2,1,3-Benzothiadiazine derivatives: Synthesis and screening versus PDE4 enzyme

Article abstract of DOI:10.1016/j.farmac.2005.05.009

A series of N-1,3 disubstituted 2,1,3-benzothiadiazine derivatives (BTDs) were synthesized and evaluated for their inhibitory activity versus enzymatic isoform PDE4 extracted from U937 cell line. Some of the tested compounds showed a high PDE4 inhibitory activity at 100:μM and the IC₅₀ value of the most interesting terms were evaluated. The structure-activity relationships of these compounds showed that the 3,5-di-tert-butyl-4-hydroxybenzyl moiety at N-1 position is important to obtain activity at micromolar level as previously reported. For the same compounds the antioxidant activity were evaluated highlighting 14 as the most significative one. The introduction of other bulky substituents in N-1 position is detrimental.

Full text of DOI:10.1016/j.farmac.2005.05.009

Il Farmaco 60 (2005) 653–663
http://france.elsevier.com/direct/FARMAC/
2,1,3-Benzothiadiazine derivatives:
synthesis and screening versus PDE4 enzyme
Annalisa Tait *, Amedeo Luppi, Rossella Avallone, Mario Baraldi
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Modena e Reggio Emilia, Via Campi 183, 41100 Modena, Italy
Received 28 January 2005; received in revised form 16 May 2005; accepted 19 May 2005
Available online 15 June 2005
Abstract
A series of N-1,3 disubstituted 2,1,3-benzothiadiazine derivatives (BTDs) were synthesized and evaluated for their inhibitory activity
versus enzymatic isoform PDE4 extracted from U937 cell line. Some of the tested compounds showed a high PDE4 inhibitory activity at
100 µM and the IC50 value of the most interesting terms were evaluated. The structure–activity relationships of these compounds showed that
the 3,5-di-tert-butyl-4-hydroxybenzyl moiety at N-1 position is important to obtain activity at micromolar level as previously reported. For the
same compounds the antioxidant activity were evaluated highlighting 14 as the most significative one. The introduction of other bulky sub-
stituents in N-1 position is detrimental.
© 2005 Elsevier SAS. All rights reserved.
Keywords: 2,1,3-Benzothiadiazine; PDE4; COPD; Antioxidant activity
1. Introduction
oped as PDE4 inhibitors [10,11]. Rolipram is the prototypic
of the PDE4 inhibitors but its clinical use has been limited by
side effects such as nausea and vomiting [12–14]. In the last
years, companies have focused their attention on the design
of new compounds with improved anti-PDE4 activity in the
attempt of dissociating beneficial anti-inflammatory profile
and emetic side effects [15]. Cilomilast (Ariflo®) can be con-
sidered a partial goal of this strategy. Developed by Glaxo-
SmithKline, as a drug for treatment of asthma and COPD,
cilomilast significantly improved health status and was gen-
erally well tolerated with the most common treatment-
related adverse events being nausea, diarrhea and abdominal
pain [16]. Cilomilast is actually is in phase III clinical trials.
The cyclic nucleotides cAMP and cGMP are intracellular
second messengers that mediate many physiological pro-
cesses. The levels of the cyclic nucleotides are mainly regu-
lated by the rate of their degradation, which is linked to phos-
phodiesterase activity. The function of many inflammatory
cells is controlled by cyclic nucleotides [1,2]. High levels of
cAMP activate protein kinase (PKA), which phosphorylates
some cytoplasmatic proteins with consequent inhibition of
chemotaxis, degranulation and oxidative neutrophil metabo-
lism. Moreover, PKA by acting on the phosphodiesterase IV
gene stimulates the synthesis of this enzyme, which in turn
transforms cAMP to inactive AMP [3,4]. The inhibition of
PDE4 enhances intracellular cAMP and decrease inflamma-
tory cells activations present in some diseases as asthma and
chronic obstructive pulmonary disease (COPD), symptom-
atically characterized by a repeated stridor and paroxysm due
to the airway contraction [5–9].
Roflumilast, synthesized by BYK Gulden, is in phase III
studies for asthma and COPD, and it seems to be safe and
well tolerated. Moreover roflumilast shows also beneficial
effects in allergic rhinitis, indicating that PDE4 inhibitors
might be useful not only in allergic asthma or allergic rhinitis
but also when the two diseases are simultaneously present
[17] (Chart 1).
In the last two decades a great variety of compounds,
belonging to very different chemical classes, have been devel-
Zardaverine is a mixed PDE3/PDE4 inhibitor whose clini-
cal development was discontinued for its side effects some of
which, as nausea and vomiting, are related to inhibition of
PDE4 [18]. More promising PDE4 phthalazinones inhibi-
* Corresponding author.
E-mail address: tait.annalisa@unimore.it (A. Tait).
0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2005.05.009

654
A. Tait et al. / Il Farmaco 60 (2005) 653–663
Chart 1
.
tors, structurally related to zardaverine, were recently de-
scribed by BYK Gulden [19].
Other potent PDE4 inhibitors were designed from nitraqua-
zone [20].
Sf21 insect cell system and others related compounds previ-
ously synthesized and characterized by antiviral [24,26] prop-
erties, were tested.
Finally the antioxidant activity of new compounds substi-
tuted with the 2,6-di-tert-butylphenol moiety was deter-
mined by UV measurements of the scavenging effect in the
presence of 2,2-diphenyl-1-picrylhydrazil (DPPH).
The compounds recently patented (US 6075016) by Euro-
celtique [21] belong to family of benzoxazoles and were char-
acterized by aryl-acetylene and antioxidant 2,6-di-tert-
butylphenol moiety. Antioxidant properties of these
compounds are interesting because they may limit the oxida-
tive damage of inflammatory chronic disorders in which
endogenous antioxidant defenses are inadequate. The pro-
gression of many of these diseases may be retarded by natu-
ral or synthetic antioxidants. These compounds exhibited
higher in vitro PDE4 activity and selectivity than rolipram
and they were also more active to providing bronchodilating
and anti-inflammatory effects.
2. Chemistry
The N-1,3 disubstituted 2,1,3-BTDs (1–29) (Table 1) were
obtained (Scheme 1) by alkylation with the appropriate alco-
hol, means Mitsunobu reaction [27], of the N-3 substituted
derivatives 30–44 (Table 2) prepared from the suitable methyl
2-aminobenzoate and sulfamoyl chloride in presence of tri-
ethylamine [28,29] to afford the intermediate N-sulfa-
moylanthranilates that were successively cyclized by sodium
methoxide [24].
The benzothiadiazine derivatives (BTDs) were described
as heterocyclic inhibitors of PDE7 with concurrent inhibi-
tory activity at PDE4 and PDE3 [22,23].
The compound 21 was also obtained in discrete yield
employing polymer-supported triphenylphosphine as reac-
tive [30–33]. The better experimental conditions found by us
require an excess of BTD 30 (3 eq.) and polymer-supported
triphenylphosphine (2 eq.) respect to 1-naphthylmethanol used
as alkylating agent, protracting the reaction time overnight at
room temperature. The polymer-supported reagent triph-
enylphosphine oxide and its by-products are easily removed
by simple filtration. The excess of BTD 30, pKa ~3.4 [34],
was removed by washing thoroughly with NaOH aq. 5% and
21 was recovered practically pure by crystallization from
methanol. The polymer-supported triphenylphosphine oxide
could be reused after regeneration employing trichlorosilane
(Cl3SiH) as reducing agent [35,36]. The method of solution-
phase chemistry above described opens the way for the rapid
generation of N,N-dialkylated 2,1,3-BTD libraries that could
represent an important tool for the discovery of new drugs
into this class of compounds.
