N,N′-Linked 1,2-benzisothiazol-3(2H)-one 1,1-dioxides: synthesis, biological activity, and derived radicals

Article abstract of DOI:10.1016/j.tet.2009.10.070

A number of N,N′-linked benzoannelated isothiazol-3(2H)-one 1,1-dioxides, not available via oxidation of isothiazolium salts, were obtained with good yields by reaction of N-amino heterocycles with 2-chlorosulfonylbenzoyl chloride and evaluated for their inhibitory activity toward human leukocyte elastase (HLE) and acetylcholinesterase (AChE). 2-(Phthalimid-1-yl)-1,2-benzisothiazol-3(2H)-one 1,1-dioxide and 2-(2-methyl-4-oxo-3(4H)-quinazolinyl)-1,2-benzisothiazol-3(2H)-one 1,1-dioxide were found to be inhibitors of HLE and tested as potential precursors of nitrogen-centered radicals using 266 nm laser flash photolysis.

Full text of DOI:10.1016/j.tet.2009.10.070

Tetrahedron 66 (2010) 379–384
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
N,N0-Linked 1,2-benzisothiazol-3(2H)-one 1,1-dioxides: synthesis, biological
activity, and derived radicals
,b, ,
Valeria M. Zakharova a
y, Ortwin Brede c, Michael Gu¨tschow d, Mikhail A. Kuznetsov b,
*
Mikhail Zibinsky b y, Joachim Sieler a, Ba¨rbel Schulze a
,
a Faculty of Chemistry and Mineralogy, University of Leipzig, Johannisallee 29, Leipzig D-04103, Germany
b St-Petersburg State University, Department of Organic Chemistry, University pr. 26, Saint-Petersburg 198504, Russia
c Interdisciplinary Group of Time-Resolved Spectroscopy, University of Leipzig, Permoserstrasse 15, Leipzig D-04303, Germany
d Pharmazeutisches Institut, Pharmazeutische Chemie I, Universita¨t Bonn, An der Immenburg 4, Bonn D-53121, Germany
a r t i c l e i n f o
a b s t r a c t
A number of N,N0-linked benzoannelated isothiazol-3(2H)-one 1,1-dioxides, not available via oxidation of
isothiazolium salts, were obtained with good yields by reaction of N-amino heterocycles with 2-chloro-
sulfonylbenzoyl chloride and evaluated for their inhibitory activity toward human leukocyte elastase (HLE)
and acetylcholinesterase (AChE). 2-(Phthalimid-1-yl)-1,2-benzisothiazol-3(2H)-one 1,1-dioxide and 2-(2-
methyl-4-oxo-3(4H)-quinazolinyl)-1,2-benzisothiazol-3(2H)-one 1,1-dioxide were found to be inhibitors of
HLE and tested as potential precursors of nitrogen-centered radicals using 266 nm laser flash photolysis.
Published by Elsevier Ltd.
Article history:
Received 1 June 2009
Received in revised form
20 October 2009
Accepted 20 October 2009
Available online 28 October 2009
Dedicated to Professor Dr. Manfred Mu¨hlsta¨dt
on the occasion of his 80th birthday
1. Introduction
a saccharin fragment, the investigation of their inhibitory properties
toward HLE and AChE and their ability to generate radicals.
1,2-Benzisothiazole-3-one 1,1-dioxide (saccharin) has been
widely incorporated into a variety of biologically active compounds.
The saccharin moiety has been identified as an important molec-
ular component in various classes of 5HT1a antagonists,1 human
2. Results and discussion
2.1. Synthesis of N,N0-linked 1,2-benzisothiazol-3(2H)-one 1,1-
dioxides
leukocyte elastase (HLE) inhibitors,2 analgesics,3 human mast cell
tryptase inhibitors,4
a
1-a adrenergic receptor antagonists and
5
aldehyde dehydrogenase inhibitors.6
It has been demonstrated that 2-aryl-3-oxosultams were easily
To our knowledge, N-substituted saccharins are limited to N-alkyl
or N-aryl compounds,7 and no N,N0-connected, heterocyclic
substituted derivatives are known. However, N,N0-linked bisazahe-
terocycles attracted significant attention because of their pharma-
cological activities8 and are reported to serve as precursors of
nitrogen-centered free radicals, which play an important role in
physiological processes and in understanding the mode of action of
many toxins.9 Recently, we have prepared a series of N,N0-linked
bisazaheterocycles through oxidation of isothiazolium salts. Some
3-hydroperoxysultames obtained in that study were found to be
inhibitors of acetylcholinesterase (AChE).10 Herein we wish to report
the synthesis of new N,N0-linked bisazaheterocycles, containing
available by reaction of b-thiocyanatovinyl aldehydes with anilines,
followed by oxidation of the resulting iminium salts.11 However, in
contrast to analogs (dimethyl-, tetrahydrobenzo-, or diphenyl
vinylaldehydes), the reaction of 2-thiocyanato benzaldehyde 1 with
N-amino heterocycles 2 gave only hydrazonium salts 3 with trans
configuration of the imine bond. Cyclization products 4 were not
obtained (Scheme 1).12
One of the described methods for the preparation of benzoan-
nelated isothiazole 1,1-dioxides is based on the reaction of
2-chlorosulfonylbenzoyl chloride with anilines.13 We applied this
approach, and the synthesis of N,N0-linked saccharin derivatives was
conducted by means of tandem acylation/sulfonylation reaction of
N-amino compounds with 2-chlorosulfonylbenzoyl chloride 6.
