Benzyl derivatives of 2,1,3-benzo- and benzothieno[3,2-a]thiadiazine 2,2-dioxides: First phosphodiesterase 7 inhibitors

Article abstract of DOI:10.1021/jm990382n

The synthesis of a new family of benzyl derivatives of 2,1,3-benzo- and benzothieno[3,2-a]-thiadiazine 2,2-dioxides was achieved. The biological data revealed the first heterocyclic family of compounds with PDE 7 inhibitory properties appearing to be a new objective for the treatment of T-cell- dependent disorders. The IC₅₀ values or percent inhibition values of the compounds against PDE 7 were calculated by testing them against human recombinant PDE 7 expressed in S. cerevisiae. In this expression system the only cyclic nucleotide hydrolyzing activity present in cell extracts corresponded to human PDE 7. Isoenzyme selectivity PDE 7 versus PDE 4 and PDE 3 was also measured. Considering simultaneously inhibition of the three different isoenzymes, monobenzyl derivatives 15 and 23 showed interesting PDE 7 potency (around 10 μM); although not statistically significant, a trend toward selectivity with respect to PDE 3 and PDE 4 was obtained. Benzothiadiazine 16, although less potent at PDE 7 (IC₅₀ = 25 μM), also showed a trend of selectivity toward PDE 3 and PDE 4. These compounds are considered the best leads for further optimization.

Full text of DOI:10.1021/jm990382n

J . Med. Chem. 2000, 43, 683-689
683
Ben zyl Der iva tives of 2,1,3-Ben zo- a n d Ben zoth ien o[3,2-a ]th ia d ia zin e
2,2-Dioxid es: F ir st P h osp h od iester a se 7 In h ibitor s
Ana Mart´ınez,*,† Ana Castro,† Carmen Gil,† Montserrat Miralpeix,‡ Victor Segarra,‡ Teresa Dome´nech,‡
J orge Beleta,‡ J ose M. Palacios,‡ Hamish Ryder,‡ Xavier Miro´,§ Carles Bonet,§ J osep M. Casacuberta,§
Ferran Azor´ın,§ Benjam´ı Pin˜a,§ and Pere Puigdome´nech§
Instituto de Quı´mica Me´dica, CSIC, J uan de la Cierva 3, 28006 Madrid, Spain, Centro de Investigacio´n,
Almirall Prodesfarma S.A., Cardener 68-74, 08024 Barcelona, Spain, and Instituto de Biolog´ıa Molecular de Barcelona,
CID-CSIC, J ordi Girona 18-26, 08034 Barcelona, Spain
Received J uly 26, 1999
The synthesis of a new family of benzyl derivatives of 2,1,3-benzo- and benzothieno[3,2-a]-
thiadiazine 2,2-dioxides was achieved. The biological data revealed the first heterocyclic family
of compounds with PDE 7 inhibitory properties appearing to be a new objective for the treatment
of T-cell-dependent disorders. The IC50 values or percent inhibition values of the compounds
against PDE 7 were calculated by testing them against human recombinant PDE 7 expressed
in S. cerevisiae. In this expression system the only cyclic nucleotide hydrolyzing activity present
in cell extracts corresponded to human PDE 7. Isoenzyme selectivity PDE 7 versus PDE 4 and
PDE 3 was also measured. Considering simultaneously inhibition of the three different
isoenzymes, monobenzyl derivatives 15 and 23 showed interesting PDE 7 potency (around 10
µM); although not statistically significant, a trend toward selectivity with respect to PDE 3
and PDE 4 was obtained. Benzothiadiazine 16, although less potent at PDE 7 (IC50 ) 25 µM),
also showed a trend of selectivity toward PDE 3 and PDE 4. These compounds are considered
the best leads for further optimization.
In tr od u ction
tissues and throughout embryonic development the
translation or stability of PDE 7 protein may be highly
The interest in identifying isoenzyme-selective phos-
phodiesterase (PDE) inhibitors has increased in the last
years. PDEs play a critical role in various biological
processes by hydrolyzing the key second messengers
adenosine and guanosine 3′,5′-cyclic monophosphate
nucleotides (cAMP and cGMP, respectively) to the
corresponding 5′-monophosphate nucleotides. Therefore,
inhibition of PDE activity produces an increase of cAMP
and cGMP intracellular levels that activates specific
protein phosphorylation pathways involved in a variety
of functional responses.1
At least nine families of mammalian PDEs have been
described, based on substrate specificity, affinity, sen-
sitivity to cofactors, sequence similarity, and sensitivity
to inhibitory drugs.2-7 The seventh phosphodiesterase
family is a cAMP-specific PDE, encoded by a single gene.
The biochemical and pharmacological characterization
of PDE 7 showed a high-affinity cAMP-specific PDE (Km
) 0.2 µM) that was not affected by cGMP and well-
known potent selective PDE isoenzyme inhibitors.8
Subsequently, the presence of a PDE 7-like activity was
described in human T-cell lines but absent in cell lines
derived from B-cells.9
regulated.8,10,11
Considering the restricted tissue expression of PDE
7, increasing cAMP levels by selective PDE 7 inhibition
appears to be a potentially promising approach to
specifically block T-cell-mediated immune responses.
Moreover, various experimental studies have clearly
demonstrated that elevation of intracellular cAMP
levels can modulate inflammatory and immunological
processes.12,13 This selective approach could presumably
be devoid of the secondary effects that plague the use
of inhibitors selective for other isoenzymes more widely
distributed (e.g. PDE 3, PDE 4). However, until now no
selective PDE 7 inhibitors have been described.
Recently a functional role of PDE 7 in T-cell activation
has been described, for the first time;14 therefore,
selective inhibitors of PDE 7 could be a new strategy to
treat T-cell-related diseases. On the other hand, the
identification of selective inhibitors of the PDE 7 iso-
enzyme could help to understand the functional role of
this cAMP-specific PDE.
Taking into account this background and continuing
with our work in the field of fused pyrimidines as PDE
inhibitors,15,16 we describe here the synthesis of benzyl
derivatives of 2,1,3-benzo- and benzothieno[3,2-a]thia-
diazine dioxides which have been proved to be the first
heterocyclic inhibitors of PDE 7. Their synthesis, bio-
logical evaluation, and structure-activity relationships
will be discussed.
PDE 7 mRNA is highly expressed in skeletal muscle
and detectable in heart, spleen, B- and T-lymphocytes,
kidney, brain, uterus, and pancreas, whereas PDE 7
activity or protein is only detected in T-cell lines and
several fetal tissues, suggesting that in the rest of the
* To whom correspondence should be addressed. Tel: 91-562-2900.
Fax: 91-564-4853. E-mail: iqmam06@pinar2.csic.es.
† Instituto de Qu´ımica Me´dica.
Ch em istr y
The benzothienothiadiazine dioxide ring 1 was pre-
pared by the sequential condensation of 2-nitrobenzoni-
‡ Almirall Prodesfarma S.A.
§ Instituto de Biolog´ıa Molecular de Barcelona.
10.1021/jm990382n CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/02/2000

684 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Mart´ınez et al.
Sch em e 1a
Sch em e 3a
a
Reagents: (i) DMF/KOH; (ii) CH3C6H5/rt; (iii) NaOH (1 N).