Recently we synthesized some 2,1,3-BTDs N-1 substi-
tuted with a 3,5-di-tert-butyl-4-hydroxybenzyl moiety and
endowed with PDE4 inhibitor activity and selectivity at micro-
molar level. These compounds are also characterized by inter-
esting scavenging properties [24].
This paper describes the synthesis and PDE4 inhibitory
activity versus the enzymatic isoform extracted from
U937 cell line of a series of new N-1,3 disubstituted 2,1,3-
BTDs.
Monocytic U937 cells express the long PDE4A4,
PDE4D5 and PDE4D3 isoforms plus the short PDE4B2 iso-
form. Immunopurification of PDE4 subfamilies identifies long
PDE4D isoforms as providing the major PDE4 activity in
U937 monocytic cells [25].
To a better evaluation of structure–activity relationships
(SAR) also the BTDs derivatives previously showing activity
on human recombinant enzyme PDE4D3 from Baculovirus/

A. Tait et al. / Il Farmaco 60 (2005) 653–663
655
Table 1
Structures and in vitro PDE4 inhibitory activity of synthesized 2,1,3-BTDs
Compounds
R1
R3
Rn
Percent
inhibition a
76
1
2
3,5-Di-tert-butyl-4-OH-benzyl
3,5-Di-tert-butyl-4-OH-benzyl
3,5-Di-tert-butyl-4-OH-benzyl
3,5-Di-tert-butyl-4-OH-benzyl
3,5-Di-tert-butyl-4-OH-benzyl
3,5-Di-tert-butyl-4-OH-benzyl
3,5-Di-tert-butyl-4-OH-benzyl
3,5-Di-tert-butyl-4-OH-benzyl
3,5-Di-tert-butyl-4-OH-benzyl
3,5-Di-tert-butyl-4-OH-benzyl
3,5-Di-tert-butyl-4-OH-benzyl
3,5-Di-tert-butyl-4-OH-benzyl
3,5-Di-tert-butyl-4-OH-benzyl
3,5-Di-tert-butyl-4-OH-benzyl
3,5-Di-tert-butylbenzyl
3-Iodobenzyl
Propyl
H
Propyl
7-Cl
72
3 [24]
4 [24]
5
6
7 [24]
8
9 [26]
10 [26]
11
12 [26]
13
14
15
16
17
18
19
20
21
22
Propyl
6,7-Dimethoxy
71
Isopropyl
Isopropyl
Benzyl
H
92
7-Cl
88
H
88
Benzyl
7-Cl
85
Benzyl
6,7-Dimethoxy
91
2-Chlorobenzyl
2-Chlorobenzyl
2-Chlorobenzyl
4-Chlorobenzyl
4-Chlorobenzyl
4-Chlorobenzyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Propyl
H
85
7-Cl
94
6,7-Dimethoxy
88
H
88
7-Cl
95
6,7-Dimethoxy
85
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
66
52
1,1′-Biphenyl-2-ylmethyl
1,1′-Biphenyl-3-ylmethyl
1,1′-Biphenyl-4-ylmethyl
1-Benzhydryl
38
13
45
50
1-Naphtylmethyl
53
2-Naphtylmethyl
40
23
4-Nitrobenzyl
54
24 [42,43]
25 [26]
26
27 [26]
28
4-Nitrobenzyl
Benzyl
43
4-Nitrobenzyl
2-Chlorobenzyl
Isopropyl
Benzyl
45
Pyridin-4-ylmethyl
68
Pyridin-4-ylmethyl
71
2-Pyridin-2-ylethyl
Isopropyl
2-Phenylethyl
58
29 [26]
Rolipram b
2-Pyridin-2-ylethyl
59
90
a All compounds were tested at 100 µM versus PDE4D extracted and purified from cell line U937.
b Rolipram was taken as PDE4 selective inhibitor of reference.
Scheme 1. Toluene, Et3N, 80 °C, 1 h (ii) Na, CH3OH, 40 °C, 2 h (iii) TPP, DIAD, THF, N2, RT.
Compound 18 was obtained by N-alkylation of 30 with
3-(bromomethyl)-1,1′-biphenyl in presence of NaH [37]
(Scheme 2).
The structures of all new intermediates (31, 40, 42 and 43)
and final compounds (1, 2, 5, 6, 8, 11, 13–23, 26 and 28)
were elucidated from their analytical and spectroscopic data
reported in Section 4. Chemical, physical and spectroscopic
Scheme 2
.
NaH, DMF, 70 °C, 3 h.

656
A. Tait et al. / Il Farmaco 60 (2005) 653–663
Table 2
substitution leads to compounds with a promising activity (3,
N-3 substituted 2,1,3-BTDs 30–44
8, 11 and 14) (71–91% inhibition) bearing in mind that the
di-alkoxy aryl moiety is a feature common to a wide number
of PDE4 inhibitors like rolipram, cilomilast, roflumilast and
zardaverine.
The N-1 replacement of 3,5-di-tert-butyl-4-hydroxybenzyl
moiety with other alkylaromatic (15–29) groups is detrimen-
tal for inhibitory activity (13–72%), independently of
N-3 and/or benzothiadiazine aromatic ring substitution.
Interestingly enough, compound 15, endowed with tert-
butyl groups in position 3,5 of N-1 benzyl moiety but devoid
of –OH in position 4, shows a relatively good inhibitory activ-
ity versus PDE4 (66%), nevertheless lower than that observed
for the compounds equipped with the 3,5-di-tert-butyl-4-
hydroxybenzyl moiety at the same position.
To evaluate if the 3,5-di-tert-butyl-4-hydroxybenzyl moi-
ety might be replaced with other unrelated bulky moieties,
we designed and synthesized 2,1,3-BTDs endowed in posi-
tion N-1 with the 3-iodobenzyl (16), biphenyl (17–19),
1-benzhydryl (20), 1-naphtylmethyl (21) and 2-naphtylmethyl
(22) moieties. This subset of compounds presents a lower
potency in comparison with that first tested; in particular the
compounds 17, 18, 19 and 22 inhibit weakly the PDE4 en-
zyme (respectively, 38%, 13%, 45% and 40% inhibition),
while the inhibitory potency of compounds 16, 20 and 21 is
greater (respectively, 52%, 50% and 53% inhibition). Con-
cerning the N-1 naphthylmethyl substitution (21, 22), the a
isomer (21) is correlated with a greater inhibitory potency
respect to b isomer (22), while, relatively to the N-1 biphenyl
substitution (17–19), it is interesting to observe that the ortho-
biphenyl is deleterious, the meta-biphenyl completely abol-
ishes the activity while a discrete anti-PDE4 activity remains
in the case of para-biphenyl moiety; probably both
1-naphtylmetyl and para-biphenyl moieties are better ar-
ranged for the interaction with the binding pocket of the
enzyme.