Compound 6 was prepared in two steps by a known procedure from
the ammonium salt of o-sulfobenzoic acid 5 and phosphorus pen-
tachloride. The initial ammonium salt was obtained via acidic hy-
drolysis of saccharin.14 The reactions of N-amino heterocycles 2a–f
with 6were carriedoutin boiling dioxane in thepresence ofpyridine
* Corresponding author. Tel.: þ1 213 477 0386.
E-mail address: valeriyz@usc.edu (V.M. Zakharova).
y
Present address: University of Southern California, Department of Chemistry,
Los Angeles, CA 90089, USA.
0040-4020/$ – see front matter Published by Elsevier Ltd.
doi:10.1016/j.tet.2009.10.070

380
V.M. Zakharova et al. / Tetrahedron 66 (2010) 379–384
+
N
-
CHO
SCN
R
ClO4
HClO4,
AcOH
H
+
N
R
1
2
S
+
SCN
3
4
H2N
R
[O]
O
O
N
O
N
N
R
N
X ;
;
R =
S
N
O
R1
R1 = H, Me
O O
Figure 1. Molecular structure of 2-(morpholin-1-yl)-1,2-benzisothiazol-3(2H)-one
1,1-dioxide (7b).
X = CH2, O, (CH2)2
Scheme 1.
The molecular structure of the saccharin derivative 7b was
confirmed by X-ray crystallography (Fig. 1). Sultam 7b is a sterically
demanding compound. The dihedral angle of 84.5ꢁ between the
two planes shows that the heterocyclic rings are almost perpen-
dicularly oriented. The C(1)–N(1) bond length of 1.388 Å indicates
partial double-bond character through electron delocalization into
the carbonyl group.
and, after simple workup, N-substituted benzisothiazol-3(2H)-one
1,1-dioxides 7a–f were obtained with moderate to good yields
(Scheme 2). For these conversions, an initial attack of the amino
group at the carbonyl group of 6 is expected, as 2-chloro-
sulfonylbenzoyl chloride primarily reacted as acylating agents with
carbon 15 and oxygen nucleophiles.16
The crystal packing is characterized by weak hydrogen bonds bet-
ween the atoms O1/H7-C7. The bond parameters are: C7–H7 0.98(2) Å,
H7/O1 2.37(2) Å, C7/O1 3.329(2) Å, and C7–H7/O1 167.0(7)ꢁ. Fig. 2
shows these one-dimensional chains along the b-axis.
H2N N
2a-c
X
;
O
pyridine
N N
X
S
7a-c
O O
52-56%
X = CH2 (a), O (b), (CH2)2 (c)
O
H2N N
;
O
O
2d
COCl
O
pyridine
N
N
S
SO2Cl
O
O
O
93%
6
7d
O
H2N N
;
O
O
N
R1
2e,f
Figure 2. Molecular chains of 7b along the b-axis (symmetry code: S1 x, y, z; S1A:
ꢀxþ0.5, yꢀ0.5, 0.6þz; S1B: x, yꢀ1, 1þz).
N N
pyridine
S
N
O
R1
O
2.2. Inhibitory properties of benzisothiazol-3(2H)-one
1,1-dioxides toward HLE and AChE
7e,f
73-83%
R1 = H (e), Me (f)
The N,N0-linked bisazaheterocycles 7a–f were evaluated as in-
hibitors of acetylcholinesterase (AChE) from Electrophorus electricus
and human leukocyte elastase (HLE). No AChE inhibition was found
for the investigated sultams. Compounds 7d and 7f showed mod-
erate inhibitory activity toward human leukocyte elastase with IC50
Scheme 2.
The structures of the previously unknown sultams 7a–f were
established by means of IR, UV, 1H, 13C NMR spectroscopies, as well
as mass spectrometry and elemental analysis. The 13C NMR spectra
of compounds 7a–f contain the characteristic signals for C-3, C-3a
and C-7a at 156–159, and 124–127 and 136–138 ppm, respectively.
Compared to the related N-heterocyclyl-substituted 4,5-dimethyl-
isothiazol-3(2H)-one 1,1-dioxides and 4,5,6,7-tetrahydro-1,2-benz-
isothiazol-3(2H)-one 1,1-dioxides,10 in the case of the sultams 7d
and 7e,f the C-3a and C-7a signals had a noticeable upfield shift
(124–125 and 136 ppm versus 134–135 and 145–147 ppm for C-4
and C-5 of the analogous 4,5-dimethylisothiazol-3(2H)-one 1,1-di-
oxides). Similar shift differences were also found for the parent
compounds, i.e., saccharin17 and 4,5-dimethylisothiazol-3(2H)-one
1,1-dioxide18 (128 and 139 ppm versus 133 and 144 ppm). The IR
spectra of the benzisothiazol-3(2H)-one 1,1-dioxides include typical
absorption bands of the carbonyl group at 1728–1773 cmꢀ1 and the
bands of symmetric and antisymmetric vibrations of the SO2 group
values of 14.1ꢂ5.9
m
M, and 23.4ꢂ8.0
m
M, respectively. The other
sultams (7a,b,c,e) had IC50 values higher than 60
mM. The re-
placement of the benzene ring of the benzisothiazolone structure of
7d (by two methyl groups) or 7f (by a tetramethylene chain) led to
a loss of HLE-inhibiting activity, as the corresponding derivatives
have been investigated in a previous study.10
2.3. Generation of N-radicals
Bisazoles connected by N–N bonds are often viewed as weakly
bound dimers, which readily homolize to two radicals.19,20 N,N0-
Linked bisazaheterocycles 7 could in fact be considered as pre-
cursors for N-radicals, provided that dissociation energy of the N–N
bond is low enough to allow its thermal or photochemical cleavage.
