.6, 156.1, 133.8, 131.4, 112.9,
109.9, 57.9, 55.9, 27.9; MS (EI, 70 eV) m/ z 191 (M+), 176 (base
peak), 162, 158, 133, 118, 103, 89.
6-Meth oxy-1,1-dim eth yl-1H-isoin dole N-Oxide (6k). Lac-
tam 14k from the previous reaction (170 mg, 0.889 mmol) was
reduced according to general procedure D to furnish the
corresponding amine as a colorless liquid which was not
purified or characterized. The crude material (177 mg, 0.889
mmol, maximum) was oxidized according to general procedure
A. The nitrone 6k was obtained as beige crystals (54 mg, 28%,
mp 119-122 °C) after FC (97:3 CH2Cl2/iPrOH): 1H NMR
(CDCl3) 7.61 (s, 1), 7.29 (d, 1, J ) 8.4), 6.90-6.85 (m, 1), 6.84
(d, 1, J ) 2.3), 3.86 (s, 3), 1.56 (s, 6); 13C NMR (CDCl3) 160.1,
147.6, 131.4, 124.9, 121.2, 113.5, 107.9, 55.6, 24.5; MS (EI, 70
eV) m/ z 191 (M+, base peak), 176, 158, 145, 131, 115, 103, 91,
89, 77. Anal. (C11H13NO2) C, H, N.
5-H yd r oxy-3,3-d im e t h yl-2,3-d ih yd r oisoin d ol-1-on e
(14m ). A 1 M solution of BBr3 in CH2Cl2 (88.0 mL, 88.0 mmol)
was dissolved in CH2Cl2. A solution of lactam 14k (7.65 g,
40.0 mmol) in CH2Cl2 (50 mL) was added to the BBr3 solution
dropwise over 10 min. The resulting mixture was stirred at
room temperature overnight. The reaction mixture was
poured into water and extracted with EtOAc (3×). The organic
phase was dried (MgSO4), filtered, and evaporated, leaving the
product 14m as a white solid (4.03 g, 57%), mp 231-233 °C,
which required no purification: 1H NMR (DMSO-d6, 2.50 ppm)
8.18 (s, 1), 7.28 (d, 1, J ) 8.5), 6.75 (d, 1, J ) 1.6), 6.67 (dd, 1,
J ) 8.5, 1.6), 1.25 (s, 6); 13C NMR (CDCl3 + DMSO-d6) 169.3,
161.1, 155.4, 124.7, 121.5, 115.4, 107.3, 58.0, 27.5; MS (EI, 70
eV) m/ z 177 (M+), 163, 162 (base peak).
6-Hyd r oxy-1,1-d im eth yl-1H-isoin d ole N-Oxid e (6m ).
Lactam 14m (1.42 g, 8.01 mmol) was reduced according to
general procedure D except that the HCl extract was simply
evaporated to furnish the amine hydrochloride salt. Residual
water was removed by repeatedly dissolving the residue in
acetonitrile and evaporating the mixture. A white solid (1.6
g, 100%) was obtained which was not purified further: 1H
NMR (CDCl3 + DMSO-d6) 8.98 (vbr s, 2), 6.95 (d, 1, J ) 9.0),
6.59 (d, 1, J ) 9.0), 6.55 (s, 1), 4.02 (s, 2), 1.32 (s, 6); 13C NMR
(DMSO-d6, 39.43 ppm) 157.0, 143.4, 122.2, 121.2, 111.6, 106.4,
66.5, 45.6, 24.3. The amine hydrochloride from above (615 mg,
3.08 mmol) was oxidized according to general procedure A

Cyclic Nitrones as Free Radical Traps
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25 4995
except that 1.0 equiv of NaOH was added to generate the free
amine in situ. The nitrone 6m was obtained as a white solid
(60 mg, 11%, mp 225-230 °C) after FC (EtOAc): 1H NMR
(DMSO-d6, 2.50 ppm) 9.48 (s, 1), 7.70 (s, 1), 7.20 (d, 1, J )
8.0), 6.82 (d, 1, J ) 2.2), 6.80-6.75 (m, 1), 1.50 (s, 6); 13C NMR
(DMSO-d6, 39.43 ppm) 157.5, 147.2, 130.0, 121.0, 115.0, 109.1,
76.0, 24.1; MS (EI, 70 eV) m/ z 177 [(M+), base peak], 162, 144,
131, 115, 91, 89, 77. Anal. (C10H11NO2) H, N; C: calcd, 67.78;
found, 67.09.