Moreover to complete the SAR studies of the previous
paper [24] we synthesized and tested 2,1,3-BTDs N-1 para-
nitrobenzyl equipped (23–25). The collected data show that
these compounds are poorly active, independently of N-3 alkyl
or aralkyl substitution (respectively, 54%, 43% and 45% inhi-
bition).
The introduction of a pyridine moiety in N-1 position was
the last structural modification evaluated (26–29). A discrete
level of activity is observed in the case of pyridin-4-ylmethyl
moiety (26 and 27) (respectively, 68% and 71% inhibition),
independently of the presence of isopropyl or benzyl in
N-3 position. The shift of pyridine nitrogen from position 4 to
position 2 and the elongation of linker chain (28 and 29) lead
to a decrease of potency (respectively, 58% and 59% inhibi-
tion).
Compounds
30 [38]
31
R3
Rn
Propyl
H
Propyl
7-Cl
32 [24]
33 [39]
34 [40]
35 [41]
36 [24]
37 [24]
38 [26]
39 [26]
40
Propyl
6,7-Dimethoxy
Isopropyl
H
Isopropyl
7-Cl
Benzyl
H
Benzyl
7-Cl
Benzyl
6,7-Dimethoxy
2-Chlorobenzyl
2-Chlorobenzyl
2-Chlorobenzyl
4-Chlorobenzyl
4-Chlorobenzyl
4-Chlorobenzyl
2-Phenylethyl
H
7-Cl
6,7-Dimethoxy
41 [26]
42
43
H
7-Cl
6,7-Dimethoxy
H
44 [26]
data of already known intermediates (30, 32–39, 41 and 44)
[24,38–41] and final compounds (3, 4, 7, 9, 10, 12, 24, 25, 27
and 29) [24,26,42,43] are in agreement with those reported
in literature.
3. Results and discussion
All compounds were tested at 100 µM concentration for
their ability to inhibit the activity of PDE4 extracted from
U937 cellular line [44–46]. Also the BTDs previously show-
ing activity on human recombinant enzyme PDE4D3 from
Baculovirus/Sf21 insect cell system (3, 4 and 7) [24] and oth-
ers related compounds (9, 10, 12, 24, 25, 27 and 29) previ-
ously synthesized and characterized by antiviral properties
[26,42,43], were tested using rolipram as reference com-
pound (Table 1). Although most of the tested compounds
showed a lower activity in comparison with rolipram, some
interesting results could be emphasized and preliminary SAR
could be commented. All BTDs submitted to screening test
are N-1,N-3-alkyl or -aralkyl disubstituted. According to our
previous report [24], the presence in position N-1 of 3,5-di-
tert-butyl-4-hydroxybenzyl moiety (1–14) plays a pivotal role,
being associated with the best percent of inhibitory activity,
comprised between 71% and 95% (rolipram 90%). The
N-3 isopropyl or aralkyl substitution (4–14) seems to favor
the potency (85–95% inhibition) compared with N-3 propyl
substitution that characterizes the less active compounds 1–3
(71–76% inhibition). As regards the substitutions at the aro-
matic ring of benzothiadiazine core, a chlorine atom in posi-
tion 7, which may favor the interaction with a lipophylic zone
of the catalytic site of the enzyme, is permitted (2, 5, 7, 10
and 13) and results particularly favorable when associated
with a 2-chloro-benzyl or 4-chloro-benzyl moiety at N-3 posi-
tion (10 and 13) (~95% inhibition). Also the 6,7-dimethoxy
All these data confirm that for BTDs, the 3,5-di-tert-butyl-
4-hydroxybenzyl moiety in N-1 plays an essential role in the
binding interaction with the PDE4 enzyme. As previously
observed and confirmed also by molecular modeling studies
[24] probably the bulky di-tert-butyl groups and the –OH pro-

A. Tait et al. / Il Farmaco 60 (2005) 653–663
657
trude at the top of S3 enzymatic sub-pocket and contribute to
better stabilize the molecule into the catalytic site of the
enzyme. All the other compounds lacking of this moiety are
unable to interact with the critical aminoacids involved in the
catalytic process.
egy for the treatment of chronic inflammatory diseases by a
double and synergic mechanism of action, on the one hand
elevating the second messenger cAMP that is able to stop the
principal inflammatory events, on the other hand scavenging
reactive oxygen species (ROS) that are involved in several
pathological conditions supported by chronic inflammatory
processes.
Moreover the IC50 values of the most interesting terms (8,
10, 13) were determined (Table 3) and fall in the micromolar
range (pIC50, respectively, 4.9, 5.1 and 5.0), about 10-fold
less potent than reference compound rolipram (pIC50 6.1).
Our previous studies [24,26] show that compounds 3, 4, 7,
9, 10 and 12, N-1 3,5-di-tert-butyl-4-hydroxybenzyl substi-
tuted, are characterized by antioxidant properties. The inter-
action of the new 1, 2, 5, 6, 8, 11, 13 and 14 derivatives with
stable free radical DPPH are shown in Table 4.All tested com-
pounds exhibited antioxidant activity. The 6,7-dimethoxy sub-
stituted derivative 14, the most active of the series, presents a
scavenging activity value of ~60% [compared to ~70% value
of reference 2,6-di-tert-butyl-4-methylphenol (BHT)].
The SAR of these compounds confirmed that the 3,5-di-
tert-butyl-4-hydroxybenzyl moiety at N-1 position is impor-
tant to obtain anti-PDE4 activity at micromolar level as well
as antioxidant properties and that the presence of 6,7-
dimethoxy substitution on the BTD aromatic ring is associ-
ated with highest antioxidant profile.
4. Experimental
4.1. Chemistry
Melting points determined in capillary tubes (Büchi
510 capillary apparatus) were uncorrected. 1H NMR spectra
were recorded with a Brucker DPX 200 spectrometer using
d6-DMSO as solvent and tetramethylsilane (TMS) as exter-
nal standard. Chemical shifts are in ppm (d) and coupling
constants (J) in Hz. Multiplicities are abbreviated as follows:
s, singlet; d, doublet; t, triplet; dd, double doublet; ddd, double
double doublet; dt, double triplet; sxt, sextet; sep, septet; b,
indicates a broadening of the signal; *, D2O changeable. IR
spectra were recorded on a Perkin–Elmer 1600 FT-IR spec-
trometer (Nujol mull) and UV spectra on a Cary 50 Bio
UV–Visible Spectrophotometer Varian. Purity was checked
The combination of PDE4 inhibition and radical scaveng-
ing activity in a single compound could be an efficient strat-
by TLC performed on aluminum sheets silica gel 60 F254
,
0.2 mm thick. Flash chromatography was performed on Silica
gel Merck (230–400 mesh). The yields of reactions were not
optimized. Elemental analyses were found within 0.4% of
the theoretical values and performed in Microanalysis Labo-
ratory of Dipartimento di Scienze Farmaceutiche of Modena
University on a Carlo Erba Elemental Analyzer 1106 appa-
ratus.
The measures of radioactivity were performed with a Top-
countTM (Packard) instrument using a 96 microvials Opti-
plate.