Sch em e 2a
a
Reagents: (i) H2O/NaHCO3/R-C6H4-CH2X; (ii) DMF/NaH/3,4-
diCl-C6H3-CH2X; (iii) DMF/NaH/2-Ph-C6H4-CH2X.
Ch a r t 1. Benzothiadiazine Derivatives Included in the
Biological Screening
thiadiazine 2 with the corresponding alkyl halides in
aqueous bicarbonate leads to the monobenzothiadia-
zines 18-23. When biphenylmethyl bromide is em-
ployed, stronger conditions were needed, using sodium
hydride as base in DMF. As a result, complex mixtures
of compounds 24-26, including O-substituted deriva-
tives, were obtained. All the isomers could be separated
by column and centrifugal circular thin-layer chroma-
tography (Scheme 3).
The structures of all new compounds were elucidated
according to analytical and spectroscopic data. Un-
equivocal assignment of all chemical shifts (1H and 13C
NMR) was done using bidimensional experimentals
such COSY or HMQC for one-bond correlation. The site
of alkylation was determined from 13C chemical shift
displacements and sequences of HMBC for long distance
proton/carbon correlation experiments.
a
Reagents: (i) H2O/NaHCO3/R-C6H4-CH2X; (ii) DMF/NaHCO3/
R-C6H4-COCH2X; (iii) DMF/NaHCO3/Ph-C6H4-COX.
trile and methyl thioglycolate17 followed by reaction
with sulfamoyl chloride and subsequent cyclization in
basic medium (Scheme 1).
Benzothienothiadiazine derivatives 3-14 were pre-
pared by reaction of the benzothienothiadiazine 1 with
the appropriate benzyl derivative in aqueous bicarbon-
ate (Scheme 2). When benzyl bromide and 2-biphenyl-
methyl bromide were used, mixtures of monoalkyl and
dialkyl compounds were obtained which could be sepa-
rated by silica gel column chromatography (Scheme 2).
In the case of compounds 15 and 16, the reaction was
achieved in DMF using an excess of sodium bicarbonate
and acetophenone halide derivatives as reactants
(Scheme 2). Derivative 17 was obtained by reaction of
benzothienothiadiazine 1 with 4-biphenylcarbonyl bro-
mide in DMF and sodium bicarbonate.
The benzothiadiazine dioxide ring 2 was obtained in
two steps following the Cohen and Klarberg procedure18
starting from methyl anthranylate and sulfamoyl chlo-
ride.
Biologica l Resu lts a n d Discu ssion
In the present work, benzothieno- and benzothiadi-
azines dioxides 3-26 were synthesized and together
with the previously reported O-derivative 2719 and N3-
derivatives 28 and 2920 (Chart 1) were tested for their
inhibitory potencies against human recombinant PDE
7 expressed in S. cerevisiae as described in the Experi-
mental Section. In this expression system the only cyclic
nucleotide hydrolyzing activity present in cell extracts
Benzyl derivatives of benzothiadiazine were obtained
following a similar procedure to previous benzothieno-
thiadiazine compounds. Thus, the reaction of benzo-

First PDE 7 Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 685
Ta ble 1. Biological Activity (PDE 7, PDE 3, and PDE 4
Inhibition) of Benzothieno- and Benzothiadiazine Dioxides
1-29a
versus 17). When the linker is a carbonylmethyl frag-
ment (compound 15) results in PDE 7 are similar to that
of methylene, but there is an increase in selectivity.
Substituents on the phenyl ring of the benzyl moiety
increase the PDE 7 potency in both benzothienothia-
diazines and benzothiadiazines, when compared to the
unsubstituted compounds (3 and 18, respectively).
Introduction of a phenyl ring (compounds 4, 7, 8, and
24) provides good results independently of the substitu-
tion position. With respect to the chloro substitution,
the ortho position (compounds 11 and 21) provides
better results than the meta or para position (com-
pounds 12, 19, and 20). However, surprisingly, the 3,4-
disubstituted compound (23) showed higher potency and
increased selectivity.
The nature of the heterocyclic framework slightly
influences the PDE 7 inhibition. The benzothienothia-
diazine moiety displays a slight increase in the inhibi-
tory enzyme potency when compared to the benzothia-
diazine (compounds 4 versus 24, 11 versus 21, etc.),
although the benzothiadiazine derivatives showed better
selectivity.
compd
PDE 7
PDE 3
PDE 4
1b
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18b
19
20
21
22
23
24
25
26
27
28b
29b
0%
14%
14
2%
0%
15
13
29%
22
(0, 0)
31%
23%
11
18%
13%
9
8
18
12
18
35%
36%
43%
27
15%
11%
16%
6%
8%
4%
6%
24
(25, 37)
0%
11%
36
0%
0%
14
16
18%
26
28%
14%
5%
23%
30
2%
1%
7%
8%
8%
9%
7%
19
(0, 0)
(7, 21)
(9, 22)
(0, 10)
(0, 0)
(14, 32)
(10, 12)
(0, 37)
(0, 33)
(7, 10)
(6, 11)
(12, 27)
(10, 15)
(14, 23)
(18, 54)
(20, 52)
(28, 58)
(23, 32)
(6, 25)
(0, 36)
(2, 31)
(0, 27)
(0, 21)
(0, 21)
(1, 11)
(17, 34)
(16, 56)
(0, 0)
(0, 25)
(24, 53)
(0, 0)
(0, 0)
(6, 35)
(10, 17)
(24, 35)
(11, 43)
(15, 53)
(18, 38)
(28, 36)
(11, 33)
(6, 22)
(18, 34)
(0, 7)
(14, 44)
(12, 36)
(24, 45)
(27, 56)
(17, 26)
(3, 22)
(17, 25)
(0, 22)
(0, 6)
(11, 18)
(13, 19)
(6, 29)
(13, 51)
(24, 32)
(0, 28)
(0, 25)
(13, 33)
(25, 36)
(0, 8)
(0, 15)
(2, 13)
(2, 15)
(5, 12)
(0, 29)
(0, 21)
(11, 34)
(11, 38)
(0, 2)
28
28%
32%
22%
11
25
2%
29%
24%
35%
41%
21%
8
21
30
20
0%
0%
40%
10%
0%
0%
0%
22%
0%
0%
0%
0%
17%
5%
4%
Con clu sion s
(0, 2)
(0, 2)
The synthesis of a new family of benzyl derivatives
of 2,1,3-benzo- and benzothieno[3,2-a]thiadiazine 2,2-
dioxides was achieved.
(37, 43)
(7, 13)
(0, 3)
(19, 24)
(0, 14)
(0, 10)
(10, 23)
(0, 11)
(0, 17)
a
The biological data revealed that these novel com-
pounds represent the first heterocyclic family of com-
pounds with PDE 7 inhibitory properties appearing to
be a new objective for the treatment of T-cell-dependent
disorders. Additionally, the fact that some of these
compounds also inhibit PDE 4 implies that this family
could be considered as new leads in the development of
drugs for asthma and other allergic airways pathologies.
