Fast atom bombardment-promoted reductive ring opening of 1,2-benzisothiazoles

Article abstract of DOI:10.1016/S0040-4039(03)00166-7

Fast atom bombardment mass spectral examination of molecules containing a 1,2-benzisothiazole ring, using a thiol reducing agent matrix, promotes reductive ring opening of the benzisothiazole ring, giving an [M+H]⁺ two daltons higher than expected. Measurements using a non-reducing matrix produce the expected [M+H]⁺. This is a general phenomenon, observed with a number of molecules containing the benzisothiazole ring. The ring-opened structure has been confirmed by chemical synthesis and observed in metabolic studies.

Full text of DOI:10.1016/S0040-4039(03)00166-7

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 2117–2120
Fast atom bombardment-promoted reductive ring opening of
1,2-benzisothiazoles
Thomas R. Sharp,a,* John F. Lambertb and Stanley W. Walinskyb
aAnalytical R&D Department, Pfizer Global Research & Development, Groton, CT 06340 USA
bChemical R&D Department, Pfizer Global Research & Development, Groton, CT 06340 USA
Received 20 December 2002; revised 15 January 2003; accepted 16 January 2003
Abstract—Fast atom bombardment mass spectral examination of molecules containing a 1,2-benzisothiazole ring, using a thiol
reducing agent matrix, promotes reductive ring opening of the benzisothiazole ring, giving an [M+H]+ two daltons higher than
expected. Measurements using a non-reducing matrix produce the expected [M+H]+. This is a general phenomenon, observed with
a number of molecules containing the benzisothiazole ring. The ring-opened structure has been confirmed by chemical synthesis
and observed in metabolic studies. © 2003 Elsevier Science Ltd. All rights reserved.
Fast atom bombardment (FAB) mass spectral charac-
terization of several drug substance candidates and
structurally related compounds, which contain the 1,2-
benzisothiazole heterocyclic ring system, produce a
mass spectrum containing an apparent [M+H]+ which is
2 daltons higher than predicted for the accepted struc-
tures of these molecules. Our investigations into the
origin of this molecular ion anomaly led to our discov-
ering the following structure-specific FAB-promoted
chemical reaction in the mass spectrometer. The com-
pound providing the focal point for this investigation is
ziprasidone (1),1,2 a recently approved antipsychotic
agent.
on the direct insertion FAB probe target. Bombard-
ment with 6 to 8 kV xenon atoms, produced by a saddle
field atom gun, generated the spectra and promoted the
chemical reactions reported here.
A FAB mass spectrum of ziprasidone from a reducing
matrix is shown in Figure 1, in which the abundance of
the peak at m/z 415 is higher than would be expected
for the simple 13C plus single chlorine isotope pattern
expected for this molecule. The phenomenon is more
dramatically illustrated by the expanded views of [M+
H]+ regions (Fig. 2) of three spectra, taken from early,
midway and late in a repetitive scan run. During the
run, the relative abundance of m/z 415 grows at the
expense of m/z 413. The shifting isotope pattern accom-
panies the apparent addition of two hydrogen atoms to
[M+H]+.
Observations were made from either a dithiothreitol–
dithioerythritol (reducing) matrix3 or an m-nitrobenzyl
alcohol (non-reducing) matrix4,5 on a VG 70VS mag-
netic sector mass spectrometer. Samples of the com-
pounds were pre-dissolved in an appropriate volatile
organic solvent. Aliquots were mixed with the matrix
The m/z 415 species corresponds to 2, formed by
reductive ring opening of the 1,2-benzisothiazole ring of
ziprasidone. We cannot currently differentiate between
2 and an isobaric ring-closed isomer in which the CꢀN
double bond has been reduced. However, accompany-
ing the FAB-promoted reduction is the appearance of
* Corresponding author. Tel.: 860-441-5932; fax: 860-441-0437;
e-mail: thomas r sharp@groton.pfizer.com
– –
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)00166-7

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T. R. Sharp et al. / Tetrahedron Letters 44 (2003) 2117–2120
Figure 1. Positive ion FAB mass spectrum of ziprasidone, recorded from a dithiothreitol–dithioerythritol reducing matrix.
the peak in Figure 1 at m/z 567. This species corresponds
to the [M+H]+ of a mixed disulfide formed from 2 and
a molecule of the dithiothreitol–dithioerythritol matrix
(3). This mass is the same as for a proton-bound cluster
of ziprasidone and a matrix molecule. However, we assign
it as 3 because its abundance grows as the experiment
proceeds.
a naphthalene moiety (5),8 does not exhibit this behavior,
showing that it is dependent upon the presence of the
benzisothiazole.
It is unclear whether the benzisothiazole gives rise directly
to both the ring-opened compound and the disulfide, or
whether the ring-opened compound is a product of the
disulfide adduct. Experiments were not carried out to
expressly probe this point.
The kinetics of the process can be followed. Disappear-
ance of the m/z 413 [M+H]+, representative of ziprasi-
done, and appearance of the m/z 415 species,
representative of the reductively ring-opened structure,
follow pseudo-first order kinetics. This behavior is in
contrast to the beam-dependent ‘instantaneous’ and
reversible reduction of oxazine dyes.6
Investigation of other structures containing the 1,2-ben-
zisothiazole moiety, including tiospirone (4), another
antipsychotic drug, reveal that this phenomenon is a
general property of the 1,2-benzisothiazole nucleus.
Tiospirone7 and other structural analogs of ziprasidone,
which contain the benzisothiazole ring, undergo the
reductive ring opening and form the apparent mixed
disulfide adduct. A structure similar to that of ziprasi-
done, in which the benzisothiazole has been replaced by
Figure 2. Expanded [M+H]+ region from three spectra taken
early (top), midway (middle) and late (bottom) during a FAB
mass spectrometric run in which spectra of ziprasidone, placed
in a dithiothreitol–dithioerythritol reducing matrix, were repet-
itively scanned.

