Synthesis, biological evaluation and molecular modelling studies on benzothiadiazine derivatives as PDE4 selective inhibitors

Article abstract of DOI:10.1016/j.bmc.2004.10.055

A series of 2,1,3- and 1,2,4-benzothiadiazine derivatives (BTDs) were synthesized and evaluated for their inhibitory activity versus enzymatic isoforms PDE3, PDE4 and PDE7. The compounds characterized by the 3,5-di-tert-butyl-4-hydroxybenzyl moiety at N1

Full text of DOI:10.1016/j.bmc.2004.10.055

Bioorganic & Medicinal Chemistry 13 (2005) 1393–1402
Synthesis, biological evaluation and molecular modelling studies on
benzothiadiazine derivatives as PDE4 selective inhibitors
Annalisa Tait,a,* Amedeo Luppi,a Armin Hatzelmann,b Paola Fossac and Luisa Mostic
a
`
Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Modena e Reggio Emilia, Via Campi 183, 41100 Modena, Italy
bALTANA Pharma AG, Byk-Gulden-Str. 2, D-78467 Konstanz, Germany
`
Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Genova, Viale Benedetto XV 3, 16132 Genova, Italy
c
Received 7 May 2004; accepted 26 October 2004
Available online 18 November 2004
Abstract—A series of 2,1,3- and 1,2,4-benzothiadiazine derivatives (BTDs) were synthesized and evaluated for their inhibitory activ-
ity versus enzymatic isoforms PDE3, PDE4and PDE7. The compounds characterized by the 3,5-di- tert-butyl-4-hydroxybenzyl moi-
ety at N1 position of 2,1,3-benzothiadiazine core (8, 13, 18), were found active and selective at micromolar level versus PDE4and
could be studied as new leads for the treatment of asthma and COPD (Chronic Obstructive Pulmonary Disease). The antioxidant
activity evaluation on the same compounds highlighted 13 as the most significative. Molecular modelling studies gave further
support to biological results and suggested targeted modifications so as to improve their potency.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
and anti-COPD properties and are currently in phase III
clinical trials.8–11
The function of many inflammatory cells is controlled
by cyclic nucleotides, such as cyclic AMP and cyclic
GMP, both of which are inactivated by phosphodiest-
erases (PDEs).1,2 This suggested the possibility that
PDE inhibitors may display beneficial anti-inflamma-
tory activity. In fact, the inhibition of PDE4and
PDE7, two of the known enzymatic isoforms, leads to
interruption of inflammatory process present in some
diseases such as asthma and COPD (Chronic Obstruc-
tive Pulmonary Disease), symptomatically characterized
by a repeated stridor and paroxysm due to the airway
contraction.3–7
It is known from the literature that benzothiadiazine
derivatives (BTDs) are heterocyclic inhibitors of PDE7
with concurrent inhibitory activity at PDE4and
PDE3.12,13
In the present study we synthesized N-3 mono (1–6) and
N-1,3 disubstituted (7–19) 2,1,3-BTDs and N-2 substi-
tuted 1,2,4-BTDs (20–25).
Some of the N-substituents here considered such as
methylphthalimide, nitrophenyl or 2,6-di-tert-butylphe-
nol were present in PDE4inhibitors such as nitra-
quazone, CDC-801 is a thalidomide analogue, and
benzoxazole derivatives recently patented by Eurocel-
tique (Chart 1).14 Then we evaluated the PDE4inhibi-
tory activity (pIC50) and isoenzyme selectivity versus
PDE3 and PDE7 of the new compounds.
Rolipram is the prototypic of the PDE4inhibitors but
nausea and emetic effects limit its therapeutic potential.
In the last years both Rolipram related and unrelated
second-generation PDE4inhibitors belonging to very
different chemical classes were developed. Roflumilast
and Cilomilast appear to display favourable anti-asthma
Moreover, it is known that the antioxidant 2,6-di-tert-
butylphenol moiety characterizes dual 5-lipoxygenase
(5-LO)/cyclooxygenase 2 (COX-2) inhibitors with anti-
inflammatory activities.15–18 COX catalysis involves
radical intermediates and a radical scavenging moiety
interferes with the cyclooxygenase reaction. To
Keywords: Benzothiadiazine; PDE4inhibitors; Antioxidant; Chronic
inflammatory diseases.
*
Corresponding author. Tel.: +39 059 2055133; fax: +39 059
371590; e-mail: tait.annalisa@unimore.it
0968-0896/$ - see front matter ꢀ 2004Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.10.055