Considering simultaneously inhibition of the three
different isoenzymes (PDE 7, PDE 4, and PDE 3)
compounds 15 and 23 showed interesting PDE 7 potency
(around 10 µM). Although not statistically significant,
a trend toward selectivity with respect to PDE 3 and
PDE 4 was obtained. Benzothiadiazine 16, although less
potent at PDE 7 (IC50 ) 25 µM), also showed a trend of
selectivity toward PDE 3 and PDE 4. These compounds
are considered the best leads for further optimization.
The inhibitory potency of the synthesized compounds on the
human PDE 7 activity was tested as described in the Experimental
Section. Isoenzyme selectivity was determined by testing their
inhibitory activity against guinea pig PDE 4 and PDE 3 enzymes.
Data are indicated as IC50 (µM) (95% confidence interval) or
percent inhibition (95% CI) at 20 µM (n ) 2-3). No statistically
significant differences were found by comparing PDE 7 values with
b
PDE 3 and PDE 4 data. Percent inhibition (95% CI) at 200 µM.
corresponded to human PDE 7. Isoenzyme selectivity
was obtained by comparing the IC50 values or percent
inhibition values of the compounds against PDE 7 with
their inhibitory activity against PDE 4 and PDE 3
(Table 1).
Some of the heterocyclic compounds evaluated exhib-
ited PDE 7 inhibitory properties (IC50 at micromolar
level), with concurrent activity, in some cases, at PDE
4 and PDE 3 (Table 1). These data revealed that benzyl
derivatives of 2,1,3-benzo- and benzothieno[3,2-a]thia-
diazine 2,2-dioxides represent the first described het-
erocyclic family of compounds with PDE 7 inhibitory
properties. The fact that some of these compounds also
inhibit PDE 4 implies that this family could be consid-
ered as new leads in the development of drugs for
asthma and other allergic airways pathologies.
Preliminary structure-activity relationships showed
that monosubstitution in the thiadiazine moiety is
required for activity against PDE 7. Dibenzyl deriva-
tives assayed (compounds 5, 6, 25, and 26) completely
lacked activity. Moreover, lack of activity found in
modified acyclonucleoside derivatives 28 and 29 should
indicate substituted benzyl compounds were required
for activity. On the other hand, monosubstitution should
be on the nitrogen atom as only residual activity was
observed in the O-substituted compound 27.
Exp er im en ta l Section
Biologica l Meth od s. Purification and characterization of
cyclic nucleotide PDE 3 and PDE 4 obtained from guinea pig
ventricular tissue were performed as described previously.21
Rolipram, SKF 94836, and zaprinast were synthesized at the
Medicinal Chemistry Department of Almirall Prodesfarma.
Reagents were purchased from commercial suppliers and used
as received.
Clon in g of h sP DE 7A cDNA. The cDNA corresponding
to hsPDE 7A was generated by ligating appropiate overlapping
fragments obtained by RT-PCR from total HeLa mRNA. The
oligonucleotides used, designed from the published sequence
information,9 were QD1 and QR1 to amplify from position 698
to position 1535 and QD2 and QR2 from position 33 to position
923, to obtain hsPDE 7A. A StyI in position 765 and XhoI
restriction sites from the polylinker of pBS were used to join
both fragments, which were sequenced twice in both directions
to verify the ORF and the absence of changes according to the
published sequence.8
The link between the heterocyclic ring and the lipo-
philic N-substituent (phenyl moiety) is important for
PDE 7 inhibition, with the methylene group being a
better spacer than the carbonyl moiety (compounds 8
hsPDE 7A was cloned into the yeast expression vector pYes2
(Invitrogen). To avoid the protein from folding improperly we
fused, by PCR, the influenza hemoglutinin epitope HA122,11

686 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Mart´ınez et al.
37 °C. The cloned PDE 7 activity was pharmacologically
characterized by using different isoenzyme selective inhibitors.
This PDE activity was cAMP-specific and was not inhibited
by 200 µM rolipram, a selective inhibitor of PDE 4, 200 µM
SKF 94836, a selective inhibitor of PDE 3, and 200 µM
zaprinast, a selective inhibitor of PDE 5. Ca2+/CaM, the
activator of CaM-PDE (PDE 1), and cGMP, the activator of
cGMP-stimulated PDE (PDE 2) and inhibitor of cGMP-
inhibited PDE (PDE 3), did not modify the cloned PDE 7
activity.
PDE 3 and PDE 4 enzyme assays were performed in 96-
well microtiter plates using a BIOMEK 2000 workstation
(Beckman). For the automatic assay, the incubation mixtures
(120 mL/well) contained 28 mM Tris-HCl (pH 8), 7 mM MgCl2,
5 mM â-mercaptoethanol, 0.19 µM AMPc, 0.16 mg/mL snake
venom 5′-nucleotidase, 0.06 µM [3H]AMPc (16 Ci/mmol), 0.1
g/L bovine serum albumin. Reaction was started by the
addition of 20 µL of the diluted enzyme preparation and
incubated for 30 min at room temperature. The incubation was
terminated by transferring 60 µL of the reaction mixture into
a Millipore 96-well filter plate (MAHVN4550) containing 200
µL of a 50% QAE-Sephadex A-25 mixture in 20 mM CAPS,
pH 10. The samples were collected by filtration into a 96-well
plate (1450-401, Wallac) containing 150 µL of liquid scintil-
lation cocktail (Supermix, Wallac) per well by using a vacuum
manifold (Millipore). The total radioactivity was measured
using a Wallac MicroBeta scintillation counter.
at the N-terminus of the protein. TAG‚PDE 7 (CT ACC GCT
CGA GCC ATG TAT CCA TAC GAT GTT CCA GAT TAT GCT
AGC TTA GGT GGT CCG GCG TAT TCA ATG GAA GTG)
was the primer used to tag protein; this primer contains a XhoI
restriction site, an ATG, the HA1 tag, a GGP linker, and 17
bases of hsPDE 7A cDNA, including the methionine. We
amplified the hsPDE 7A tag using pBS(hsPDE 7A) as tem-
plate, the TAG‚PDE 7 and QR1 primers, and Vent polymerase,
under the following conditions: 951C 5 min, 33 cycles of 951C
1 min, 551C 30 s, and 721C 90 s. We cloned the PCR product
into the pBS(hsPDE 7A) in XhoI-StyI restriction sites. Finally
we cloned the hsPDE 7A tag into pYes using XhoI-XbaI
restriction sites.
Disr u p tion of S. cer evisia e P DE Gen es. The two yeast
PDE genes, PDE1 and PDE2, were obtained from the yeast
genome by PCR. The amplified fragments were cloned into
pUC18. Large deletions of both genes (positions 577-1744 for
the PDE1 gene and positions 925-1325 for the PDE2 gene)
were originated by amplifying these constructs with primers
Pde1‚AU and Pde1‚AL (PDE1) and Pde2‚AU and Pde2‚AL
(PDE2). The missing sequences were then replaced by either
the LEU2 gene (PDE1) or the TRP1 gene (PDE2), obtained
from plasmidsYDp-L and YDp-W, respectively.23 The ampli-
fication products of these disrupting plasmids with oligos Pde1‚
U and Pde1‚L and Pde2‚U and Pde2‚L were ultimately used
to disrupt both PDE genes on the protease-deficient strain
BJ 5459,24 following standard procedures.25 The double knock
out (dPDEKO) was confirmed by the absence of PDE activity.