Table 3
In vitro pIC50 values versus PDE4 a
b
Compounds
pIC50
4.9
8
10
5.1
13
5.0
Rolipram c
6.1
a Data represent mean values for two separate experiments.
b pIC50 = –log IC50 versus PDE4D extracted from cell line U937.
c Rolipram was taken as PDE4 selective inhibitor of reference.
Reagents and solvents were purchased from common com-
mercial suppliers and used as received.
Table 4
Absorbance decrease at 514 nm versus control (DA) and (S.A.%) for 50 µM
solutions after 6 h
4.1.1. 7-Chloro-3-propyl-1H-2,1,3-benzothiadiazin-4(3H)-
one 2,2-dioxide (31)
Compounds
DA a
S.A.% b
30.8
32.1
33.1
40.9
53.5
41.0
42.8
60.4
73.9
1
2
5
6
8
11
13
14
0.1538 0.0018
0.1602 0.0016
0.1556 0.0329
0.1921 0.0182
0.2670 0.0028
0.2046 0.0028
0.2013 0.0201
0.2840 0.0191
0.3475 0.0170
4.1.1.1. General procedure. To a stirred and cooled (0 °C)
solution of propylamine (5.00 g, 85 mmol) in dichloromethane
(50 ml), chlorosulfonic acid (3.30 g, 28.3 mmol) was added
drop by drop and the reaction mixture was stirred for an addi-
tional hour after being warmed to room temperature gradu-
ally. After evaporation of the solvent in vacuo, the resultant
salt between propylsulfamic acid and propylamine was dis-
solved in toluene (50 ml) and treated with phosphorus pen-
tachloride (7.06 g, 33.9 mmol). The mixture was refluxed for
1 h and the inorganic by-products removed by filtration. The
filtrate was evaporated in vacuo to give the N-propylsulfamoyl
chloride as an oily residue that was used in the next synthetic
step without further purification.
BHT c
a Difference of absorbance after 6 h between DPPH solution (50 µM)
(control) and DPPH solution (50 µM) in the presence of tested compound
(50 µM). The mean values standard deviation were obtained from quadru-
plicate experiments.
b Scavenging activity percent of tested compounds calculated according
to equation: S.A.% = 100 × DA/At, where At is the absorbance value at 514 nm
of DPPH solution (control) after 6 h.
c BHT (2,6-di-tert-butyl-4-methylphenol) was taken as antioxidant com-
pound of reference.
To a solution of methyl 2-amino-4-chlorobenzoate (3.51 g,
18.9 mmol) and triethylamine (2.87 g, 28.4 mmol) in toluene

658
A. Tait et al. / Il Farmaco 60 (2005) 653–663
(50 ml) was slowly added a solution of N-propylsulfamoyl
chloride (3.57 g, 22.6 mmol) in toluene (10 ml) and the result-
ing mixture was heated at 80 °C for 1 h. The resultant triethy-
lamine hydrochloride was filtered and the filtrate was evapo-
rated in vacuo to give an oily residue. The residue was
dissolved in 75 ml of a 0.5 M sodium methoxide methanolic
solution, freshly prepared. After stirring for 2 h at 40 °C the
solvent was evaporated under reduced pressure and the resi-
due dissolved in water. After cooling the aqueous solution
supplied 31 on acidification with hydrochloric acid as a white
solid, yield 2.44 g (47%), m.p. 160–161 °C (DMF/water). 1H
NMR (d6-DMSO) d 7.96 (dd, 1H, J = 0.3, 8.5 Hz, H-5), 7.27
(dd, 1H, J = 2.0, 8.5 Hz, H-6), 7.14 (dd, 1H, J = 0.3, 2.0 Hz,
H-8), 3.78 (t, 2H, J = 7.4 Hz, NCH2), 1.67 (sxt, 2H,
J = 7.4 Hz, CH2), 0.89 (t, 3H, J = 7.4 Hz, CH3). IR(Nujol)
3132, 1644, 1354, 1170, 1006, 760 cm−1. Anal.
(C10H11ClN2O3S): C, H, N.
tetrahydrofuran (10 ml), at 0 °C under N2 atmosphere, were
added 3-propyl-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-
dioxide (30) (0.76 g, 3.0 mmol), triphenylphosphine (1.57 g,
6.0 mmol) and, over a period of 5 min, diisopropylazodicar-
boxylate (DIAD) (1.21 g, 6.0 mmol): the orange–red color of
DIAD disappears immediately with slight liberation of heat.
The mixture was stirred at room temperature overnight in N2
atmosphere. The solvent was evaporated under reduced pres-
sure and the residue was purified by flash column chromatog-
raphy (cyclohexane/ethyl acetate 6:4). 1 was obtained as a
white solid, yield 0.40 g (30%); m.p. 110 °C (trituration with
petrol ether 40–60 °C). 1H NMR (d6-DMSO) d 7.95 (dd, 1H,
J = 1.6, 7.9 Hz, H-5), 7.84 (ddd, 1H, J = 1.6, 7.4, 8.0 Hz,
H-6), 7.71 (dd, 1H, J = 1.2, 8.0 Hz, H-8), 7.50 (ddd, 1H,
J = 1.2, 7.4, 8.0 Hz, H-7), 6.91 (s*, 1H, OH), 6.62 (s, 2H,
aromatic), 4.92 (s, 2H, NCH2Ph), 3.44 (t, 2H, J = 7.3 Hz,
NCH2), 1.45 (sxt, 2H, J = 7.3 Hz, CH2), 1.19 (s, 18H, tert-
butyl), 0.78 (t, 3H, J = 7.3 Hz, CH3). IR(Nujol) 3637, 1677,
1603, 1329, 1171, 1003 cm−1. Anal. (C25H34N2O4S): C, H,
N.
4.1.2. 3-(2-Chlorobenzyl)-6,7-dimethoxy-1H-2,1,3-ben-
zothiadiazin-4(3H)-one 2,2-dioxide (40)
This compound was obtained starting from methyl
2-amino-4,5-dimethoxybenzoate by reaction with 2-chloro-
benzylsulfamoyl chloride as a white solid in 70% yield; m.p.
190–191 °C. 1H NMR (d6-DMSO) d 7.48–7.43 (m, 1H, aro-
matic), 7.34 (s, 1H, H-5), 7.33–7.22 (m, 3H, aromatic), 6.64
(s, 1H, H-8), 5.04 (s, 2H, NCH2Ph), 3.85 (s, 3H, OCH3), 3.77
(s, 3H, OCH3). IR(Nujol) 3113, 1668, 1651, 1340, 1189, 1013,
762 cm−1. Anal. (C16H15ClN2O5S): C, H, N.
4.1.6. 7-Chloro-1-(3,5-di-tert-butyl-4-hydroxybenzyl)-3-
propyl-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (2)
This compound was obtained starting from 7-chloro-3-
propyl-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (31)
by reaction with 3,5-di-tert-butyl-4-hydroxybenzyl alcohol.