1394
A. Tait et al. / Bioorg. Med. Chem. 13 (2005) 1393–1402
F
F
O
O
O
O
O
O
O
Cl
HN
Cl
O
N
H
NC
N
COOH
Rolipram
Roflumilast
Cilomilast
N
O
N
N
O
N
O
O
Cl
O
O
N
(H3C)3C
C(CH3)3
NO2
NH2
O
OH
Nitraquazone
CDC-801
US-6075016
Chart 1.
acquire preliminary information about the antioxidant
activity of synthesized compounds, the radical scaveng-
ing effect was determined in the presence of DPPH.
hyde in aqueous solution22 and subsequent Mitsunobu
alkylation of intermediate 26 (Scheme 2, Table 2).
NOESY experiments showed that the alkylation of
26 took place at N-2; thus, proton of NH in position-4
presents NOE effect with aromatic H-5.
Finally docking studies were carried out on the most ac-
tive compounds as further investigation on the molecu-
lar interactions that lead to the PDE4inhibition.
The structures of all new compounds were elucidated
from their analytical and spectroscopic data reported
in Experimental. Unequivocal assignments of 1H
NMR chemical shifts were done using bi-dimensional
experiments such as COSY, HMBC and HMQC.
2. Results
2.1. Chemistry
2.2. Biology
Scheme 1 outlines the synthesis of 2,1,3-BTDs. The N-3
substituted 2,1,3-BTDs (1–6) (Table 1) were obtained
starting from the suitable methyl 2-aminobenzoate and
sulfamoyl chloride.19,20 The intermediate N-sulfamoyl-
anthranilates were cyclized by sodium methoxide. By
alkylation with the appropriate alcohol, via Mitsunobu
reaction,21 the N,N-disubstituted derivatives (7–19)
(Table 1) were obtained.
Table 3 shows the in vitro inhibitory activity of BTDs
with respect to PDE3 from cytosol of human platelets
and to human recombinant enzymes PDE4D3 and
PDE7A1 from Baculovirus/Sf21 insect cell system. The
3-isobutyl-1-methylxanthine (IBMX) was taken as non-
selective reference compound. All data are expressed as
pIC50.
The 1,2,4-BTDs were prepared by condensation of 2-
amino-4-chlorobenzensulfonamide with chloroacetalde-
Among the tested compounds, only the 2,1,3-benzothia-
diazines with the 3,5-di-tert-butyl-4-hydroxybenzyl moi-
O
O
O
COCH3
O
S
O
R
COCH3
N
i
ii
R''
R''
+
R''
R
NH
Cl
O
S
O
SO2
N
H
1 - 6
NH
NHR
R
NH2
+
R' OH
O
N
SO2
R''
iii
N
R'
7 - 19
Scheme 1. Reagents and conditions: (i) toluene, Et3N, 80ꢁC, 1h; (ii) Na, CH3OH, 40ꢁC, 2h; (iii) TPP, DIAD, THF, N2, rt.