The inhibition effect of each drug on the PDE activities was
evaluated using 4-5 different concentrations with duplicate
determinations. IC50 values were obtained by nonlinear re-
gression by use of SAS on a DEC AXP computer. Drugs were
dissolved in dimethyl sulfoxide (DMSO) and the effects of this
solvent on the enzyme activities were taken into consideration
in the calculations.
Ch em ica l P r oced u r es. Melting points were determined
with a Reichert-J ung Thermovar apparatus and are uncor-
rected. Flash column chomatography was carried out at
medium pressure using silica gel (E. Merck, grade 60, particle
size 0.040-0.063 mm, 230-240 mesh ASTM) with the indi-
cated solvent as eluent. 1H NMR spectra were obtained on
Varian XL-300 and Gemini-200 spectrometers working at 300
and 200 MHz, respectively. Typical spectral parameters were
spectral width 10 ppm, pulse width 9 µs (57°), data size 32 K.
13C NMR experiments were carried out on the Varian Gemini-
200 spectrometer operating at 50 MHz. The acquisiton pa-
rameters were spectral width 16 kHz, acquisition time 0.99 s,
pulse width 9 µs (57°), data size 32 K. Chemical shifts are
reported in δ values (ppm) relative to internal Me4Si and J
values are reported in hertz. Elemental analyses were per-
formed by the analytical departement at C.N.Q.O. (CSIC) and
the results obtained were within (0.4% of the theoretical
values.
Ben zoth ien o[3,2-a ]-1,2,6-th ia d ia zin -4(1H,3H)-on e 2,2-
Dioxid e (1). Sulfamoyl chloride (3.46 g, 30 mmol) was added
to a solution of methyl 3-aminobenzo[b]thiophene-2-carboxy-
late17 (2.07 g, 10 mmol) in toluene (30 mL). The reaction
mixture was heated at 60 °C for 4 h. After the mixture was
cooled to room temperature, the precipitated solid was collected
by filtration and dissolved in 1 N NaOH (25 mL). The solution
was stirred for 4 h at room temperature. After that time, the
mixture was made acidic with concentrated HCl and the
precipitate collected by filtration. Compound 1 was obtained
(1.16 g, 45%) as a white solid: mp 234-236 °C.
Gen er a l P r oced u r e for th e Syn th esis of Ben zyl De-
r iva tives of Ben zoth ien oth ia d ia zin e. The corresponding
benzyl halide derivative (1 mmol) was added to a solution of
benzothienothiadiazine dioxide 1 (1 mmol) in a sodium bicar-
bonate aqueous solution (10 mL). The reaction mixture was
stirred in the indicated conditions in each case. After that, the
aqueous phase was acidified using concentrated HCl and
extracted with AcOEt (5 × 10 mL). The combined organic
phases were dried over sodium sulfate and the solvent was
eliminated under reduced pressure. The residue was chro-
E xp r ession of h P DE 7A cDNA in t h e d P DE KO S.
cer evisia e Str a in . The construct described above, hsPDE 7A
tag into pYes, was transformed into the dPDEKO yeast strain
by lithium acetate precipitation.26 Positive clones were selected
in SD medium agar plates. The colonies were grown in YPD
until 0.8 OD595, then were spun down, and resuspended in
SD medium supplemented with galactose as carbon source.
The yeast were collected and powered under liquid nitrogen
to prepare protein extracts using standard protocols.
S. cer evisia e str a in s u sed in th is stu d y: BJ 5459, MATa
pep4::HIS3 pbr1D1.6R ura3-52 trp1 lys2-801 leu2D1 his3D200
can1 GAL; dPdeKO, MATa pep4::HIS3 pbr1D1.6R ura3-52
trp1 lys2-801 leu2D1 his3D200 can1 GAL; pde1::LEU2
pde2::TRP1.
Mea su r em en t of P DE Activities. Cyclic nucleotide PDE
7 activity from yeast extracts was measured by a two-step
procedure according to Thompson and Strada27 at a cAMP
concentration of 0.25 µM. The incubations were performed at

First PDE 7 Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 687
matographed on silica gel column using as eluents mixtures
of solvents in the portions indicated.
Conditions: room temperature, 36 h. Purification: CH2Cl2:
MeOH (20:1); yield 0.10 g (28%); mp 228-230 °C; 1H NMR
(DMSO-d6) δ 5.19 (s, 2H, N-CH2-Ph), 7.26-7.98 (m, 8H, Ar-
H); 13C NMR (DMSO-d6) δ 51.55 (N-CH2-Ph), 109.46 (C-4a),
117.02, 122.24, 123.95, 124.67, 126.78, 127.84, 127.98, 132.27,
132.91, 133.48, 133.56, 137.75, 139.47 (Ar-C), 142.41 (C-9b),
164.53 (C-4). Anal. (C17H11N3S2O3) C, H, N, S.
1-[(2-Nitr oph en yl)m eth yl]ben zoth ien o[3,2-a ]-1,2,6-th ia-
d ia zin -4(3H)-on e 2,2-Dioxid e (10). Reagents: benzothieno-
thiadiazine dioxide 1 (0.13 g, 0.5 mmol), H2O/NaHCO3 (10 mL),
2-nitrophenylmethyl bromide (0.11 g, 0.5 mmol). Conditions:
room temperature, 12 h. Purification: CH2Cl2:MeOH (20:1);
yield 0.04 g (26%); mp 245-246 °C; 1H NMR (DMSO-d6) δ 5.29
(s, 2H, N-CH2-Ph), 7.20-8.17 (m, 8H, Ar-H); 13C NMR
(DMSO-d6) δ 50.65 (N-CH2-Ph), 121.23 (C-4a), 122.10,
123.90, 124.60, 124.87, 126.65, 128.43, 129.24, 132.02, 134.12,
134.63, 137.80, 139.61, (Ar-C), 146.93 (C-9b), 164.23 (C-4).
Anal. (C16H11N3S2O5) C, H, N, S.
1-[(2-Ch lor op h en yl)m et h yl]b en zot h ien o[3,2-a ]-1,2,6-
t h ia d ia zin -4(3H)-on e 2,2-Dioxid e (11). Reagents: benzo-
thienothiadiazine dioxide 1 (0.06 g, 0.25 mmol), H2O/NaHCO3
(10 mL), 2-chlorophenylmethyl bromide (0.08 g, 0.25 mmol).
Conditions: reflux, 12 h. Purification: CH2Cl2:MeOH (9:1);
yield 0.04 g (40%); mp 205-206 °C; 1H NMR (DMSO-d6) δ 5.02
(s, 2H, N-CH2-Ph), 7.23-7.87 (m, 8H, Ar-H); 13C NMR
(DMSO-d6) δ 50.97 (N-CH2-Ph), 100.00 (C-4a), 122.15,
123.97, 124.57, 126.70, 127.43, 128.61, 128.80, 129.19, 130.74,
132.16, 136.38, 137.86 (Ar-C), 139.73 (C-9b), 164.38 (C-4).
Anal. (C16H11ClN2S2O3) C, H, N, S.