Flash column chromatography: eluent cyclohexane/ethyl
acetate (9:1, v/v). Yield 21%; m.p. 135–138 °C (trituration
with petrol ether 40–60 °C). 1H NMR (d6-DMSO) d 8.00 (d,
1H, J = 8.5 Hz, H-5), 7.96 (d, 1H, J = 2.0 Hz, H-8), 7.58 (dd,
1H, J = 2.0, 8.5 Hz, H-6), 6.98 (s*, 1H, OH), 6.72 (s, 2H,
aromatic), 5.02 (s, 2H, NCH2Ph), 3.54 (t, 2H, J = 7.3 Hz,
NCH2), 1.49 (2H, sxt, J = 7.3 Hz, CH2), 1.25 (s, 18H, tert-
butyl), 0.83 (t, 3H, J = 7.3 Hz, CH3). IR(Nujol) 3582, 1679,
1592, 1308, 1185, 1006 cm−1. Anal. (C25H33ClN2O4S): C,
H, N.
4.1.3. 7-Chloro-3-(4-chlorobenzyl)-1H-2,1,3-benzothiadi-
azin-4(3H)-one 2,2-dioxide (42)
This compound was obtained starting from methyl
2-amino-4-chlorobenzoate by reaction with 4-chlorobenzyl-
sulfamoyl chloride as a white solid in 31% yield; m.p. 184–
185 °C (DMF/water). 1H NMR (d6-DMSO) d 7.93 (dd, 1H,
J = 0.3, 8.5 Hz, H-5), 7.38 (m, 4H, aromatic), 7.20 (dd, 1H,
J = 2.0, 8.5 Hz, H-6), 7.11 (dd, 1H, J = 0.3, 2.0 Hz, H-8),
4.98 (s, 2H, NCH2Ph). IR(Nujol) 3165, 1633, 1602, 1304,
1180, 928, 765 cm−1. Anal. (C14H10Cl2N2O3S): C, H, N.
4.1.7. 7-Chloro-1-(3,5-di-tert-butyl-4-hydroxybenzyl)-3-
isopropyl-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide
(5)
This compound was obtained starting from 7-chloro-3-
isopropyl-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide
(34) by reaction with 3,5-di-tert-butyl-4-hydroxybenzyl alco-
hol. Flash column chromatography: eluent cyclohexane/ethyl
acetate (6:4, v/v). Yield 45%; m.p. 118–120 °C (methanol/
water). 1H NMR (d6-DMSO) d 8.02 (d, 1H, J = 8.5 Hz, H-5),
7.89 (d, 1H, J = 2.0 Hz, H-8), 7.56 (dd, 1H, J = 2.0, 8.5 Hz,
H-6), 6.99 (s*, 1H, OH), 6.77 (s, 2H, aromatic), 5,05 (s, 2H,
NCH2Ph), 4.62 (sep, 1H, J = 6.9 Hz, NCH), 1.25 (s, 18H,
tert-butyl), 1.22 (d, 6H, J = 6.9 Hz, CH3). IR(Nujol) 3634,
1679, 1595, 1292, 1195, 1005 cm−1.Anal. (C25H33ClN2O4S):
C, H, N.
4.1.4. 3-(4-Chlorobenzyl)-6,7-dimethoxy-1H-2,1,3-ben-
zothiadiazin-4(3H)-one 2,2-dioxide (43)
This compound was obtained starting from methyl
2-amino-4,5-dimethoxybenzoate by reaction with 4-chloro-
benzylsulfamoyl chloride as a white solid in 26% yield; m.p.
182–185 °C (methanol/water). 1H NMR (d6-DMSO) d 7.37
(m, 4H, aromatic), 7.30 (s, 1H, H-5), 6.55 (s, 1H, H-8), 4.92
(s, 2H, NCH2Ph), 3.81 (s, 3H, OCH3), 3.74 (s, 3H, OCH3).
IR(Nujol) 3113, 1681, 1613, 1321, 1152, 1005, 847 cm−1.
Anal. (C16H15ClN2O5S): C, H, N.
4.1.5. 1-(3,5-Di-tert-butyl-4-hydroxybenzyl)-3-propyl-1H-
2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (1)
4.1.8. 3-Benzyl-1-(3,5-di-tert-butyl-4-hydroxybenzyl)-1H-
2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (6)
4.1.5.1. General procedure. To a solution of 3,5-di-tert-butyl-
4-hydroxybenzyl alcohol (0.66 g, 3.0 mmol) in anhydrous
This compound was obtained starting from 3-benzyl-1H-
2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (35) by reac-

A. Tait et al. / Il Farmaco 60 (2005) 653–663
659
tion with 3,5-di-tert-butyl-4-hydroxybenzyl alcohol. Flash
column chromatography: eluent cyclohexane/ethyl acetate
(6:4, v/v).Yield 38%; m.p. 153–155 °C (trituration with petrol
ether 40–60 °C). 1H NMR (d6-DMSO) d 7.97 (dd, 1H, J = 1.4,
7.9 Hz, H-5), 7.85 (ddd, 1H, J = 1.4, 7.3, 8.1 Hz, H-7), 7.69
(dd, 1H, J = 1.0, 8.1 Hz, H-8), 7.52 (ddd, 1H, J = 1.0, 7.9,
8.1 Hz, H-6), 7.29 (m, 5H, aromatic), 6.99 (s*, 1H, OH), 6.69
(s, 2H, aromatic), 4.94 (s, 2H, NCH2Ph), 4.59 (s, 2H,
NCH2Ph), 1.23 (s, 18H, tert-butyl). IR(Nujol) 3563, 1679,
1603, 1326, 1177, 1052 cm−1. Anal. (C29H34N2O4S): C, H,
N.
1H, OH), 6.74 (s, 2H, aromatic), 5.01 (s, 2H, NCH2Ph), 4.69
(s, 2H, NCH2Ph), 1.23 (s, 18H, tert-butyl). IR(Nujol) 3600,
1677, 1596, 1292, 1186 cm−1. Anal. (C29H33Cl2N2O4S): C,
H, N.
4.1.12. 3-(4-Chlorobenzyl)-1-(3,5-di-tert-butyl-4-hydroxy-
benzyl)-6,7-dimethoxy-1H-2,1,3-benzothiadiazin-4(3H)-one
2,2-dioxide (14)
This compound was obtained starting from 3-(4-
chlorobenzyl)-6,7-dimethoxy-1H-2,1,3-benzothiadiazin-
4(3H)-one 2,2-dioxide (43) by reaction with 3,5-di-tert-butyl-
4-hydroxybenzyl alcohol. Flash column chromatography:
eluent cyclohexane/ethyl acetate (6:4, v/v). Yield 25%; m.p.
151–152 °C (DMF/water). 1H NMR (d6-DMSO) d 7.40 (bd,
2H, J = 8.5 Hz, 4-chlorobenzyl H-3 and H-5), 7.35 (s, 1H,
H-5), 7.27 (bd, 2H, J = 8.5 Hz, 4-chlorobenzyl H-2 and H-6),
7.12 (s, 1H, H-8), 7.02 (s*, 1H, OH), 6.73 (s, 2H, aromatic),
4.89 (s, 2H, NCH2Ph), 4.62 (s, 2H, NCH2Ph), 3.89 (s, 3H,
OCH3), 3.23 (s, 3H, OCH3), 1.27 (s, 18H, tert-butyl). IR(Nu-
jol) 3565, 1666, 1609, 1281, 1176 cm−1. Anal.
(C31H37ClN2O6S): C, H, N.