A. Tait et al. / Bioorg. Med. Chem. 13 (2005) 1393–1402
1395
Table 1. Compounds 1–19
O
R
N
R''
SO2
N
R'
Compd
R
R0
R00
H
130
2
331
4
Isopropyl
Propyl
Benzyl
H
H
H
H
H
H
6,7-Dimethoxy
H
7-Cl
Benzyl
Benzyl
5
6,7-Dimethoxy
H
632
7
2-Phenylethyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Propyl
4-OH-Benzyl
H
8
3,5-Di-tert-butyl-4-OH-benzyl
3-Nitro-benzyl
Cyclohexyl
H
9
H
10
1133
12
13
14
15
1632
1732
18
19
H
N-Methylphthalimide
Ethyl-pyrrolydine-2,5-dione
3,5-Di-tert-butyl-4-OH-benzyl
4-OH-Benzyl
H
H
6,7-Dimethoxy
Benzyl
Benzyl
H
3-Nitro-benzyl
2-Furylmethyl
H
Benzyl
Benzyl
H
N-Methylphthalimide
3,5-Di-tert-butyl-4-OH-benzyl
2-Nitro-benzyl
H
Benzyl
2-Phenylethyl
7-Cl
H
O
O
O O
SO2NH2
O
S
S
R
NH
CH2Cl
N
i
ii
+
HCCH2Cl
+ R OH
Cl
NH2
Cl
N
Cl
N
H
CH2Cl
H
26
20 - 25
Scheme 2. Reagents and conditions: (i) NH4Cl, DMF, H2O, 100ꢁC, 1h; (ii) TPP, DIAD, THF, N2, rt, overnight.
2.3. Molecular modelling studies
Table 2. Compounds 20–25
In order to further rationalize the biological results, a
molecular docking study was carried out on compounds
8, 13, 18, the most active as PDE4inhibitors among
those newly synthesized. The compounds were docked
into PDE4D catalytic domain23 (PDB entry 1MKD)
using the FlexX24 module as implemented in Sybyl.25
FlexX is a widely used docking algorithm in drug design
whose ability in predicting a conformation of the ligand
very close to its X-ray structure has been widely de-
scribed in literature.26
O
O
S
R
N
Cl
N
H
CH2Cl
Compd
R
20
21
22
23
24
25
4-OH-Benzyl
4-Pyridylmethyl
2-Nitro-benzyl
3-Nitro-benzyl
N-Methylphthalimide
N-Ethyl-pyrrolydine-2,5-dione
According to our FlexX calculations, compounds 8, 13,
18 could bind to the PDE4D catalytic site occupying
part of the pocket where zardaverine, the co-crystallized
ligand, binds. This region, defined by Lee et al.23 as the
inhibitor binding pocket, is delimited mainly by the fol-
lowing hydrophobic residues: Phe469, Leu416, Met370,
Met434, Ile433, Phe437 and Met454. Zardaverine fills
approximately half of the active site-pocket, while more
selective and chemically different PDE4inhibitors such
as roflumilast, according to Lee, are able to better occu-
py this site. For clarity, in the description of interactions
of our compounds with PDE4D catalytic site, we have
ety at N1 and an isopropyl (8), propyl (13) and benzyl
(18) group at N3 showed PDE inhibitory activity (IC50
between 1 and 10lM) with an interesting PDE4selectiv-
ity. The removal of 3,5-di-tert-butyl group (7, 14) leads
to a marked decrease of the activity so as the substitu-
tion of 3,5-di-tert-butyl-4-hydroxybenzyl moiety with
other aromatic (9, 15, 19) or aliphatic groups (10, 12,
16).