1-[(4-Ch lor op h en yl)m et h yl]b en zot h ien o[3,2-a ]-1,2,6-
t h ia d ia zin -4(3H)-on e 2,2-Dioxid e (12). Reagents: benzo-
thienothiadiazine dioxide 1 (0.06 g, 0.25 mmol), H2O/NaHCO3
(10 mL), 4-chlorophenylmethyl chloride (0.08 g, 0.25 mmol).
Conditions: reflux, 12 h. Purification: CH2Cl2:MeOH (20:1);
yield 0.04 g (48%); mp 244-245 °C; 1H NMR (DMSO-d6) δ 5.07
(s, 2H, N-CH2-Ph), 7.31-7.46 (m, 6H, Ar-H), 7.73 (d, 1H, J
) 8.0, Ar-H), 7.92 (d, 1H, J ) 7.7, Ar-H); 13C NMR (DMSO-
d6) δ 51.98 (N-CH2-Ph), 122.95 (C-4a), 123.83, 124.54, 126.60,
128.17, 128.98, 131.59, 132.46, 137.35, 137.64, 139.69 (Ar-
C), 144.26 (C-9b), 164.47 (C-4). Anal. (C16H11ClN2S2O3) C, H,
N, S.
1-Be n zylb e n zot h ie n o[3,2-a ]-1,2,6-t h ia d ia zin -4(3H )-
on e 2,2-Dioxid e (3) a n d 1,3-Diben zylben zoth ien o[3,2-a ]-
1,2,6-t h ia d ia zin -4-on e 2,2-Dioxid e (5). Reagents: benzo-
thienothiadiazine dioxide 1 (0.25 g, 1 mmol), H2O/NaHCO3 (10
mL), benzyl bromide (0.17 g, 1 mmol). Conditions: room
temperature, 12 h. Purification: CH2Cl2:MeOH (20:1), first
fraction, yield of dibenzyl derivative 5; 0.19 g (4%); mp 120-
121 °C; 1H NMR (DMSO-d6) δ 4.79 (s, 2H, N1-CH2-Ph), 4.99
(s, 2H, N3-CH2-Ph), 6.92 (d, 1H, J ) 7.1, Ar-H), 7.07-7.60
(m, 11H, Ar-H), 7.84 (d, 1H, J ) 6.6, Ar-H), 7.90 (d, 1H, J )
7.9, Ar-H); 13C NMR (DMSO-d6) δ 46.54 (N3-CH2-Ph), 56.95
(N1-CH2-Ph), 111.72 (C-4a), 123.12, 123.98, 125.79, 128.08,
128.61, 128.92, 129.04 129.59, 129.83, 130.30, 131.81, 132.54,
135.45, 137.27 (Ar-C), 140.17 (C-9b), 158.47 (C-4). Anal.
(C23H18N2S2O3) C, H, N, S.
Purification: second fraction, CH2Cl2:MeOH (20:1), yield of
monobenzyl derivative 3; 0.28 g (81%); mp 219-221 °C; 1H
NMR (DMSO-d6) δ 5.12 (s, 2H, N-CH2-Ph), 7.14-7.43 (m,
7H, Ar-H), 7.75 (d, 1H, J ) 8.1, Ar-H), 7.92 (d, 1H, J ) 8.1,
Ar-H); 13C NMR (DMSO-d6) δ 52.54 (N-CH2-Ph), 121.40 (C-
4a), 123.14, 123.83, 124.48, 126.64, 127.00, 128.26, 132.52,
137.73, 138.45 (Ar-C), 139.53 (C-9b), 164.93 (C-4). Anal.
(C16H12N2S2O3) C, H, N, S.
1-[(2-Bip h en yl)m eth yl]ben zoth ien o[3,2-a ]-1,2,6-th ia d i-
a zin -4(3H)-on e 2,2-Dioxid e (4) a n d 1,3-Di[(2-bip h en yl)-
m et h yl]b en zot h ien o[3,2-a ]-1,2,6-t h ia d ia zin -4-on e 2,2-
Dioxid e (6). Reagents: benzothienothiadiazine dioxide 1 (0.25
g, 1 mmol), H2O/NaHCO3 (10 mL), 2-biphenylmethyl bromide
(0.24 g, 1 mmol). Conditions: room temperature, 60 h.
Purification: CH2Cl2:MeOH (20:1), first fraction, yield of
compound 6; 0.13 g (13%); mp 143-144 °C; 1H NMR (DMSO-
d6) δ 4.86 (s, 2H, N1-CH2-Ph), 4.99 (s, 2H, N3-CH2-Ph),
6.87-7.80 (m, 22 H, Ar-H); 13C NMR (DMSO-d6) δ 44.75 (N3-
CH2-Ph), 54.35 (N1-CH2-Ph), 122.81 (C-4a), 123.47, 125.40,
126.54, 126.96, 127.29, 127.71, 128.17, 128.30, 128.40, 128.49,
129.31, 132.92, 138.37, 139.46, 140.05, 140.49, 141.11 (Ar-
C), 141.93 (C-9b), 158.91 (C-4). Anal. (C35H26N2S2O3) C, H, N,
S.
Purification: second fraction, yield of monobenzyl derivative
1
4; 0.07 g (18%); mp 212-215 °C; H NMR (DMSO-d6) δ 4.90
1-[(4-Cya n op h en yl)m et h yl]b en zot h ien o[3,2-a ]-1,2,6-
t h ia d ia zin -4(3H)-on e 2,2-Dioxid e (13). Reagents: benzo-
thienothiadiazine dioxide 1 (0.06 g, 0.25 mmol), H2O/NaHCO3
(10 mL), 4-cyanophenylmethyl bromide (0.10 g, 0.25 mmol).
Conditions: reflux, 12 h. Purification: CH2Cl2:MeOH (9:1);
yield 0.03 g (29%); mp 255-256 °C; 1H NMR (DMSO-d6) δ 5.14
(s, 2H, N-CH2-Ph), 7.27-7.98 (m, 8H, Ar-H); 13C NMR
(DMSO-d6) δ 52.42 (N-CH2-Ph), 109.82 (C-4a), 118.85,
122.77, 123.44, 123.91, 124.62, 126.67, 127.90, 132.29, 132.64,
137.70, 139.13 (Ar-C), 144.55 (C-9b), 164.33 (C-4). Anal.
(C17H11N3S2O3) C, H, N, S.
(s, 2H, N-CH2-Ph), 7.16-7.44 (m, 11H, Ar-H), 7.80 (d, 1H,
J ) 8.1, Ar-H), 7.87 (d, 1H, J ) 7.3, Ar-H); 13C NMR (DMSO-
d6) δ 51.28 (N-CH2-Ph), 120.20 (C-4a), 122.47, 123.84, 124.35,
126.59, 127.07, 127.16, 127.43, 127.72, 128.44, 129.05, 129.70,
132.09, 136.12, 137.75, 139.76, 139.79 (Ar-C), 139.86 (C-9b),
164.47 (C-4). Anal. (C22H16N2S2O3) C, H, N, S.