Ack n ow led gm en t. We thank David Hay and Drs.
Ron Bernotas, Bert Carr, Bob Cregge, and Thaddeus
Nieduzak for helpful discussions and Dr. Roy Vaz for
performing the molecular modeling studies and linear
regression analyses.
Su p p or tin g In for m a tion Ava ila ble: Full experimental
details and spectral data for compounds 2a -f, 3a ,b,d -f, 5a -
f,h , 6a -f,h ,j, 7g, 7-11h , and 12-14j (18 pages). Ordering
information is given on any current masthead page.
3,3-Dim eth ylisoin d ol-1-on e (14n ). A solution of lactam
14m (1.77 g, 10.0 mmol) and 5-chloro-1-phenyl-1H-tetrazole
(2.17 g, 12.0 mmol) in DMF (50 mL) was treated with solid
K2CO3 (2.07 g, 15.0 mmol). The mixture was stirred overnight
at room temperature, then poured into water, and extracted
with EtOAc (2×). The organic phase was washed with water
(3×) and brine (2×), then dried (MgSO4), filtered, and evapo-
rated. The residue was crystallized from CH2Cl2 to give the
intermediate tetrazolyl ether as a white solid (3.10 g, 97%
yield): mp 202-204 °C; 1H NMR (CDCl3) 7.90 (d, 1, J ) 8.3),
7.80 (m, 2), 7.60-7.55 (m, 4), 7.50-7.45 (m, 1), 6.78 (s, 1), 1.59
(s, 6); 13C NMR (CDCl3) 168.5, 158.8, 156.2, 155.3, 132.8, 129.8,
128.8, 125.8, 122.3, 119.4, 112.1, 59.1, 53.4, 27.6; MS (EI, 70
eV) m/ z 321 (M+), 306, 293, 278, 261, 250, 236, 222, 208, 187,
176, 161 (base peak), 145, 133, 117, 103, 91, 77. The product
from above (3.10 g, 9.65 mmol) was dissolved in EtOH (80 mL)
and hydrogenated over 5% Pd/C (400 mg) at 50 psi of H2 on a
Parr shaker overnight at room temperature. The catalyst was
filtered off and the solvent evaporated. The residue was
purified by FC (Et2O) to furnish 14n as a white solid (1.03 g,
Refer en ces
(1) Floyd, R. A. Role of Oxygen Free Radicals in Carcinogenesis and
Brain Ischemia. FASEB J . 1990, 4, 2587-2597.
(2) Watson, B. D.; Busto, R.; Goldberg, W. J .; Santiso, M.; Yoshida,
S.; Ginsberg, M. D. Lipid Peroxidation in Vivo Induced by
Reversible Global Ischemia in Rat Brain. J . Neurochem. 1984,
42, 268-274.
(3) Watson, B. D. Evaluation of the Concomitance of Lipid Peroxi-
dation in Experimental Models of Cerebral Ischemia and Stroke.
In Progress in Brain Research; Kogure, K., Hossman, K.-A.,
Siesjo, B. K., Eds., Elsevier Science Publishers: Amsterdam,
1993; Vol. 96, pp 69-95.
(4) Hall, E. D.; Andrus, P. K.; Althaus, J . S.; VonVoigtlander, P. F.