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A. Tait et al. / Bioorg. Med. Chem. 13 (2005) 1393–1402
Table 3. In vitro PDE3/4/7 inhibitory activity
Table 5. Molecular interactions displayed by compounds 8, 13 and 18
in the top-ranked docked conformations
Compd
PDE3a pIC50
PDE4b pIC50
PDE7b pIC50
c
c
c
Compound
H-bonds
IBMX
3
5.3
<4.0
<4.0
<4.0
<4.0
<4.0
<4.0
<4.0
<4.0
<4.0
<4.0
<4.0
<5.0
<5.0
<5.0
<4.0
<5.0
<4.0
<4.0
<4.0
<4.0
<4.0
<4.0
5.0
<4.0
<4.0
<4.0
<4.0
5.3
4.0
<4.0
<4.0
<4.0
<4.0
<4.0
<4.0
<4.0
<4.0
<4.0
4.2
Roflumilast
Cilomilast
His301, Met370
His261, His297, Asp298, His301
His301, Asn306
4
5
8
7
13
18
Asn306, Gln307, Met370
His301, Asn306, Met370
8
9
4.4
10
11
12
13
14
15d
16d
17d
18
19d
20
21
22
23
24
25
<4.0
<4.0
<4.0
5.4
a H-bond between carbonyl group on position-4of the
ligand and the NH of Gln307 backbone, plus two H-
bonds between the methoxy groups on positions-6 and
-7 and the NH of Asn306 and Met370 backbone, respec-
tively. An alternative binding mode proposed by FlexX
for 13 is similar to the one of 8, via two H-bonds be-
tween the carbonyl group on position-4of the ligand
and the NH of His301 side chain and between an oxygen
on the sulfonic moiety and the NH of Met370 backbone.
According to the biological data available in the SWISS-
PROT database27 and the results of multiple sequence
alignments of PDE isozymes performed by us employing
CLUSTALW,28 Met370 is one of few residues in the
catalytic site with significant sequence variation in
known PDEs. A specific interaction of our compounds
via H-bond with this residue, could thus help in explain-
ing their selectivity towards PDE4.
<4.0
<5.0
<5.0
<5.0
5.3
<4.0
<5.0
<5.0
<5.0
<4.0
<5.0
<4.0
<4.0
<4.0
<4.0
<4.0
<4.0
<5.0
4.3
4.3
<4.0
<4.0
<4.0
<4.0
a Inhibition of PDE3 was investigated in the cytosol of human
platelets.
b The activity against PDE4and PDE7 was determined versus human
recombinant enzymes PDE4D3 and PDE7A1.
c pIC50 = ꢀlog IC50
.
d 10ꢀ4 insoluble.
In our model, the benzothiadiazine 18, which presents a
bulkier substituent on N-3 in comparison with 8 and 13,
follows the same binding pattern, displaying three
hydrogen bonds with His301, Asn306 and Met370.
Interestingly, two of these H-bonds involve the oxygens
of the sulfonic group. Surprisingly, even if the inhibitor
binding pocket presents several hystidine and phenylala-
nine residues and the new BTDs possess two or three
aromatic rings, no p–p interactions were observed.
divided the inhibitor binding site into three main sub-
pockets, S1, S2 and S3, as proposed by Lee et al.23
The S1 sub-pocket is formed by Met434, Met454 and
Phe437. The sub-pocket S2 consists of Met370, Ser371,
Glu327, Asn306, Asp369 and metal ions. The S3 sub-
pocket branches from the middle of the main active site
pocket and is characterized by residues Phe437, Glu436,
Gln440, Ser305, Cys455 and Ser452.
In order to point out significant differences in the bind-
ing mode among 8, 13, 18 and more potent PDE4inhib-
itors, Cilomilast (Ariflo) and Roflumilast were docked
into the enzyme active site (Table 5). In this way we were
able also to compare FlexX results with Lee hypothesis
on their binding pose.
Compounds 8, 13, 18 mainly interact with S2 sub-pock-
et, in particular with Met370, a key residue for the selec-
tivity towards PDE4D, and with Asn306. Molecular
interactions displayed by 8, 13, 18 are reported in Table
5. More in details, the main features of the binding
mode proposed for compound 8 are two hydrogen
bonds, the first between the carbonyl group on posi-
tion-4of 2,1,3-BTDs and the NH of His301 side chain,
the second between an oxygen of the sulfonic function
and the NH of Asn306 backbone. In the case of 13
(Fig. 2), the best solution proposed by FlexX presents
In agreement with Lee, Roflumilast and Cilomilast,
characterized by bulkier groups replacing the methoxy
moiety on the dialkoxy pharmacophore, an essential
feature of all PDE4inhibitors, were found to occupy
S1 area of the catalytic site. Besides, having a more elon-
gated structure than zardaverine, they resulted able to
fill also the sub-pocket S2, displaying a hydrophobic
interaction with Met370. In this sub-region, a good
superimposition of roflumilast and compound 13 has
however been observed (Fig. 2).
Table 4. Absorbance decrease at 514nm versus control (DA) and
(S.A.%) for 1.0 · 10ꢀ4 M solutions after 6h
Compd
DAa
S.A.%b
8
0.2007 0.00527
0.3131 0.00546
0.2213 0.01359
39.02
60.87
43.02
3. Discussion
13
18
The biological results show the important role of the
3,5-di-tert-butyl-4-hydroxybenzyl moiety in N1 of
2,1,3-benzothiadiazines in eliciting PDE4inhibitory
activity. The molecular docking study into PDE4D cat-
a The means values were obtained from quadruplicate experiments.
b S.A.% = 100 · DA/At where At is absorption at 514nm of DPPH
solution (control) after 6h.