The geometries of compounds 7b, 7d, and 7f were optimized using
at 1178–1193 and 1330–1358 cmꢀ1
.

V.M. Zakharova et al. / Tetrahedron 66 (2010) 379–384
381
B3LYP method with 6-31G basis set. Calculated N–N bond lengths
*
are 1.370 Å, 1.361 Å, and 1.380 Å correspondingly. The calculated
values indicate that compound 7f has the highest propensity to give
N-radicals. We tried to generate N-radicals from N,N0-linked ben-
zisothiazol-3(2H)-one 1,1-dioxides by laser flash photolysis with
detection by emission and absorption spectroscopy. The experi-
ments were carried out with 2-(phthalimid-1-yl)- and 2-(2-
methyl-4-oxo-3(4H)-quinazolinyl)-1,2-benzisothiazol-3(2H)-one
1,1-dioxides (7d,f) since both parts of the molecules of sultams 7d,f
contain carbonyl groups, on which unpaired electrons can be
delocalized, stabilizing the radicals formed. Calculation of Mulliken
atomic spin densities for the phthalimid-1-yl radical using the
B3LYP/6-31G method confirmed that approximately 77% of spin
*
density is located at the nitrogen atom, while 20% are divided
among the two neighboring carbonyl groups. A similar spin density
distribution is assumed for the corresponding benzisothiazol-2-yl
radical and has to be confirmed in future investigations. The UV
spectra of the initial sultams 7d,f show absorption bands in the
range below 266 nm (see Experimental). Their transient absorption
spectra were measured from 280 nm upwards.
After excitation of the intermediate singlet state, the competing
reactions 2–6 (M¼investigated sultam, E¼excess heat) should be
considered. Under our experimental conditions, self-quenching 6
does not play a significant role because of the low concentration of
parent sultams 7d and 7f chosen.
Figure 3. Transient absorption spectra of acetonitrile solution of pure sultam 7d
purged with N2 (d) and with addition of 1,3-cyclohexadiene (2ꢃ10ꢀ5 M) (– – –) or
ferrocene (10ꢀ6 M) ($$$$$$) taken 1
ms and 20 ms after the pulse.
MDh
n
1/MðS1Þ
(1)
n
MðS1Þ/MðS0ÞDh 2; fluorescence
(2)
MðS1Þ/MðS0ÞDE; internal conversion
MðS1Þ/MðT1Þ; intersystem crossingðISCÞ
MðS1Þ/ > N$D$N <; fragmentation
MðS1ÞDM/2MðS0Þ; self ꢀ quenching
(3)
(4)
(5)
(6)
Figure 4. Time profiles of the acetonitrile solution of pure 7d and with addition of 1,3-
cyclohexadiene (2ꢃ10ꢀ5 M and 6ꢃ10ꢀ5 M) or ferrocene (10ꢀ6 M) at
¼330 nm.
l
triplet state and, therefore, it can be concluded that the first sub-
stance 7d does not produce marked amounts of N-radicals under
the present conditions of laser flash photolysis.
The fluorescence spectrum of the second compound, sultam 7f,
in acetonitrile solution showed three maxima lF(max) at 430, 490,
and 560 nm. All of them exhibit same order decay kinetics. The
lifetime of the first excited singlet state S1 of 29 ns measured at
490 nm is comparable to those data for compound 7d.
The transient absorption spectra obtained in oxygen-free solution in
the 266 nm laserflash photolysisof7fare shown in Figure 5.Thespectra
exhibit three bands with the maxima at 290, 340, and 420 nm. This
absorption behavior is similar to that described for the succinimidyl
radical, which reveals absorbance peaking at 270, 330, and 380 nm.21
All three peaks revealed uniform kinetics and, therefore, the
most characteristic peak at 340 nm is discussed. In contrast to the
data of sultam 7d, the decay monitored at 340 nm (Fig. 6) showed
mixed kinetics, which might indicate the existence of more than
one transient with different lifetimes.
This assumption was verified by quenching experiments with
O2, 1,3-cyclohexadiene and ferrocene. In their presence, the ab-
sorption band at 340 nm was partly quenched (Figs. 5 and 6)
(k¼2.1ꢃ107 (O2), 6.0ꢃ108 (1,3-cyclohexadiene), 7.0ꢃ108 (ferrocene)
dm3 molꢀ1 sꢀ1) remaining the decay of the second long-lived
In the 266 nm laser photolysis the fluorescence spectrum of
sultam 7d measured in deoxygenated acetonitrile solution exhibits
a maximum lF(max) at 320 nm. The decay at 320 nm showed first
order kinetics with a lifetime of 33 ns and hence indicates a long-
living singlet state S1. Non-radiative processes of the deactivation of
the first excited singlet statedintersystem crossing (ISC) and
fragmentationdwere investigated by absorption spectroscopy.
The time dependent transient absorption spectra of 7d, mea-
sured in acetonitrile 1 ms and 20 ms after the pulse, display one
broad and intense band with a maximum at 330 nm (Fig. 3). The
decay monitored at 330 nm shows pseudo-first order kinetics
corresponding to a lifetime of 16.6
ms (Fig. 4). This could be caused
by a triplet state as well as radicals.
In order to clarify the nature of the species formed, additional
measurements with typical triplet quenchersdoxygen, 1,3-cyclo-
hexadiene and ferrocenedwere performed. In their presence the
absorption band observed at 330 nm was markedly quenched with
reaction constants k¼4.9ꢃ108 (O2), 1.4ꢃ109 (1,3-cyclohexadiene)
and kꢄ2.4ꢃ1010 (ferrocene) dm3 molꢀ1 sꢀ1 (Figs. 3 and 4).