Hydroxyl Radical Production and Lipid Peroxidation Parallels
Selective Post-Ischemic Vulnerability in Gerbil Brain. J . Neu-
rosci. Res. 1993, 34, 107-112.
(5) (a) Colado, M. I.; Green, A. R. The Spin Trap Reagent R-Phenyl-
N-tert-butyl Nitrone Prevents 'Ecstasy'-Induced Neurodegen-
eration of 5-Hydroxytryptamine Neurones. Eur. J . Pharm. 1995,
280, 343-346. (b) Oliver, C. N.; Starke-Reed, P. E.; Stadtman,
E. R.; Liu, G. J .; Carney, J . M.; Floyd, R. A. Oxidative Damage
to Brain Proteins, Loss of Glutamine Synthetase Activity, and
Production of Free Radicals During Ischemia/Reperfusion-
Induced Injury to Gerbil Brain. Proc. Natl. Acad. Sci. U.S.A.
1990, 87, 5144-5147. (c) Yue, T.-L.; Gu, J .-L.; Lysko, P. G.;
Cheng, H.-Y.; Barone, F. C.; Feuerstein, G. Neuroprotective
Effects of Phenyl-t-butyl-nitrone in Gerbil Global Brain Ischemia
and in Cultured Rat Cerebellar Neurons. Brain Res. 1992, 574,
193-197 and references cited therein.
(6) (a) Bernotas, R. C.; Thomas, C. E.; Carr, A. A.; Nieduzak, T. R.;
Adams, G.; Ohlweiler, D. F.; Hay, D. A. Synthesis and Radical
Scavenging Activity of 3,3-Dialkyl-3,4-Dihydro-isoquinoline 2-Ox-
ides. Bioorg. Med. Chem. Lett. 1996, 6, 1105-1110. (b) Bernotas,
R. C.; Adams, G.; Carr, A. A. Synthesis of Benzazepine-based
Nitrones as Radical Traps. Tetrahedron 1996, 52, 6519-6526.
(7) Structures for 1 and PBN were constructed by using SYBYL6.0
(Tripos, Inc.). The structures were optimized with the TRIPOS52
Force Field (Clark, M.; Cramer, R. D. III; Van Opdenbosh, N.
Validation of the General Purpose Tripos 5.2 Force Field. J .
Comput. Chem. 1989, 10, 982-1012) and subjected to full energy
optimization by using the AM1 Hamiltonian in the semiempiri-
cal quantum program MOPAC (QCPE, Indiana University), and
the HOMO and LUMO were plotted for both structures. The
structures were subjected to further geometry refinement with
the program SPARTAN3.0 (Wavefunction, Inc.) by using a split
valence basis set (6-31G*) with the unrestricted Hartree-Fock
method. The dihedral angles between the nitrone double bond
and the aromatic ring calculated for 1 and PBN were 13.5° and
<1°, respectively. Compound 1 is 8-fold more potent than PBN
in the lipid peroxidation assay and 25-fold more potent in the
hydroxyl radical trapping assay. See: Thomas, C. E.; Ohlweiler,
D. F.; Carr, A. A.; Nieduzak, T. R.; Hay, D. A.; Adams, G.; Vaz,
R.; Bernotas, R. C. Characterization of the Radical Trapping
Activity of a Novel Series of Cyclic Nitrone Spin Traps. J . Biol.
Chem. 1996, 271, 3097-3104.
(8) (a) J anzen, E. G.; Zhang, Y.-K.; Haire, D. L. Synthesis of a Novel
Nitrone, 2-Phenyl-5,5-dimethyl-1-pyrroline N-Oxide (nitronyl-
13C), for Enhanced Radical Addend Recognition and Spin Adduct
Persistence. J . Am. Chem. Soc. 1994, 116, 3738-3743. (b) Yang,
L.; Xiuhua, W.; Benming, C.; Guangzhi, X. Crystal Structure of
Phenyl-t-butyl Nitrone. J iegou Huaxue (J . Struct. Chem.) 1987,
6, 17-19.