1-[(3-Bip h en yl)m eth yl]ben zoth ien o[3,2-a ]-1,2,6-th ia d i-
a zin -4(3H)-on e 2,2-Dioxid e (7). Reagents: benzothienothia-
diazine dioxide 1 (0.06 g, 0.25 mmol), H2O/NaHCO3 (10 mL),
3-biphenylmethyl bromide (0.12 g, 0.25 mmol). Conditions:
reflux, 12 h. Purification: CH2Cl2:MeOH (10:1); yield 0.02 g
(25%); mp 220-222 °C; 1H NMR (DMSO-d6) δ 5.19 (s, 2H,
N-CH2-Ph), 7.26-7.94 (m, 13H, Ar-H); 13C NMR (DMSO-
d6) δ 52.60 (N-CH2-Ph), 121.35 (C-4a), 123.08, 123.76, 124.45,
125.32, 125.52, 126.08, 126.53, 126.59, 127.41, 128.79, 128.86,
132.59, 137.64, 139.00, 139.35, 139.96 (Ar-C), 140.05 (C-9b),
164.54 (C-4). Anal. (C22H16N2S2O3) C, H, N, S.
1-[(4-Bip h en yl)m eth yl]ben zoth ien o[3,2-a ]-1,2,6-th ia d i-
a zin -4(3H)-on e 2,2-Dioxid e (8). Reagents: benzothienothia-
diazine dioxide 1 (0.06 g, 0.25 mmol), H2O/NaHCO3 (10 mL),
4-biphenylmethyl chloride (0.10 g, 0.25 mmol). Conditions:
reflux, 12 h. Purification: CH2Cl2:MeOH (10:1); yield 0.03 g
(26%); mp 227-229 °C; 1H NMR (DMSO-d6) δ 5.14 (s, 2H,
N-CH2-Ph), 7.28-7.94 (m, 13H, Ar-H); 13C NMR (DMSO-
d6) δ 52.17 (N-CH2-Ph), 123.01 (C-4a), 123.75, 124.39, 126.28,
126.43, 126.50, 126.61, 127.27, 127.54, 128.83, 132.54, 137.64,
137.81, 138.64, 139.28 (Ar-C), 139.71 (C-9b), 164.37 (C-4).
Anal. (C22H16N2S2O3) C, H, N, S.
1-[(4-Meth oxyp h en yl)m eth yl]ben zoth ien o[3,2-a ]-1,2,6-
t h ia d ia zin -4(3H)-on e 2,2-Dioxid e (14). Reagents: benzo-
thienothiadiazine dioxide 1 (0.06 g, 0.25 mmol), H2O/NaHCO3
(10 mL), 4-methoxyphenylmethyl bromide (0.05 g, 0.25 mmol).
Conditions: reflux, 12 h. Purification: CH2Cl2:MeOH (10:1);
1
yield 0.01 g (13%); mp 222-225 °C; H NMR (CDCl3) δ 3.68
(s, 3H, OCH3), 4.88 (s, 2H, N-CH2-Ph), 6.67 (d, 2H, J ) 7.7,
Ar-H), 6.82 (d, 2H, J ) 7.7, Ar-H), 7.53 (m, 3H, J ) 7.7, Ar-
H), 7.89 (t, 1H, J ) 8.2, Ar-H); 13C NMR (CDCl3) δ 52.22 (N-
CH2-Ph), 56.87 (OCH3), 113.97 (C-4a), 123.48, 124.06, 124.25,
124.33, 125.94, 126.60, 128.93, 130.36, 131.96 (Ar-C), 139.66
(C-9b), 159.27 (C-4), 166.08 (CdO). Anal. (C17H14N2S2O4) C,
H, N, S.
1-[(4-Met h oxyp h en yl)ca r b on ylm et h yl]b en zot h ien o-
[3,2-a ]-1,2,6-th ia d ia zin -4(3H)-on e 2,2-Dioxid e (15). To a
solution of benzothienothiadiazine dioxide 1 (0.06 g, 0.25
mmol) and sodium bicarbonate in excess in DMF (15 mL) was
added 2-bromo-4′-methoxyacetophenone (0.12 g, 0.25 mmol).
The reaction mixture was stirred at room temperature for 48
h. After that time, the reaction mixture was put over water
and extracted with AcOEt (5 × 10 mL). The organic phase was
dried over sodium sulfate and the solvent eliminated under
1-[(2-Cya n op h en yl)m et h yl]b en zot h ien o[3,2-a ]-1,2,6-
t h ia d ia zin -4(3H )-on e 2,2-Dioxid e (9). Reagents: ben-
zothienothiadiazine dioxide 1 (0.25 g, 1 mmol), H2O/NaHCO3
(10 mL), 2-cyanophenylmethyl bromide (0.19 g, 1 mmol).

688 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Mart´ınez et al.
reduced pressure. The residue was purified by silica gel column
chromatography CH2Cl2:MeOH (20:1) yielding 0.03 g (36%) of
derivative 15: mp 217-218 °C; 1H NMR (DMSO-d6) δ 3.84 (s,
3H, OCH3), 5.05 (s, 2H, N-CH2-COPh), 7.04 (d, 1H, J ) 8.9,
Ar-H), 7.42 (m, 5H, Ar-H), 7.82 (d, 1H, J ) 8.9, Ar-H), 8.02
(d, 1H, Ar-H); 13C NMR (DMSO-d6) δ 41.30 (N-CH2-Ph),
55.62 (OCH3), 113.97 (C-4a), 121.37, 123.27, 123.87, 130.19,
130.29, 130.58, 139.33 (Ar-C), 144.34 (C-9b), 165.39 (C-4),
167.32 (CdO). Anal. (C18H14N2S2O5) C, H, N, S.
1-[(4-Biph en yl)car bon ylm eth yl]ben zoth ien o[3,2-a ]-1,2,6-
th ia d ia zin -4(3H)-on e 2,2-Dioxid e (16). Following the above
procedure a solution of benzothienothiadiazine dioxide 1 (0.06
g, 0.25 mmol) and 2-bromo-4′-phenylacetophenone (0.07 g, 0.25
mmol) in DMF was stirred for 48 h. at room temperature. After
workup, compound 16 was obtained (0.04 g, 32%): mp 249-
251 °C; 1H NMR (DMSO-d6) δ 4.09 (s, 2H, N-CH2-COPh),
7.36-8.14 (m, 13H, Ar-H); 13C NMR (DMSO-d6) δ 40.33 (N-
CH2-COPh), 123.27 (C-4a), 123.89, 126.95, 127.02, 127.93,
128.43, 128.54, 128.66, 128.98, 129.12, 133.94, 134.51, 138.87,
138.87, 139.38, 144.68 (Ar-C), 150.69 (C-9b), 160.57(C-4),
192.67 (CdO). Anal. (C23H16N2S2O4) C, H, N, S.