Moreover, the saturation of the solution with quencher (in-
creasing of concentration) has led to the acceleration of the tran-
sient decay (Fig. 4). These quenching reactions are typical for the

382
V.M. Zakharova et al. / Tetrahedron 66 (2010) 379–384
transient, which does not react with the scavenger in such a short
time range. Furthermore, the decay is accelerated with increasing
quencher concentrations (Fig. 6).
Thus, we identified for the first time saccharin-derived N,N0-linked
bisazaheterocycles as inhibitors of the therapeutically relevant
protease HLE.
We obtained a preliminary picture of photophysical properties of
sultams 7d,f and quantitative information about the deactivation
mechanism of the first excited singlet state of these saccharin ana-
logs. The common schema proposed for the transformations gen-
erated by the laser flash photolysis is as follows. Excitation brings the
molecule from the ground state S0 to a vibrationally excited state S1.
Besides fluorescence, the intersystem crossing and dissociation are
main deactivation channels rapidly leading to either triplet state or
radical formation. Sultam 7d did not produce a significant amount of
radical species under conditions used. In the case of 7f we have
found parallel rapid formation of both transients, which are formed
in competition rather than in subsequent reactions. The nature of the
radicals was not fully examined and requires further investigation.
However, their properties (such as insensibility to oxygen) indicated
the possible formation of N-centered radicals.
4. Experimental
4.1. General
Figure 5. Transient absorption spectra of acetonitrile solution of pure sultam 7f
purged with N2 (d) and with O2 (– – –) taken 890 ns and 33 ms after pulse.
Mp: Boetius micro-melting-point apparatus; corrected. Ele-
mental analysis: Heraeus CHNO Rapid Analyser. UV/VIS Spectra:
Beckman DU650; lmax in nm (log
Analytical System (ATI Mattson); KBr pellets, values in cmꢀ1
(400 MHz) and 13C- (100 MHz) Spectra: Varian Mercury Plus 400
spectrometer; in ppm rel to Me4Si as standard, J in Hertz. MS:
3
). IR Spectra: Genesis FTIR Unicam
.
1H-
d
Quadrupole-MS VG 12-250; 70 eV.
4.2. General procedure for the preparation of 1,2-
benzisothiazol-3(2H)-one 1,1-dioxides
The N-amino heterocycle 2a–f (4 mmol) was added under argon
atmosphere to a stirred solution of 2-chlorosulphonylbenzoyl
chloride 6 (0.96 g, 4 mmol) and pyridine (0.63 g, 8 mmol) in dry
dioxane (3 ml). The mixture was stirred for 15 min at room tem-
perature, then at 80 ꢁC for 1.5 h and poured into water or ethanol.
The precipitate was filtered off and recrystallized from ethanol to
obtain 7a–f.
Figure 6. Time profiles of acetonitrile solution of pure 7f and with addition of
1,3-cyclohexadiene (12ꢃ10ꢀ5 M) and ferrocene (5ꢃ10ꢀ5 M and 2ꢃ10ꢀ5 M) at
4.2.1. 2-(Piperidin-1-yl)-1,2-benzisothiazol-3(2H)-one
(7a). Yield: 56%. Beige solid. Mp 148–150 ꢁC. IR (KBr) 1730 (CO),
1330 (SO2), 1178 (SO2) cmꢀ1. UV (EtOH): lmax (log
)¼214 nm (4.11),
245 nm (3.53). 1H NMR (400 MHz, DMSO-d6)
8.24 (d, J¼8.0 Hz,1H,
H-4), 7.94–8.05 (m, 3H, 3 arom. CH), 3.31 (m, 4H, 2CH2N), 1.65 (m,
4H, 2CH2), 1.42 (m, 2H, CH2). 13C NMR (100 MHz, DMSO-d6)
157.9
(CO), 135.9 (arom. CH), 135.6 (C-7a), 135.1 (arom. CH), 125.1 (C-3a),
124.8 (arom. CH), 121.8 (arom. CH), 53.7 (2CH2N), 25.9 (2CH2), 22.6
(CH2). Anal. Calcd for C12H14N2O3S: C, 54.12; H, 5.30; N, 10.52; S,
12.04. Found: C, 53.79; H, 5.51; N, 10.13; S, 12.21. MS (EI) 266 (Mþ).
1,1-dioxide
l
¼340 nm.
3
d
Because of this typical behavior, the short-lived transient was
assigned to be a triplet state. The second long-lived species is at-
tributed to the radical products of the fragmentation process. For
the long-lived transient species, we suggest the structure of an N-
radical. This is supported by its low sensitivity against oxygen. And,
certainly, the dissociation appears to be the most probable process
in consideration of the relative weakness of N–N bonds. The anal-
ysis of the long-lived species’ behavior shows good agreement with
the data for the succinimidyl radical, which has relatively long
lifetime of 10ꢀ4 s as well.21–23
d
4.2.2. 2-(Morpholin-1-yl)-1,2-benzisothiazol-3(2H)-one 1,1-dioxide
(7b). Yield: 54%. Beige solid. Mp 166–168 ꢁC. IR (KBr) 1728 (CO),
1344 (SO2), 1183 (SO2) cmꢀ1. UV (EtOH): lmax (log
3
)¼211 nm (4.29),
218 nm (4.26), 225 nm (4.20), 243 nm (3.80). 1H NMR (400 MHz,
acetone-d6) 7.99–8.14 (m, 4H, 4 arom. CH), 3.80 (m, 4H, 2CH2O),
3.48 (m, 4H, 2CH2N). 13C NMR (100 MHz, acetone-d6)
159.0 (CO),
3. Conclusions
d
d
A number of N,N0-linked 1,2-benzisothiazol-3(2H)-one 1,1-di-
oxides have been synthesized by acylation/sulfonylation of N-amino
heterocycles with 2-chlorosulfonylbenzoyl chloride. New saccharin
analogs were evaluated for their inhibitory activity toward HLE and
AChE. Two sultams, 2-(N-phthalimidyl)-1,2-benzisothiazol-3(2H)-
one 1,1-dioxide and 2-(2-methyl-4-oxo-3(4H)-quinazolinyl)-1,2-
benzisothiazol-3(2H)-one 1,1-dioxide were found to inhibit HLE.