4.1.9. 3-Benzyl-1-(3,5-di-tert-butyl-4-hydroxybenzyl)-6,7-
dimethoxy-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-
dioxide (8)
This compound was obtained starting from 3-benzyl-6,7-
dimethoxy-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide
(37) by reaction with 3,5-di-tert-butyl-4-hydroxybenzyl alco-
hol. Flash column chromatography: eluent cyclohexane/ethyl
acetate (6:4, v/v). Yield 57%; m.p. 140–142 °C (methanol/
1
water). H NMR (d6-DMSO) d 7.33 (s, 1H, H-5), 7.30 (m,
5H, aromatic), 7.10 (s, 1H, H-8), 6.98 (s*, 1H, OH), 6.70 (s,
2H, aromatic), 4.85 (s, 2H, NCH2Ph), 4.58 (s, 2H, NCH2Ph),
3.86 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 1.24 (s, 18H, tert-
butyl). IR(Nujol) 3621, 1678, 1603, 1259, 1175, 1007 cm−1.
Anal. (C31H38N2O6S): C, H, N.
4.1.13. 1-(3,5-Di-tert-butylbenzyl)-3-isopropyl-1H-2,1,3-
benzothiadiazin-4(3H)-one 2,2-dioxide (15)
This compound was obtained starting from 3-isopropyl-
1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (33) by
reaction with 3,5-di-tert-butylbenzyl alcohol. Flash column
chromatography: eluent cyclohexane/ethyl acetate (8.5:1.5,
v/v). Yield 68%; m.p. 75–77 °C (DMF/water). 1H NMR (d6-
DMSO) d 8.02 (dd, 1H, J = 1.6, 7.8 Hz, H-5), 7.79 (ddd, 1H,
J = 1.6, 7.5, 8.2 Hz, H-7), 7.62 (dd, 1H, J = 1.1, 8.2 Hz, H-8),
7.47 (ddd, 1H, J = 1.1, 7.5, 7.8 Hz, H-6), 7.26 (t, 1H,
J = 1.7 Hz, H-4′ di-tert-butylbenzyl), 6.80 (d, 2H, J = 1.7 Hz,
H-2′ and H-6′ di-tert-butylbenzyl), 5.08 (s, 2H, NCH2Ph),
4.57 (sep, 1H, J = 6.9 Hz, NCH), 1.17 (d, 6H, J = 6.9 Hz,
CH3). 1.13 (s, 18H, tert-butyl), IR(Nujol) 1679, 1600, 1316,
1251, 1194, 1171, 1138, 1100, 1046, 977, 890, 855, 696,
610 cm−1. Anal.(C25H34N2O3S): C, H, N.
4.1.10. 3-(2-Chlorobenzyl)-1-(3,5-di-tert-butyl-4-hydroxy-
benzyl)-6,7-dimethoxy-1H-2,1,3-benzothiadiazin-4(3H)-one
2,2-dioxide (11)
This compound was obtained starting from 3-(2-
chlorobenzyl)-6,7-dimethoxy-1H-2,1,3-benzothiadiazin-
4(3H)-one 2,2-dioxide (40) by reaction with 3,5-di-tert-butyl-
4-hydroxybenzyl alcohol. Flash column chromatography:
eluent cyclohexane/ethyl acetate (8:2, v/v). Yield 42%; m.p.
157–160 °C (trituration with petrol ether 40–60 °C). 1H NMR
(d6-DMSO) d 7.49 (m, 1H, aromatic), 7.36 (s, 1H, H-5), 7.31
(m, 2H, aromatic), 7.27 (s, 1H, H-8), 7.15 (m, 1H, aromatic),
7.01 (s*, 1H, OH), 6.70 (s, 2H, aromatic), 4.91 (s, 2H,
NCH2Ph), 4.65 (s, 2H, NCH2Ph), 3.95 (s, 3H, OCH3), 3.84
(s, 3H, OCH3), 1.27 (s, 18H, tert-butyl). IR(Nujol) 3590, 1677,
1605, 1284, 1183, 1010 cm−1. Anal. (C31H37ClN2O6S): C,
H, N.
4.1.14. 1-(3-Iodobenzyl)-3-isopropyl-1H-2,1,3-benzothiadi-
azin-4(3H)-one 2,2-dioxide (16)
This compound was obtained starting from 3-isopropyl-
1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (33) by
reaction with (3-iodophenyl)methanol. Flash column chro-
matography: eluent cyclohexane/ethyl acetate (7:3, v/v).Yield
67%; m.p. 97–100 °C. 1H NMR (d6-DMSO) d 8.00 (dd, 1H,
J = 1.6, 7.7 Hz, H-5), 7.76 (ddd, 1H, J = 1.6, 7.4, 8.0 Hz,
H-7 aromatic), 7.66–7.54 (m, 3H, aromatic), 7.44 (ddd, 1H,
J = 1.2, 7.7, 8.0 Hz, H-6 aromatic), 7.14–7.03 (m, 2H, aro-
matic), 5.10 (s, 2H, NCH2Ph), 4.80 (sep, 1H, J = 6.9 Hz,
NCH), 1.39 (d, 6H, J = 6.9 Hz, CH3). IR(Nujol) 1682, 1603,
1567, 1319, 1301, 1248, 1170, 1046, 996, 859, 793, 743 cm−1.
Anal. (C17H17IN2O3S): C, H, N.
4.1.11. 7-Chloro-3-(4-chlorobenzyl)-1-(3,5-di-tert-butyl-4-
hydroxybenzyl)-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-
dioxide (13)
This compound was obtained starting from 7-chloro-3-(4-
chlorobenzyl)-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-
dioxide (42) by reaction with 3,5-di-tert-butyl-4-hydroxy-
benzyl alcohol. Flash column chromatography: eluent
chloroform. Yield 40%; m.p. 138–142 °C (methanol/water).
1H NMR (d6-DMSO) d 7.95 (d, 1H, J = 8.5 Hz, H-5), 7.88
(d, 1H, J = 2.0 Hz, H-8), 7.56 (dd, 1H J = 2.0, 8.5 Hz, H-6),
7.38 (bd, 2H, J = 8.5 Hz, 4-chlorobenzyl H-3 and H-5), 7.27
(bd, 2H, J = 8.5 Hz, 4-chlorobenzyl H-2 and H-6), 7.02 (s*,

660
A. Tait et al. / Il Farmaco 60 (2005) 653–663
1
4.1.15. 1-(1,1′-Biphenyl-2-ylmethyl)-3-isopropyl-1H-2,1,3-
benzothiadiazin-4(3H)-one 2,2-dioxide (17)
m.p. 112–114 °C (methanol). H NMR (d6-DMSO) d 7.87
(ddd, 1H, J = 0.5, 1.7, 7.6 Hz, H-5) 7.49 (ddd, 1H, J = 1.7,
7.9, 8.0 Hz, H-7), 7.41–7.28 (m, 7H, aromatic), 7.21–7.12
(m, 4H, aromatic), 6.92 (ddd, 1H, J = 0.5, 1.1, 7.9 Hz H-8),
6.78 (s, 1H, NCHPh2), 4.75 (sep, 1H, J = 6.9 Hz, NCH), 1.36
(d, 6H, J = 6.9 Hz, CH3). IR(Nujol) 2976, 1714, 1682, 1602,
1495, 1479, 1372, 1304, 1255, 1196, 1137, 1096, 988, 831,
696 cm−1. Anal. (C23H22N2O3S): C, H, N.