T. R. Sharp et al. / Tetrahedron Letters 44 (2003) 2117–2120
2119
Compound 2 has been identified as a metabolite of
ziprasidone,9,10 and an intermediate in formation of
other metabolites.11 Metabolic ring openings of risperi-
done,12 iloperidone13 and zonisamide14 (all benzisoxa-
zoles) have been reported. However, the nature of the
metabolites suggests a different mechanism, namely
opening of the benzisoxazole ring to an ortho-phenolic
imine, followed by hydrolysis to an ortho-phenolic
ketone.
90% isolated yield. See Scheme 1. Thin-layer chromato-
graphic monitoring of the reaction suggests intermedi-
ate formation of the mixed disulfide with benzyl
mercaptan. This evidence supports the assignment of
the m/z 567 species in the FAB mass spectrum as the
mixed disulfide 3 with the matrix, and that it is an
obligate intermediate. Benzyl disulfide was isolated and
identified as a reaction by-product. If less than 2 equiv.
of thiol were used, the ring opening reaction was
incomplete. 2, dissolved in methanol, slowly re-oxidizes
and closes the ring back to ziprasidone. Ring closure
can be driven synthetically by heating in dimethyl sulf-
oxide for 24 h at 100°C, regenerating ziprasidone in
high yield and dimethyl sulfide. Similar conversions
could be done with 1,2-benzisothiazole-piperazine.
We refer to this reaction as a FAB-promoted reaction
rather than a FAB-induced reaction because we could
detect conversion of ziprasidone by dispersing a sample
of the material in the dithiothreitol–dithioerythritol
matrix prior to introducing it into the mass spectrome-
ter and bombarding it with fast atoms.
A sample of synthetically produced 2 was subjected to
FAB mass spectral examination, along with a sample of
ziprasidone, in both the dithiothreitol–dithioerythritol
(reducing) and m-nitrobenzyl alcohol (non-reducing)
matrices. The expected behavior was observed. While
ziprasidone converted upon exposure to the thiol
matrix and fast atom bombardment, 2 remained
unchanged. Neither compound changed when examined
from the non-reducing m-nitrobenzyl alcohol matrix.
Observation of this ring opening in the mass spectrom-
eter prompted further investigation of the chemistry in
solution. Reviews of the chemistry of benzisoxazoles15
and benzisothiazoles16 have appeared. The benzisoxa-
zole ring is reported to be opened by a number of
treatments. Benzisothiazoles, by contrast are reported
to be quite stable. However, hydrazine can open a
benzisothiazole. Benzisothiazole quaternary salts can be
formed, and produce ring-opened products under acid
or alkali reflux treatment.
FAB-induced chemical reactions of analytes in the
matrix and on the insertion probe target have been
documented a number of times in the literature, and
have been reviewed.20,21 Reduction of disulfides in pep-
tides has been shown to be analytically useful.22 Appar-
ent oxidations of certain platinum organometallic
compounds have been offered23 as a rationale for the
appearance of peaks in the spectrum 16 daltons above
molecular ions and certain fragments. Dehydrohalo-
genation of halogenated nucleosides and nucleotides
has been reported.24 A number of aromatic oximes can
be reduced on the probe to their corresponding
imines.25 An investigation of the mechanism and struc-
tures produced by the condensation of hydroxyl-con-
taining matrices with carbonyl-containing analytes has
been presented.26
Bo¨shagen and Geiger, however, have shown17 that
diphenyl disulfides can be induced to disproportionate
into the corresponding benzisothiazole and ortho-sub-
stituted thiophenol, particularly when an amidine is
attached ortho to the sulfur. The reaction is reversible,
and can be driven in one direction or the other by
addition of HCl. Compound 2 is an amidine-substi-
tuted ortho-thiophenol. Ring opening of 3-chloro-1,2-
benzisothiazole with thiol reagents has also been
reported.18
The structure of 2 has been confirmed synthetically.19
Treatment of ziprasidone dissolved in isopropanol with
two equivalents of benzyl mercaptan, and heated to
approximately 80°C for 24 h, produced 2 cleanly in
Scheme 1. Cyclic opening and closing of the benzisothiazole ring.

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T. R. Sharp et al. / Tetrahedron Letters 44 (2003) 2117–2120
Mass Spectrometry and Allied Topics, Chicago, IL, 27–31
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The authors thank J. A. Lowe III and H. R. Howard
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