137.5 (C-7a), 136.5 (arom. CH), 135.6 (arom. CH), 126.7 (C-3a), 125.6
(arom. CH), 122.3 (arom. CH), 67.7 (2CH2O), 54.0 (2CH2N). Anal.
Calcd for C11H12N2O4S: C, 49.25; H, 4.51; N, 10.44; S, 11.95. Found: C,
49.16; H, 4.47; N, 10.37; S, 12.34. MS (EI) 268 (Mþ).
4.2.3. 2-(Azepan-1-yl)-1,2-benzisothiazol-3(2H)-one
1,1-dioxide
(7c). Yield: 52%. White solid. Mp 127–129 ꢁC. IR (KBr) 1737 (CO),

V.M. Zakharova et al. / Tetrahedron 66 (2010) 379–384
383
1339 (SO2), 1184 (SO2) cmꢀ1. UV (EtOH): lmax (log
226 nm (3.83), 240 nm (3.45). 1H NMR (400 MHz, acetone-d6)
7.94–8.12 (m, 4H, 4 arom. CH), 3.64 (m, 4H, 2CH2N), 1.79 (m, 4H,
2CH2), 1.68 (m, 4H, 2CH2). 13C NMR (100 MHz, acetone-d6)
158.9
(CO), 137.5 (C-7a), 136.4 (arom. CH), 135.5 (arom. CH), 126.8 (C-3a),
125.5 (arom. CH), 122.3 (arom. CH), 57.6 (2CH2N), 29.0 (2CH2), 27.9
(2CH2). Anal. Calcd for C13H16N2O3S: C, 55.70; H, 5.75; N, 9.99; S,
11.44. Found: C, 55.35; H, 5.83; N, 10.24; S, 11.17. MS (EI): 280 (Mþ).
3
)¼209 nm (4.15),
acetonitrile. Into a cuvette containing 825
the DTNB solution, 55 l acetonitrile, 10
and 10 l of an enzyme solution (w3 U/ml) were added and thor-
oughly mixed. After incubation for 15 min at 25 ꢁC, the reaction was
initiated by adding 50 l of the ATCh solution and was followed
over 5 min. The reactions were performed with 7a–f at a single
concentration (12.5 or 25 M), and IC50 values were calculated from
m
l assay buffer, 50
m
l of
m
ml of an inhibitor solution,
d
m
d
m
m
the linear steady-state turnover of the substrate.
Table 1
4.2.4. 2-(Phthalimid-1-yl)-1,2-benzisothiazol-3(2H)-one 1,1-dioxide
(7d). Yield: 93%. White solid. Mp 257–260 ꢁC. IR (KBr) 1753 (CO),
Crystal data and structure refinement for 7b
1354 (SO2), 1193 (SO2) cmꢀ1. UV (CH3CN): lmax (log
3
)¼221 nm
Empirical formula
C11H12N2O4S
(4.44), 277 nm (3.50). 1H NMR (400 MHz, DMSO-d6)
d 8.02–8.11 (m,
Formula weight
Temperature [K]
Crystal system
Space group
a [Å]
268.29
213(2)
5H, 5 arom. CH), 8.21 (m, 1H, arom. CH), 8.27 (d, J¼8.0 Hz, 1H, arom.
CH), 8.54 (d, J¼8.0 Hz, 1H, arom. CH). 13C NMR (100 MHz, DMSO-d6)
Orthorhombic
Pna2 (1)
11.642 (1)
9.699 (1)
10.744 (1)
90
d
162.8 (2CO),156.4 (CO), 137.5 (arom. CH), 136.3 (2 arom. CH), 136.1
(arom. CH), 135.9 (C-7a), 128.9 (2 arom. C), 126.4 (arom. C), 124.8 (2
arom. CH), 123.7 (C-3a), 122.9 (arom. CH). Anal. Calcd for
C15H8N2O5S: C, 54.88; H, 2.46; N, 8.53; S, 9.77. Found: C, 54.61; H,
2.21; N, 8.48; S, 9.90. MS (EI): 328 (Mþ).
b [Å]
c [Å]
a
b
g
[ꢁ]
[ꢁ]
[ꢁ]
90
90
Volume [Å3]
1213.2 (2)
4
1.469
4.2.5. 2-[4-Oxo-3(4H)-quinazolinyl]-1,2-benzisothiazol-3(2H)-one
1,1-dioxide (7e). Yield: 83%. White solid. Mp 275–278 ꢁC. IR (KBr)
1773 (CO), 1720 (CO), 1608 (C]N), 1358 (SO2), 1192 (SO2) cmꢀ1. UV
Z
Density [g/cm3]
Absorption coeff. [mmꢀ1
Crystal size [mm]
]
0.275
(CH3CN): lmax (log
3
)¼225 nm (4.64), 260 nm (4.13). 1H NMR
0.4ꢃ0.3ꢃ0.3
2.73–26.07
ꢀ14ꢄhꢄ14, 11ꢄkꢄ11,
ꢀ13ꢄlꢄ12
7535
2361 [Rint¼0.0361]
0.9219/0.8978
2361/1/211
0.587
q
range for data collect. [ꢁ]
(400 MHz, DMSO-d6)
d
8.57 (m, 2H, 2 arom. CH), 8.31 (m, 1H, arom.