(9) Fleming, I. Frontier Orbitals and Organic Chemical Reactions;
J ohn Wiley & Sons; Chichester, 1976; pp 182-191.
(10) Thomas, C. E.; Ohlweiler, D. F.; Kalyanaraman, B. Multiple
Mechanisms for Inhibition of Low Density Lipoprotein Oxidation
by Novel Cyclic Nitrone Spin Traps. J . Biol. Chem. 1994, 269
(45), 28055-28061.
(11) Krimen, L. I.; Cota, D. J . The Ritter Reaction. In Organic
Reactions; J ohn Wiley & Sons: New York, 1969; Vol. 17, pp 213-
325.
(12) Larsen, R. D.; Reamer, R. A.; Corley, E. G.; Davis, P.; Grabowski,
E. J . J .; Reider, P. J .; Shinkai, I. A Modified Bischler-Napieralski
Procedure for the Synthesis of 3-Aryl-3,4-dihydroisoquinolines.
J . Org. Chem. 1991, 56, 6034-6038.
1
66%): mp 159-160 °C; H NMR (CDCl3) 7.83 (d, 1, J ) 7.6),
7.57 (t, 1, J ) 7.6), 7.45-7.40 (m, 2), 1.57 (s, 6); 13C NMR
(CDCl3) 169.9, 153.2, 132.0, 130.7, 127.9, 123.8, 120.8, 59.1,
27.7; MS (EI, 70 eV) m/ z 161 (M+), 146 (base peak), 128, 103,
91, 77.
1,1-Dim eth yl-1H-isoin d ole N-Oxid e (6n ). Lactam 14n
(1.42 g, 8.01 mmol) was reduced according to general procedure
D, except that the HCl extract was simply evaporated to
furnish the amine hydrochloride salt. Residual water was
removed by repeatedly dissolving the residue in acetonitrile
and evaporating the mixture. A white solid (918 mg, 100%)
was obtained which was not purified further: 1H NMR (CDCl3
+ DMSO-d6) 10.30 (br s, 2), 7.40-7.35 (m, 3), 7.25-7.20 (m,
1), 4.55 (s, 2), 1.76 (s, 6); 13C NMR (CDCl3 + DMSO-d6) 132.0,
128.1, 127.9, 122.3, 120.3, 110.4, 61.0, 46.8, 25.5. The amine
hydrochloride from above (918 mg, 5.00 mmol) was oxidized
according to general procedure A except that 1.0 equiv of
NaOH was added to generate the free amine in situ. The
nitrone 6n was obtained as a white solid (113 mg, 14%, mp
64-65 °C) after FC (8:2 CH2Cl2/CH3CN): 1H NMR (CDCl3)
7.66 (s, 1), 7.36 (m, 3), 7.27 (m, 1), 1.57 (s, 6); 13C NMR (CDCl3)
145.4, 132.3, 131.5, 128.4, 127.5, 120.7, 120.1, 77.6, 24.5; MS
(CI/CH4, 70 eV) m/ z 162 [(M + H)+, base peak], 144, 128. Anal.
(C10H11NO) C, H, N.