This compound was obtained starting from 3-isopropyl-
1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (33) by
reaction with 1,1′-biphenyl-2-ylmethanol. Flash column chro-
matography: eluent cyclohexane/ethyl acetate (7:3, v/v).Yield
91%; m.p. 122–125 °C. 1H NMR (d6-DMSO) d 7.97 (dd, 1H,
J = 1.7, 7.8 Hz, H-5), 7.56 (ddd, 1H, J = 1.7, 7.4, 8.1 Hz,
H-7), 7.46–7.33 (m, 7H, aromatic), 7.26–7.22 (m, 3H, aro-
matic), 6.87 (dd, 1H, J = 1.1, 8.0 Hz, H-8), 4.96 (s, 2H,
NCH2Ph), 4.80 (sep, 1H, J = 6.9 Hz, NCH), 1.36 (d, 6H,
J = 6.9 Hz, CH3). IR(Nujol) 1682, 1599, 1481, 1295, 1263,
1243, 1195, 1170, 1145 cm−1. Anal. (C23H22N2O3S): C, H,
N.
4.1.19. 3-Isopropyl-1-(1-naphthylmethyl)-1H-2,1,3-ben-
zothiadiazin-4(3H)-one 2,2-dioxide (21)
This compound was obtained starting from 3-isopropyl-
1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (33) by
reaction with 1-naphthylmethanol. Flash column chromatog-
raphy: eluent cyclohexane/ethyl acetate (8:2, v/v).Yield 88%;
m.p. 118–119 °C. 1H NMR (d6-DMSO) d 8.17–8.12 (m, 1H,
aromatic), 8.02 (dd, 1H, J = 1.7, 7.8 Hz, H-5), 8.00–7.88 (m,
2H, aromatic), 7.63–7.32 (m, 6H, aromatic), 7.00 (dd, 1H,
J = 1.1, 8.0 Hz, H-8), 5.51 (s, 2H, NCH2), 4.91 (sep, 1H,
J = 6.9 Hz, NCH), 1.45 (d, 6H, J = 6.9 Hz, CH3). IR(Nujol)
1682, 1599, 1511, 1295, 1265, 1244, 1195, 990, 874,
753 cm−1. Anal. (C21H20N2O3S): C, H, N.
4.1.16. 1-(1,1′-Biphenyl-3-ylmethyl)-3-isopropyl-1H-2,1,3-
benzothiadiazin-4(3H)-one 2,2-dioxide (18)
To a suspension of sodium hydride (0.036 g, 1.5 mmol) in
DMF (10 ml) were added 3-isopropyl-1H-2,1,3-benzo-
thiadiazin-4(3H)-one 2,2-dioxide (33) (0.240 g, 1.0 mmol)
and, drop by drop, stirring the mixture in N2 atmosphere at
0 °C, a solution of 3-(bromomethyl)-1,1′-biphenyl dissolved
5 ml of DMF. The resulting mixture was warmed to 70 °C for
3 h. After cooling the solvent was evaporated under reduced
pressure and the oily residue was chromatographed using
chloroform/acetone (9.5:0.5, v/v) as eluent. Compound 18
The compound 21, was also synthesized by the use of
polymer-supported triphenylphosphine, as follows:
to a solution of 1-naphthylmethanol (0.158 g, 1.0 mmol)
in anhydrous tetrahydrofuran (10 ml), at 0 °C under N2 atmo-
sphere, were added 3-isopropyl-1H-2,1,3-benzothiadiazin-
4(3H)-one 2,2-dioxide (33) (1.0 g, 4.0 mmol), polystyrene
crosslinked with 2% divinylbenzene triphenylphosphine
(3.0 mmol P/g resin) (0.67 g, 2.0 mmol) and, over a period of
5 min, diisopropylazodicarboxylate (DIAD) (0.40 g,
2.0 mmol). The mixture was gently stirred at room tempera-
ture overnight in N2 atmosphere. The filtration of polymeric
phase of triphenylphosphine oxide and washing with small
portions of THF gave a solution that was evaporated in vacuo.
The oil obtained is dissolved in chloroform (20 ml) and shaken
with 5% aq. NaOH (20 ml × 5). Evaporation of the dried
(Na2SO4) organic layer in vacuo afforded the crude title com-
pound 21, which was crystallized by methanol (0,14 g, yield
39%).
1
was obtained (0.3 g, yield 74%) as an yellow oil. H NMR
(d6-DMSO) d 8.01 (ddd, 1H, J = 0.5, 1.7, 7.9 Hz, aromatic),
7.72 (ddd, 1H, J = 1.7, 7.2, 7.3 Hz, aromatic), 7.51 (m, 10H,
aromatic), 7.12 (d, 1H, J = 8.0 Hz, aromatic), 5.17 (s, 2H,
CH2Ph), 4.76 (sep 1H, J = 6.9 Hz, NCH), 1.32 (d, 6H,
J = 6.9 Hz, CH3). IR 3032, 2927, 1688, 1682, 1674, 1603,
1480, 1462, 1372, 1296, 1193, 1171, 1041, 988, 755, 695,
608 cm−1. Anal. (C23H22N2O3S): C, H, N.
4.1.17. 1-(1,1′-Biphenyl-4-ylmethyl)-3-isopropyl-1H-2,1,3-
benzothiadiazin-4(3H)-one 2,2-dioxide (19)
This compound was obtained starting from 3-isopropyl-
1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (33) by
reaction with 1,1′-biphenyl-4-ylmethanol. Flash column chro-
matography: eluent cyclohexane/ethyl acetate (6:4, v/v).Yield
76%; m.p. 38–40 °C (methanol/water). 1H NMR (d6-DMSO)
d 8.01 (dd, 1H, J = 1.6, 7.8 Hz, H-5), 7.77 (ddd, 1H, J = 1.6,
7.6, 7.8 Hz, H-7), 7.64–7.56 (m, 5H, aromatic), 7.48–7.30
(m, 4H, aromatic), 7.25 (d, 2H, J = 8.4 Hz, aromatic), 5.16
(s, 2H, NCH2Ph), 4.80 (sep, 1H, J = 6.9 Hz, NCH), 1.39 (d,
6H, J = 6.9 Hz, CH3). IR(Nujol) 1678, 1601, 1435, 1408,
1336, 1291, 1242, 1172, 1160, 1142, 1109, 1070, 974, 871,
758 cm−1. Anal.(C23H22N2O3S): C, H, N.
4.1.20. 3-Isopropyl-1-(2-naphthylmethyl)-1H-2,1,3-ben-
zothiadiazin-4(3H)-one 2,2-dioxide (22)
This compound was obtained starting from 3-isopropyl-
1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (33) by
reaction with 2-naphthylmethanol. Flash column chromatog-
raphy: eluent cyclohexane/ethyl acetate (7:3, v/v).Yield 76%
1
as oil. H NMR (d6-DMSO) d 7.98 (ddd, 1H, J = 0.4, 1.5,
7.8 Hz, aromatic), 7.89–7.70 (m, 5H, aromatic), 7.60–7.38
(m, 4H, aromatic), 7.23 (dd, 1H, J = 1.8, 8.5 Hz, H-8), 5.28
(s, 2H, NCH2), 4.80 (sep, 1H, J = 6.9 Hz, NCH), 1.35 (d, 6H,
J = 6.9 Hz, CH3). IR 3056, 2973, 2938, 1732, 1694, 1682,
1667, 1606, 1580, 1510, 1045, 987, 895, 821, 695 cm−1.Anal.