Index ranges
CH), 8.22–8.14 (m, 3H, 2 arom. CHþCH-20), 8.01 (m, 1H, arom. CH),
8.84 (m, 1H, arom. CH), 7.71 (m, 1H, arom. CH). 13C NMR (100 MHz,
Reflections collected
Independent reflections
Max./min. transmission
Data/parameters
DMSO-d6)
d
156.8, 156.3, 147.3 (C-20), 146.5 (C-8a0), 137.3, 136.3,
136.1 (3 arom. CH), 135.6 (C-7a), 128.8, 128.1, 127.0, 126.3 (4 arom.
CH), 124.5 (C-3a), 122.9 (arom. CH), 121.5 (C-4a0). Anal. Calcd for
C15H9N3O4S: C, 55.04; H, 2.77; N, 12.84; S, 9.80. Found: C, 54.88; H,
2.89; N, 12.81; S, 9.71. MS (EI): 327 (Mþ).
Goodness-of-Fit on F2
Final R indices [I>2
R indices (all data)
s
(I)]
R1¼0.0239,
u
R2¼0.0682
R1¼0.0256,
uR2¼0.0700
Lgst diff. Peak/hole [e Åꢀ3
]
0.152/ꢀ0.195
4.2.6. 2-[2-Methyl-4-oxo-3(4H)-quinazolinyl]-1,2-benzisothiazol-
3(2H)-one 1,1-dioxide (7f). Yield: 73%. Yellow solid. Mp 235–238 ꢁC.
IR (KBr) 1773 (CO), 1700 (CO), 1614 (C]N), 1350 (SO2), 1187 (SO2)
Human leukocyte elastase (HLE) was assayed spectrophoto-
metrically at 405 nm at 25 ꢁC in duplicate experiments. Assay
buffer was 50 mM sodium phosphate buffer, 500 mM NaCl, pH 7.8.
Inhibitor stock solutions were prepared in DMSO. A stock solution
of the chromogenic substrate MeOSuc-Ala-Ala-Pro-Val-pNA was
prepared in DMSO and diluted with assay buffer. Final concentra-
tion of the chromogenic substrate MeOSuc-Ala-Ala-Pro-Val-pNA
cmꢀ1. UV (EtOH): lmax (log
NMR (400 MHz, DMSO-d6)
3
)¼213 nm (4.05), 223 nm (4.06). 1H
d
7.59–8.54 (m, 8H, 8 arom. CH), 2.63 (s,
1H, CH3). 13C NMR (100 MHz, DMSO-d6)
d 157.1, 156.8, 155.2, 145.9
(C-8a0), 137.4, 136.4, 136.2 (3 arom. CH), 135.8 (C-7a), 128.0, 127.5,
127.0, 126.5 (4 arom. CH), 124.7 (C-3a), 122.8 (arom. CH), 119.8 (C-
4a0), 21.0 (CH3). Anal. calcd for C16H11N3O4S: C, 56.30; H, 3.25; N,
12.31; S, 9.39. Found: C, 56.01; H, 3.17; N,12.42; S, 9.49. MS (EI): 341
(Mþ).
was 100
performed with a final HLE concentration of 50 ng/ml. Into a cu-
vette containing 890 l assay buffer, 10 l of an inhibitor solution,
50 l of the substrate solution were added and thoroughly mixed.
The reaction was initiated by adding 50 l of the HLE solution and
mM, final concentration of DMSO was 1.5%. Assays were
m
m
m
m
4.3. X-ray crystal structure analysis of 7b
was followed over 10 min. IC50 values were calculated from the
linear steady-state turnover of the substrate as described.27 The
reactions were performed with 7a,b,c,e at a single concentration
(12.5 or 25 mM). Compounds 7d,f were measured at five different
inhibitor concentrations between 5 and 40 mM.
Crystals were obtained from DMSO. The intensities were mea-
sured on a STOE IPDA-1 diffractometer. Data collection and cell
refinement parameters are listed in the Table 1. The structure was
solved by direct methods, and refinement was performed with
SHELX-97.24 Crystallographic data have been deposited with the
Cambridge Crystallographic Center, CCDC 725591. Copies of the
data can be obtained, free of charge, from CCDC, 12 Union Road,
Cambridge, CB21EZ, UK (fax: þ44-1233-336033; e-mail: deposit@
ccdc.cam.ac.uk; internet: http://www.ccdc.cam.ac.uk).
4.5. ns-Laser flash photolysis
Acetonitrile of the highest spectroscopic grade was used as
solvent (99.9%, VWR). 1,3-Cyclohexadiene and ferrocene were
obtained from Aldrich.