4-Meth oxy-1,1-dim eth yl-1H-isoin dole N-Oxide (6l). Lac-
tam 14l (1.19 g, 6.22 mmol) was reduced according to general
procedure D, except that the HCl extract was simply evapo-
rated to furnish the amine hydrochloride salt. Residual water
was removed by repeatedly dissolving the residue in acetoni-
trile and evaporating the mixture. A white solid (1.33 g, 100%)
was obtained which was not purified further: 1H NMR (CDCl3
+ DMSO-d6) 10.26 (br s, 2), 7.35 (t, 1, J ) 7.7), 6.85-6.75 (m,
2), 4.47 (br s, 2), 3.86 (s, 3), 1.74 (s, 6); 13C NMR (CDCl3
+
DMSO-d6) 143.6, 129.5, 125.0, 118.9, 111.6, 108.9, 67.4, 53.9,
44.2, 24.2. The amine hydrochloride from above (1.33 g, 6.21
mmol) was oxidized according to general procedure A except
that 1.0 equiv of NaOH was added to generate the free amine
in situ. The nitrone 6l was obtained (190 mg, 16%) as a pale
yellow solid, mp 149-152 °C, after FC (EtOAc, the 9:1 CH2-
Cl2/MeOH) and crystallization from CH2Cl2/hexane: 1H NMR
(CDCl3 + CD3OD) 7.74 (s, 1), 7.35-7.25 (m, 1), 6.88 (d, 1, J )
8.9), 6.84 (d, 1, J ) 8.9), 3.90 (s, 3), 1.55 (s, 6); 13C NMR (CDCl3
+ CD3OD) 152.1, 147.2, 129.3, 129.1, 113.3, 110.2, 77.8, 55.5,
24.5; MS (EI, 70 eV) m/ z 191 [(M+), base peak], 176, 158, 134,
131, 128, 115, 91, 77. Anal. (C11H13NO2) C, H, N.

4996 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25
Fevig et al.
(13) Murahashi, S-I.; Shiota, T.; Imada, Y. Oxidation of Secondary
Amines to Nitrones. 6-Methyl-2,3,4,5-tetrahydropyridine N-
Oxide. In Organic Syntheses; Wiley: New York, 1991; Vol. 70,
pp 265-271.
(14) Compound 7g was prepared by esterification (MeOH, H2SO4) of
the corresponding acid, and 7h was prepared by esterification
(MeOH, H2SO4) and catalytic hydrogenation (H2, Pd/C) of the
corresponding cinnamic acid. See the Supporting Information.
(15) Deamination of phenolic Mannich bases often requires extremely
harsh conditions, even for simple, unhindered substrates. See,
for example: Caldwell, W. T.; Thompson, T. R. Nuclear Methy-
lation of Phenols. A New Synthesis of Intermediates in the
Preparation of Antisterility Factors. J . Am. Chem. Soc. 1939,
61, 765-767.
(16) Poppelsdorf, F.; Holt, S. J . Reactions of Thiols and Thioethers.
Part I. An Analogue of the Mannich Reaction Involving Thiols,
Formaldehyde, and Active Methylene or Methylidyne Com-
pounds. J . Chem. Soc. 1954, 1124-1130.
(17) Umezawa, B.; Hoshino, O.; Sawaki, S.; Mori, K. A Cyclization
of â-Arylethylisocyanates to 3,4-Dihydroisocarbostyrils. Chem.
Pharm. Bull. 1980, 28, 1003-1005.
Liquid Chromatography: Theory, Experimental Techniques, and
Application in Studies on Quantitative Structure-Activity Re-
lationships. J . Chromatogr. 1986, 373, 191-225. (b) Hsieh, M.
M.; Dorsey, J . G. Bioavailability Estimation by Reversed-Phase
Liquid Chromatography: High Bonding Density C-18 Phases
for Modeling Biopartitioning Processes. Anal. Chem. 1995, 67,
48-57. (c) Kugel, C.; Heintzelmann, B.; Wagner, J . Determina-
tion of Distribution Coefficients for Some 5-HT3 Receptor
Antagonists by Reversed-Phase High-Performance Liquid Chro-
matography. J . Chromatogr. A 1994, 667, 29-35.
(22) Thomas, C. E.; McLean, L. R.; Parker, R. A.; Ohlweiler, D. F.
Ascorbate and Phenolic Antioxidant Interactions in Prevention
of Liposomal Oxidation. Lipids 1992, 27, 543-550.
(23) Minotti, G.; Aust, S. D. Redox Cycling of Iron and Lipid
Peroxidation. Lipids 1992, 27, 219-226.