(C21H20N2O3S): C, H, N.
4.1.18. 1-Benzhydryl-3-isopropyl-1H-2,1,3-benzothiadi-
azin-4(3H)-one 2,2-dioxide (20)
This compound was obtained starting from 3-isopropyl-
1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (33) by
reaction with diphenylmethanol. Flash column chromatogra-
phy: eluent cyclohexane/ethyl acetate (9:1, v/v). Yield 63%;

A. Tait et al. / Il Farmaco 60 (2005) 653–663
661
4.1.21. 1-(4-Nitrobenzyl)-3-propyl-1H-2,1,3-benzothiadi-
azin-4(3H)-one 2,2-dioxide (23)
tion of DMSO in the assays (< 1% v/v) does not substantially
affect PDE activity.
This compound was obtained starting from 3-propyl-1H-
2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (30) by reac-
tion with (4-nitrophenyl)methanol. Flash column chromatog-
raphy: eluent cyclohexane/ethyl acetate (8:2, v/v).Yield 57%;
m.p. 95 °C (trituration with petrol ether 40–60 °C). 1H NMR
(d6-DMSO) d 8.20 (bd, 2H, J = 8.8 Hz, 4-nitrobenzyl H-3 and
H-5), 8.03 (dd, 1H, J = 1.6, 7.9 Hz, H-5), 7.78 (ddd, 1H,
J = 1.7, 7.7, 7.9 Hz, H-7), 7.49 (m, 2H, aromatic), 7.48 (bd,
2H, J = 8.8 Hz, 4-nitrobenzyl H-2 and H-6), 5.26 (s, 2H,
NCH2Ph), 3.78 (t, 2H, J = 7.3 Hz, NCH2), 1.65 (sex, 2H,
J = 7.3 Hz, CH2), 0.88 (t, 3H, J = 7.3 Hz, CH3). IR(Nujol)
1683, 1604, 1519, 1460, 1184, 1000, 706 cm−1. Anal.
(C17H17N3O5S): C, H, N.
PDE activity was measured by a modified scintillation
proximity assay (SPA) test from Amersham Biosciences (see
procedural instructions “phosphodiesterase [3H]cAMP SPA
enzyme assay, code TRKQ 7090”), carried out in 96 well
microplates (MTPs). The test volume is 100 µl and contains
20 mM Tris buffer (pH 7.4), 0.1 mg ml–1 BSA, 5 mM Mg2+,
0.5 µM of the substrate cAMP (including about 50,000 cpm
of the corresponding [3H]-labeled cyclic nucleotide), 1 µl of
the respective substance dilution and sufficient enzyme to
ensure that 10–20% of the substrate is converted.
After a preincubation of 40 min at 37 °C in the presence of
the compounds, the reaction is started by adding the sub-
strate and the assays are incubated for a further 15 min; after
that, they are stopped by adding SPA beads (50 µl). In accor-
dance with the manufacturer’s instructions, the SPA beads
have previously been resuspended in water. After the beads
have been sedimented (18 h), the MTPs are analyzed with a
TopcountTM (Packard). IC50 values were calculated from the
concentration–inhibition curves by non-linear regression
analysis using Graph Pad Prism program.
4.1.22. 3-Isopropyl-1-(pyridin-4-ylmethyl)-1H-2,1,3-ben-
zothiadiazin-4(3H)-one 2,2-dioxide (26)
This compound was obtained starting from 3-isopropyl-
1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (33) by
reaction with 4-hydroxymethylpyridine. Flash column chro-
matography: eluent chloroform/acetone (8:2, v/v).Yield 86%
as oil. 1H NMR (d6-DMSO) d 8.55 (bd, 2H, J = 6.0 Hz, pyri-
dine H-2 and H-6), 8.07 (dd, 1H, J = 1.6, 7.9 Hz, H-5), 7.78
(ddd, 1H, J = 1.6, 7.8, 8.2 Hz, H-7), 7.50 (dd, 1H, J = 1.1,
7.8 Hz, H-8), 7.48 (ddd, 1H, J = 1.1, 7.9, 8.2 Hz, H-6), 7.23
(bd, 2H, J = 6.0 Hz, pyridine H-3 and H-5), 5.19 (s, 2H,
NCH2Ph), 4.87 (sep, 1H, J = 6.9 Hz, NCH), 1.43 (d, 6H,
J = 6.9 Hz, CH3). IR(Nujol) 1681, 1602, 1193, 752, 722 cm−1.
The final product presents a 10% of triphenylphosphine as
impurity.
4.3. Radical scavenging effect on DPPH radical
Two milliliters of an ethanolic solution (1 × 10−4 M) of the
tested compounds was added to 2 ml of a DPPH solution (1 ×
10−4 M), and the reaction mixture was shaken vigorously and
kept at 37 °C 0.02 (Haake F 3C Thermocriostat) in air.
DPPH absorption was measured at 514 nm every 15 min.
The mean values were obtained from quadruplicate experi-
ments.
4.1.23. 3-Isopropyl-1-(2-pyridin-2-ylethyl)-1H-2,1,3-ben-
zothiadiazin-4(3H)-one 2,2-dioxide (28)
This compound was obtained starting from 3-isopropyl-
1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (33) by
reaction with 2-(2-hydroxyethyl)-pyridine. Flash column
chromatography: eluent chloroform/acetone (9.5:0.5, v/v).
Yield 39% as oil. 1H NMR (d6-DMSO) d 8.42 (m, 1H, aro-
matic), 7.99 (dd, 1H, J = 1.5, 7.8 Hz, H-5), 7.72 (ddd, 1H,
J = 1.8, 7.4, 7.5 Hz, aromatic), 7.70 (m, 2H, aromatic), 7.49
(bd, 1H, J = 8 Hz, aromatic), 7.63 (ddd, 1H, J = 1.8, 7.4,
7.6 Hz, aromatic), 7.41 (ddd, 1H, J = 1.0, 7.3, 7.9 Hz, aro-
matic), 7.17 (m, 2H, aromatic), 4.89 (sep 1H, J = 6.9 Hz,
NCH), 4.27 (t, 2H, J = 7.2 Hz, NCH2), 3.03 (t, 2H, J = 7.2 Hz,
CH2-pyridine), 1.46 (d, 6H, J = 6.9 Hz, CH3). IR(Nujol) 1686,
1681, 1603, 1592, 1570, 1461, 1377, 1192, 1170, 991 cm−1.
Anal. (C17H19N3O3S): C, H, N.
Acknowledgements
Thanks are due to Mrs. Rossella Gallesi who performed
elemental analyses of synthesized compounds.
We thank Inpharzam Ricerche SA (Gruppo Zambon) for
the gentle gift of PDE4 enzyme, and finally the laboratory of
Oncoematologia Istituto Seragnoli Policlinico S. Orsola-
Malpighi Bologna for the use of the TopcountTM (Packard)
instrument.
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