4.4. Enzyme inhibition
Real-time nanosecond laser photolysis was applied with
detection by emission and absorption spectroscopy. The solutions
of compounds 7d,f (with concentrations 2ꢃ10ꢀ4 M and 6ꢃ10ꢀ5 M
correspondingly) were deoxygenated by bubbling with nitrogen or
purged with oxygen and photolyzed at 20 ꢁC in a flow system
(suprasil quartz cell) by the fourth harmonics (266 nm) of
a Quanta-Ray GCR-11 Nd:YAG laser (Spectra Physics). Pulses of
<3 ns duration (FWHM) with energies of 1–4 mJ were used. The
Acetylcholinesterase from Electrophorus electricus was assayed
spectrophotometrically at 412 nm at 25 ꢁC in duplicate experi-
ments.25,26 Assay buffer was 100 mM sodium phosphate, 100 mM
NaCl, pH 7.3. Acetylthiocholine (ATCh) (10 mM) and 5,50-dithio-bis-
(2-nitrobenzoic acid) (DTNB) (7 mM) were dissolved in assay
buffer. Stock solutions of the inhibitors were prepared in

384
V.M. Zakharova et al. / Tetrahedron 66 (2010) 379–384
Appl. EP 509400, 1991. Chem. Abstr. 1993, 118, 101970; (c) Bhalla, M.; Srivastava,
V. K.; Bhalla, T. N.; Shanker, K. Arzneimittelforschung 1993, 43, 595; (d) Tyagy, R.;
Goel, B.; Srivastava, V. K.; Kumar, A. Indian J. Pharm. Sci. 1998, 60, 283; (e) Hoda,
A. A.; Sayed, A. S.; Momen, A. E. I. K.; Ahmed, S. A. Y. Phosphorous, Sulfur, Silicon
Relat. Elem. 1992, 72, 237; (f) Ibrahim, M. K. Egyptian J. Pharm. Sci. 1998, 39, 519;
(g) Srivastava, V. K.; Singh, K. S.; Singh, S. N.; Fatima, N.; Chaterjee, R. K. Indian J.
Chem., Sect. B 1991, 30, 859; (h) Angerbauer, R.; Huebsch, W.; Fey, P.; Bischoff,
H.; Petzinna, D.; Schmidt, D.; Thomas, G. Eur. Pat. Appl. EP 339342, 1989. Chem.
Abstr. 1990, 113, 115074; (i) Srivastava, V. K.; Singh, K. S.; Gulati, A.; Shanker, K.
Indian J. Chem., Sect. B 1987, 26, 652.
optical detection system consisted of a pulsed xenon lamp (XBO
150, Osram), monochromator (SpectraPro-275, Acton Research),
R955 photomultiplier tube (Hamamatsu Photonics) or fast
Si-photodiode with 1 Ghz amplification, and 500 MHz digitizing
oscilloscope (DSA 602 A, Tektronix). The laser power was moni-
tored for every pulse using bypath with a fast Si-photodiode. The
laser flash photolysis set-up is reported elsewhere in more detail.28
9. (a) Reddy, G. M.; Bhavani, A. K. D.; Reddy, P. P.; Reddy, P. S. N. Synthesis 2002,
1311; (b) Wang, J. H. Acc. Chem. Res. 1970, 3, 90; (c) Inbaraj, J. J.; Bilski, P.;
Chignell, C. F. Photochem. Photobiol. 2002, 75, 107; (d) Hawkins, C. L.; Davies, M.
J. Chem. Res. Toxicol. 2002, 15, 183.
Acknowledgements
The authors wish to thank Stephanie Hautmann for technical
assistance. The work was supported by the Deutsche For-
schungsgemeinschaft
‘Mechanistische und Anwendungsaspekte nichtkonventioneller
Oxidationsreaktionen’.
10. Zakharova, V. M.; Gonza´lez Tanarro, C. M.; Gu¨ tschow, M.; Hennig, L.; Sieler,
J.; Schulze, B. Synthesis 2008, 1133.
and
the
Graduiertenkolleg
378
11. (a) Schulze, B.; Kirrbach, S.; Illgen, K.; Fuhrmann, P. Tetrahedron 1996, 52, 783;
(b) Hartung, C.; Illgen, K.; Sieler, J.; Schneider, B.; Schulze, B. Helv. Chim. Acta
1999, 82, 685; (c) Taubert, K.; Sieler, J.; Hennig, L.; Findeisen, M.; Schulze, B.
Helv. Chim. Acta 2002, 85, 183; (d) Taubert, K.; Siegemund, A.; Eilfeld, A.; Bau-
mann, S.; Sieler, J.; Schulze, B. Synthesis 2004, 2265; (e) Siegemund, A.; Hartung,
C.; Eilfeld, A.; Sieler, J.; Schulze, B. Z. Naturforsch. 2004, 59b, 478.
12. Zakharova, V.; Siegemund-Eilfeld, A.; Sieler, J.; Schulze, B. Z. Naturforsch. 2006,
61b, 464.
13. Ashe, B. M.; Clark, R. L.; Jones, H.; Zimmerman, M. J. Biol. Chem. 1981, 256, 11603.
14. Li, P.; Wang, B.; Ma, N.; Li, Z.; Wang, S. Huaxue Shiji 2005, 27, 445.
15. Dart, E.C.; Holt, G. J. Chem. Soc. Perkin Trans 1 1974, 1403.
16. (a) Leov, B.; Kormendy, M. J. Org. Chem. 1962, 27, 1703; (b) Lepore, S. D.; Bhunia,
A. K.; Cohn, P. J. Org. Chem. 2005, 70, 8117; (c) Lepore, S. D.; Bhunia, A. K.;
Mondal, D.; Cohn, P.; Lefkowitz, C. J. Org. Chem. 2006, 71, 3285.