(24) The equation was derived by linear regression analysis of the
data using SYBYL software (Tripos, Inc.).
(25) For a discussion of the relationship between lipophilicity, lipid
peroxidation inhibition, and cytoprotection within the context
of a different class of antioxidants, see: Yu, M. J .; McCowan, J .
R.; Phebus, L. A.; Towner, R. D.; Ho, P. P. K.; Keith, P. T.;
Luttman, C. A.; Saunders, R. D.; Ruterbories, K. J .; Lindstrom,
T. D.; Wikel, J . H.; Morgan, E.; Hahn, R. A. Benzylamine
Antioxidants: Relationship between Structure, Peroxyl Radical
Scavenging, Lipid Peroxidation Inhibition, and Cytoprotection.
J . Med. Chem. 1993, 36, 1262-1271 and references cited therein.
(26) The compositions of these staining solutions can be found in the
following: Thin-Layer Chromatography, a Laboratory Handbook;
Stahl, E., Ed.; Springer-Verlag: Berlin, 1969.
(18) Compound 14l (not shown) results from cyclization ortho to the
methoxy substituent. The use of AlCl3 gave 25-30% yields of
the 5-membered ring lactams, whereas protic acids (MeSO3H,
MeSO3H/P2O5, PPA) or other Lewis acids (BF3‚Et2O, SnCl4,
TiCl4, TMSOTf) afforded little or none of the desired products.
(19) Musliner, W. J .; Gates, J . W., J r. The Replacement of Phenolic
Hydroxyl Groups by Hydrogen. J . Am. Chem. Soc. 1966, 88,
4271-4273.
(20) (a) After our work was completed, an acyclic version of 6g,h was
reported in the patent literature. See: J anzen, E. G.; Wilcox, A.
L.; Hinton, R. D. Novel Spin Trap Nitronyl Hindered Phenols.
Int. Pat. Publ. Number WO 95/11227, 27 April 1995. (b) Bowry,
V. W.; Ingold, K. U. Extraordinary Kinetic Behavior of the
R-Tocopheroxyl (Vitamin E) Radical. J . Org. Chem. 1995, 60,
5456-5467. (c) van Acker, S. A. B. E.; Koymans, L. M. H.; Bast,
A. Molecular Pharmacology of Vitamin E: Structural Aspects
of Antioxidant Activity. Free Radical Biol. Med. 1993, 15, 311-
328. (d) Grisar, J . M.; Bolkenius, F. N.; Petty, M. A.; Verne, J .
2,3-Dihydro-1-benzofuran-5-ols as Analogues of R-Tocopherol
that Inhibit in Vitro and ex Vivo Lipid Autoxidation and Protect
Mice Against Central Nervous System Trauma. J . Med. Chem.
1995, 38, 453-458 and references cited therein.
(27) Still, W. C.; Kahn, M.; Mitra, A. Rapid Chromatographic
Technique for Preparative Separation with Moderate Resolution.
J . Org. Chem. 1978, 43, 2923-2925.
(28) The structure of the side product is tentatively assigned as i.
(29) Blicke, F. F.; Burckhalter, J . H. R-Thienylaminoalkanes. J . Am.
Chem. Soc. 1942, 64, 477-480.
(30) Ku¨ndig, E. P.; Desobry, V.; Simmons, D. P.; Wenger, E. Ther-
modynamic Control of Regioselectivity in the Addition of Car-
banions to (Arene)tricarbonylchromium Complexes. J . Am.
Chem. Soc. 1989, 111, 1804-1814.
(21) In our previous studies, clog
P values were used for the
lipophilicity/activity correlation. However, similar calculations
for several of the compounds reported here (those containing
cyclic ethers) were obviously erroneous. Therefore, log k′w values
were measured according to a literature procedure as described
briefly in the Experimental Section. See: (a) Braumann, T.
Determination of Hydrophobic Parameters by Reversed-Phase
J M960243V