1-[(4-Bip h en yl)ca r bon yl]ben zoth ien o[3,2-a ]-1,2,6-th ia -
d ia zin -4(3H)-on e 2,2-Dioxid e (17). According to the method
described for derivative 15 a solution of benzothienothiadiazine
dioxide 1 (0.06 g, 0.25 mmol) and 2-bromo-4′-biphenylcarbonyl
(0.05 g, 0.25 mmol) in DMF was stirred for 48 h. After workup
compound 17 was obtained (0.01 g, 11%): mp 233-235 °C;
1H NMR (DMSO-d6) δ 7.39-8.02 (m, 13H, J ) 8.9, Ar-H);
13C NMR (DMSO-d6) δ 121.35 (C-4a), 126.56, 126.91, 127.05,
128.10, 129.05, 129.89, 137.75, 139.27, 144.32, 144.78, 161.47
(C-9b), 162.97(C-4), 187.07 (CdO). Anal. (C22H14N2S2O4) C, H,
N, S.
Gen er a l P r oced u r e for th e Syn th esis of Ben zyl De-
r iva tives of Ben zoth ia d ia zin e. To a solution of benzothia-
diazine dioxide 218 (1 mmol) in a sodium bicarbonate aqueous
solution (20 mL) was added the corresponding benzyl halide
(1.5 mmol). The reaction mixture was refluxed for 2 h. After
cooling to room temperature, the aqueous phase was washed
with CH2Cl2 (2 × 10 mL). The aqueous phase was cooled at 4
°C and the product was isolated and purified in each particular
case.
) 7.0, H-6), 7.28 (t, 1H, J
) 1.7, H-7), 7.85 (dd, 1H, H-5),
H5H7
7.22-7.41 (m, 4H, Ar-H); 13C NMR (DMSO-d6) δ 45.97 (CH2),
113.61 (C-8), 119.37 (C-4a), 119.40 (C-6), 125.59, 126.61,
126.89, 130.21, 133.05, 140.55 (Ar-C), 128.83 (C-5), 132.06
(C-7), 141.91 (C-8a), 165.54 (C-4). Anal. (C14H11N2O3SCl) C,
H, N, S.
1-[(2-Ch lor oph en yl)m eth yl]-2,1,3-ben zoth iadiazin -4(3H)-
on e 2,2-Dioxid e (21). Reagents: benzothiadiazine dioxide 2
(0.50 g, 2.5 mmol), 2-chlorophenylmethyl chloride (0.61 g, 3.7
mmol). Isolation: filtration of aqueous phase. Purification:
recrystallization from toluene/MeOH; yield 0.58 g (72%) as a
white solid; mp 292-294 °C; 1H NMR (DMSO-d6) δ 4.99 (s,
2H, N-CH2), 6.54 (dd, 1H, J
) 1.6, J
) 8.0, H-8),
H6H8
H7H8
6.92 (t, 1H, J
) 7.7, J
) 7.7, H-6), 7.29 (t, 1H, J
H5H6
H6H7
H5H7
) 1.7, H-7), 7.93 (dd, 1H, H-5), 7.24-7.32 (m, 4H, Ar-H); 13
C
NMR (DMSO-d6) δ 44.66 (CH2), 113.10 (C-8), 119.43 (C-6),
119.55 (C-4a), 127.22, 128.04, 128.64, 128.93, 131.35, 134.38
(Ar-C), 129.19 (C-5), 132.07 (C-7), 142.01 (C-8a), 165.56 (C-
4). Anal. (C14H11N2O3SCl) C, H, N, S.
1-[(4-Me t h ylp h e n yl)m e t h yl]-2,1,3-b e n zot h ia d ia zin -
4(3H)-on e 2,2-Dioxid e (22). Reagents: benzothiadiazine
dioxide 2 (0.32 g, 1.6 mmol), 4-methylphenylmethyl chloride
(0.34 g, 2.4 mmol). Isolation: filtration of aqueous phase.
Purification: filtration of acidified aqueous phase. Purifica-
tion: silica gel column chromatrography, eluent CH2Cl2/MeOH
(50:1); yield 0.20 g (50%) as a white solid; mp 290-292 °C; 1H
NMR (DMSO-d6) δ 2.24 (s, 3H, CH3),4.91 (s, 2H, N-CH2), 6.77
(dd, 1H, J
) 0.6, J
) 8.3, H-8), 6.88 (t, 1H, J
)
H6H8
H7H8
H5H6
7.8, J H6H7 ) 7.3, H-6), 7.26 (t, 1H, J H5H7 ) 1.7, H-7), 7.90 (dd,
1H, H-5), 7.07-7.30 (m, 4H, Ar-H); 13C NMR (DMSO-d6) δ
20.71 (CH3), 46.27 (CH2), 113.97 (C-8), 119.31 (C-6), 119.42
(C-4a), 126.93, 128.99, 136.05, 134.67 (Ar-C), 128.83 (C-5),
132.07 (C-7), 142.21 (C-8a), 166.01 (C-4). Anal. (C15H14N2O3S)
C, H, N, S.
1-[(3,4-Dich lor op h en yl)m eth yl]-2,1,3-ben zoth ia d ia zin -
4(3H)-on e 2,2-Dioxid e (23). Reagents: benzothiadiazine
dioxide 2 (0.26 g, 1.3 mmol), 3,4-dichlorophenylmethyl chloride
(0.40 g, 1.9 mmol). Isolation: filtration of aqueous phase.
Purification: recrystallization from toluene/MeOH; yield 0.36
1
g (76%) as a white solid; mp 250-252 °C; H NMR (DMSO-
d6) δ 4.96 (s, 2H, N-CH2), 6.74 (dd, 1H, J
) 0.8, J
)
H6H8
H7H8
8.1, H-8), 6.89 (t, 1H, J
) 7.7, J
) 7.2, H-6), 7.29 (t,
1-Ben zyl-2,1,3-ben zoth ia d ia zin -4(3H)-on e 2,2-Dioxid e
(18). Reagents: benzothiadiazine dioxide 2 (0.67 g, 3.3 mmol),
benzyl bromide (0.86 g, 4.9 mmol). Isolation: filtration of
aqueous phase. Purification: recrystallization from toluene/
MeOH; yield 0.80 g (81%) as a white solid; mp 288-290 °C;
1H NMR (DMSO-d6) δ 4.95 (s, 2H, N-CH2), 6.74 (dd, 1H, J
H5H6
H6H7
1H, J
) 1.6, H-7), 7.89 (dd, 1H, H-5), 7.41-7.67 (m, 3H,
H5H7
Ar-H); 13C NMR (DMSO-d6) δ 45.42 (CH2), 113.08 (C-8),
119.49 (C-6), 119.63 (C-4a), 127.40, 128.95, 129.48, 131.01,
139.42 (Ar-C), 130.64 (C-5), 132.08 (C-7), 141.81 (C-8a), 165.57
(C-4). Anal. (C14H10N2O3SCl2) C, H, N, S.
) 1.0, J
) 7.7, H-8), 6.87 (t, 1H, J
) 7.7, J
H6H8
H7H8
H5H6
H6H7
1-[(2-Bip h en yl)m et h yl]-2,1,3-b en zot h ia d ia zin -4(3H )-
on e 2,2-Dioxid e (24), 1,3-Di[(2-bip h en yl)m eth yl]-2,1,3-
b en zot h ia d ia zin -4-on e 2,2-Dioxid e (25), a n d 1-[(2-Bi-
p h en yl)m et h yl]-4-[(2-b ip h en yl)m et h yloxy]-2,1,3-b en zo-
th ia d ia zin e 2,2-Dioxid e (26). To a suspension of sodium
hydride (0.04 g, 1.6 mmol) in DMF were added benzothiadi-
azine dioxide 2 (0.21 g, 1 mmol) and 2-biphenylmethyl bromide
(0.38 g, 1.5 mmol). The reaction mixture was refluxed for 3 h.