17. Stassinopoulou, C. I.; Catsoulacos, P.; Camoutsis, C. Org. Magn. Reson. 1983,
21, 187.
References and notes
1. (a) Hilbert, M. F.; Mir, A. K.; Fozard, J. R. In Comprehensive Medicinal Chemistry;
Sammes, P. G., Taylor, J. B., Eds.; Pergamon: Oxford, 1990; Vol. 3, p 578; (b)
Romero, A. G.; Darlington, W. H.; Piercey, M. F.; Lahti, R. A. Bioorg. Med. Chem.
Lett. 1992, 2, 1703.
2. (a) Martyn, D. C.; Moore, M. J.; Abell, A. D. Curr. Pharm. Des. 1999, 5, 405; (b)
Groutas, W. C.; Epp, J. B.; Venkataraman, R.; Kuang, K.; Truong, T. M.; McCle-
nahan, J. J.; Prakash, O. Bioorg. Med. Chem. 1996, 4, 1393; (c) Groutas, W. C.;
Huang, H.; Venkataraman, R.; Houser-Archield, N.; Epp, J. B. Bioorg. Med. Chem.
1993, 1, 273; (d) Hlasta, D. J.; Bell, M. R.; Boaz, N. W.; Court, J. J.; Desai, R. C.;
Franke, C. A.; Mura, A. J.; Subramanyam, C.; Dunlap, R. P. Bioorg. Med. Chem. Lett.
1994, 4, 1801; (e) Hlasta, D. J.; Bell, M. R.; Court, J. J.; Cundy, K. C.; Desai, R. C.;
Ferguson, E. W.; Gordon, R. J.; Kumar, V.; Maycock, A. L.; Subramanyam, C.
Bioorg. Med. Chem. Lett. 1995, 5, 331; (f) Hlasta, D. J.; Subramanyam, C.; Bell, M.
R.; Carabateas, P. M.; Court, J. J.; Desai, R. C.; Drozd, M. L.; Eickhoff, W. M.; Fer-
guson, E. W.; Gordon, R. J.; Johnson, J. A.; Kumar, V.; Maycock, A. L.; Mueller, K. R.;
Pagani, E. D.; Robinson, D. T.; Saindane, M. T.; Silver, P. J.; Subramanian, S.;
Dunlap, R. P.; Franke, C. A.; Mura, A. J.; Rowlands, A. G. J. Med. Chem. 1995, 38, 739.
3. Gonzalez-Martin, G.; Lyndon, C.; Sunkel, C. Eur. J. Pharm. Biopharm. 1998, 46, 293.
4. Combrink, K. D.; Gulgeze, H. B.; Meanwell, N. A.; Pearce, B. C.; Zulan, P.; Bissacci,
G. S.; Roberts, D. G. M.; Stanley, P.; Seiler, S. M. J. Med. Chem. 1998, 41, 4854.
5. Patane, M. A.; Dipardo, R. M.; Price, R. P.; Chang, R. S. L.; Ransom, R. W.;
O’Malley, S. S.; De Salvo, J.; Bock, M. G. Bioorg. Med. Chem. Lett. 1998, 8, 2495.
6. Nagasawa, H. T.; Kawle, S. P.; Elberling, J. A.; DeMaster, E. G.; Fukuto, J. M. J. Med.
Chem. 1995, 38, 1865.
18. Schulze, B.; Kirsten, G.; Kirrbach, S.; Rahm, A.; Heimgartner, H. Helv. Chim. Acta
1991, 74, 1059.
19. Stella, L. In Radicals in Organic Synthesis; Renaud, P., Sibi, M. P., Eds.; Willey-
VCH: Weinheim, 2001; Vol. 2, p 407.
20. Zimmerman, H.; Baumgartel, H.; Bakke, F. Angew. Chem. 1961, 73, 808.
21. Lind, J.; Jonsson, M.; Eriksen, T. E.; Merenyi, G.; Eberson, L. J. Phys. Chem. 1993,
97, 1610.
22. Lind, J.; Merenyi, G. In The Chemistry of Free Radicals: N-Centered Radicals;
Alfassi, Z. B., Ed.; John Wiley & Sons Ltd: Chichester, UK, 1998; p 563.
23. Yip, R. W.; Chow, Y. L.; Beddard, C. J.C.S. Chem. Commun. 1981, 955.
24. Sheldrick, G.M. SHELX-97, a Program System for Solution and the Refinement of
X-ray Crystal Structures, Univ. of Go¨ttingen: Germany, 1997.
25. Ellman, G. L.; Courtney, K. D.; Andres, V.; Feather-Stone, R. M. Biochem. Phar-
macol. 1961, 7, 88.
26. Pietsch, M.; Gu¨ tschow, M. J. Med. Chem. 2005, 48, 8270.
27. Gu¨tschow, M.; Pietsch, M.; Themann, A.; Fahrig, J.; Schulze, B. J. Enz. Inhib. Med.
Chem. 2005, 20, 341.
7. (a) Hettler, H. Adv. Heterocycl. Chem. 1973, 15, 233; (b) Schulze, B.; Illgen, K.
J. Prakt. Chem. 1997, 339, 1.
28. Hermann, R.; Mahalaxmi, G. R.; Jochum, T.; Naumov, S.; Brede, O. J. Phys. Chem.
A 2002, 106, 2379.
8. (a) Obniska, J.; Jurczyk, S.; Zeic, A.; Kaminski, K.; Tatarczynska, E.; Stachowicz,
K. Pharmcol. Rep. 2005, 57, 170; (b) Effland, R.C.; Davis, L.; Olsen, G.E. Eur. Pat.