The solvent was evaporated under reduced pressure. The
residue was dissolved in water and the aqueous phase was
extracted with CH2Cl2 (2 × 10 mL). The organic phase was
dried over sodium sulfate and the solvent evaporated under
reduced pressure. The residue was chromatographed on silica
gel column using CH2Cl2/MeOH (50:1) as eluent. From the first
fraction was isolated derivative 25: yield 0.06 g (11%) as a
) 7.4, H-6), 7.23 (t, 1H, J
) 1.7, H-7), 7.88 (dd, 1H, H-5),
H5H7
7.27-7.42 (m, 5H, Ar-H); 13C NMR (DMSO-d6) δ 46.47 (CH2),
113.80 (C-8), 119.28 (C-6), 119.70 (C-4a), 126.93, 126.98,
128.44, 137.88 (Ar-C), 128.85 (C-5), 131.93 (C-7), 142.22 (C-
8a), 165.91 (C-4). Anal. (C14H12N2O3S) C, H, N, S.
1-[(4-Ch lor oph en yl)m eth yl]-2,1,3-ben zoth iadiazin -4(3H)-
on e 2,2-Dioxid e (19). Reagents: benzothiadiazine dioxide 2
(0.40 g, 2.0 mmol), 4-chlorophenylmethyl chloride (0.60 g, 3.0
mmol). Isolation: filtration of aqueous phase. Purification:
recrystallization from toluene/MeOH; yield 0.41 g (63%) as a
white solid; mp 285-287 °C; 1H NMR (DMSO-d6) δ 4.93 (s,
2H, N-CH2), 6.75 (dd, 1H, J
) 1.0, J
) 8.2, H-8),
H6H8
H7H8
6.92 (t, 1H, J
) 7.7, J
) 7.4, H-6), 7.30 (t, 1H, J
H5H6
H6H7
H5H7
) 1.7, H-7), 7.86 (dd, 1H, H-5), 7.27-7.39 (m, 4H, Ar-H); 13
C
1
white solid; mp 67-68 °C; H NMR (CDCl3-d6) δ 4.87 (s, 2H,
NMR (DMSO-d6) δ 46.51 (CH2), 114.54 (C-8), 119.74 (C-4a),
N1-CH2), 5.02 (s, 2H, N3-CH2) 6.62 (dd, 1H, J
) 1.4, J
120.64 (C-6), 129.01, 129.37, 129.52, 132.35 (Ar-C), 133.17
H6H8
) 7.5, H-8), 8.04 (dd, 1H, J
) 7.6, J
) 1.5, H-5),
(C-5), 137.05 (C-7), 142.30 (C-8a), 167.29 (C-4). Anal. (C14H11
N2O3SCl) C, H, N, S.
-
H7H8
H5H6
H5H7
7.04-7.65 (m, 20H, Ar-H, H-6, H-7); 13C NMR (CDCl3-d6) δ
44.64 (N3-CH2), 52.44 (N1-CH2), 121.00 (C-8), 125.81 (C-6),
127.72 (C-4a), 126.23, 127.23, 127.30, 127.50, 127.62, 127.97,
128.11, 128.32, 128.39, 128.45, 128.96, 129.26, 130.07, 130.38,
131.84, 132.96, 139.74, 139.86, 141.21, 141.61 (Ar-C), 130.44
1-[(3-Ch lor oph en yl)m eth yl]-2,1,3-ben zoth iadiazin -4(3H)-
on e 2,2-Dioxid e (20). Reagents: benzothiadiazine dioxide 2
(0.21 g, 1.0 mmol), 3-chlorophenylmethyl chloride (0.26 g, 1.5
mmol). Isolation: filtration of acidified aqueous phase. Puri-
fication: silica gel column chromatrography, eluent CH2Cl2/
MeOH (50:1); yield 0.14 g (42%) as a white solid; mp 195-197
(C-5), 134.52 (C-7), 140.50 (C-8a), 162.23 (C-4). Anal. (C33H26
N2O3S) C, H, N, S.
From the second fraction was isolated derivative 26: yield
0.02 g (4%) as a white solid; mp 160-162 °C; 1H NMR (CDCl3-
-
1
°C; H NMR (DMSO-d6) δ 4.92 (s, 2H, N-CH2), 6.71 (dd, 1H,
J
H6H8 ) 1.2, J H7H8 ) 8.3, H-8), 6.86 (t, 1H, J H5H6 ) 7.9, J
H6H7

First PDE 7 Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 689
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d6) δ 5.11 (s, 2H, N1-CH2), 5.43 (s, 2H, O-CH2), 6.51 (dd, 1H,
J
H6H8 ) 1.2, J H7H8 ) 8.5, H-8), 6.98 (t, 1H, J H5H6 ) 7.9, J
H6H7
) 7.3, H-6), 7.38 (t, 1H, J
) 1.6, H-7), 7.78 (dd, 1H, H-5),
H5H7
7.24-7.61 (m, 18H, Ar-H); 13C NMR (CDCl3-d6) δ 47.11 (N1-
CH2), 68.88 (O-CH2), 112.17 (C-4a), 115.75 (C-8), 121.66 (C-
6), 126.82, 127.23, 127.53, 127.57, 127.68, 127.73, 127.79,
128.14, 128.42, 128.61, 128.96, 129.09, 130.04, 130.07, 131.65,
132.37, 140.10, 140.35, 142.57, 142.87 (Ar-C), 130.43 (C-5),
135.52 (C-7), 140.00 (C-8a), 165.41 (C-4). Anal. (C33H26N2O3S)
C, H, N, S.
From the third fraction was isolated derivative 24: yield 0.10
g (27%) as a syrup; 1H NMR (DMSO-d6) δ 4.77 (s, 2H, N-CH2),
6.26 (dd, 1H, J
) 7.8, J
) 1.0, J
) 7.8, H-8), 6.84 (t, 1H, J
H6H8
H7H8
) 7.3, H-6), 7.17 (t, 1H, J
) 1.7, H-7),
H5H6
H6H7
H5H7
7.85 (dd, 1H, H-5), 7.25-7.55 (m, 8H, Ar-H); 13C NMR
(DMSO-d6) δ 44.73 (CH2), 113.12 (C-8), 119.24 (C-6), 119.51
(C-4a), 126.62, 126.97, 127.54, 127.63, 128.56, 129.13, 134.32,
139.99, 140.44 (Ar-C), 128.84 (C-5), 131.88 (C-7), 142.08 (C-
8a), 165.57 (C-4). Anal. (C20H16N2O3S) C, H, N, S.
Ack n ow led gm en t. These investigations were sup-
ported by Laboratorios Almirall-Prodesfarma and by
Fondo de Investigaciones Sanitarias (Project No. FIS
98/253). One of us (C. Gil) acknowledges a grant from
Comunidad de Madrid. We also thank the excellent
technical assistance of M. C. Cabello, J . E. Diaz, and L.
Estrella.
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J M990382N