A. Tait et al. / Bioorg. Med. Chem. 13 (2005) 1393–1402
1397
alytic domain indicates that, probably, the bulky di-tert-
butyl groups, which protrude at the top of S3 sub-pock-
et, 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
amino acid residues of S3. Furthermore, the 6,7-dimeth-
oxy substitution at the aromatic ring of benzothiadi-
azine core (13) leads to the most potent compound
(pIC50 5.4); the structure of part of this molecule mimics
the well known di-alkoxy aryl moiety common to a wide
number of PDE4inhibitors like Rolipram, Cilomilast
and Roflumilast.
The 2,1,3-BTD derivatives 11 and 17, with a N1-methyl-
phthalimide substitution, as in some PDE4inhibitors
structurally related to Thalidomide,14 completely lacked
of activity. This could be explained, on the basis of our
computational results, taking into account the high
hydrophobicity of S3 site where N1 substituent should
be allocated. The hydrophilicity of the methylphthal-
imide moiety could probably determine unfavourable
interactions. All novel synthesized compounds are una-
ble to inhibit PDE3 or PDE7.
Figure 2. Overlay of the docked orientations for roflumilast (blue) and
compound 13 in the active site of PDE4D. The most significant amino
acid residues are reported and labelled accordingly.
The 6,7-dimethoxy substituted derivative 13, the most
significative as PDE4inhibitor, showed the best antioxi-
dant property. On the basis of these experimental data,
we can deduce that the 3,5-di-tert-butyl-4-hydroxy-
benzyl moiety at N1 produces anti-PDE4compounds
characterized by antioxidant properties.
The 2,1,3-BTDs N3 substituted (3–5) and the 1,2,4-
BTDs N2 substituted (20–25) are unable to significantly
inhibit the PDE4enzyme. For this reason we can assert
that, in the case of 2,1,3-BTD heterocyclic system, the
double N,N substitution is necessary for activity, while
the synthesis of N,N disubstituted 1,2,4-BTDs must be
performed to elucidate the S.A.R. of this class of benzo-
thiadiazine derivatives.
It is worth mentioning that the combination of PDE4
inhibition and radical scavenging activity in a single
compound could prove an efficient strategy for the treat-
ment of chronic inflammatory diseases by a double and
synergic mechanism of action.
4. Experimental
4.1. Chemistry
The 2,1,3-BTD derivatives 8, 13, 18 are also character-
ized by antioxidant properties. UV measurement of free
radical scavenging activity (S.A.%)29 showed that these
compounds scavenge the DPPH radical. The DPPH
absorbance shows a nonlinear exponential decay (Fig.
1).
Melting points were determined in capillary tubes
(Buchi 510 capillary apparatus) and are uncorrected. H
¨
1
NMR and 13C NMR spectra were recorded on a Bruc-
ker DPX 200 spectrometer using DMSO-d6 as solvent.
Chemical shifts were reported in d (ppm) units relative
to internal reference tetramethylsilane (TMS). Coupling
constants (J) values were given in Hertz. Multiplicities
are abbreviated as follows: s, singlet; d, doublet; t, tri-
plet; 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 spectro-
meter (Nujol mull) and UV spectra on a Cary 50 Bio
UV–VISIBLE Spectrophotometer Varian. Mass spectra
were obtained using a Finnigan Mat SSQ710A spec-
trometer. Thin layer chromatography (TLC) performed
on aluminium silica gel sheets 60 F254 was used for con-
firming the purity on analytical samples. Flash chroma-
tography was performed on Silica gel Merck (230–400
mesh). Elemental analyses for C, H, N were performed
by a Carlo Erba Elemental Analyzer 1106 apparatus.
Reagents and solvents were purchased from common
commercial suppliers.
The S.A.% of each compound was expressed by the ratio
of absorbance decrease of DPPH solution in presence of
compound (DA) versus control (absorbance of DPPH
solution in the absence of compound) (Table 4).
0.6
0.5
0.4
0.3
13
0.2
18
8
0.1
DPPH
0
0
30
60
90
120
150
180
210
240
270
300
330
360
Time (minutes)
4.1.1. General procedure for the synthesis of 3-substituted
2,1,3-BTDs (1–6). To a stirred and cooled (0ꢁC) solution
Figure 1. Time courses of decrease in DPPH concentration for
compound 8, 13 and 18.

1398
A. Tait et al. / Bioorg. Med. Chem. 13 (2005) 1393–1402
of appropriate amine (85mmol) in dichloromethane
(50mL), chlorosulfonic acid (3.30g, 28.3mmol) was
added drop by drop and the reaction mixture was stirred
for an additional hour at room temperature. After evap-
oration under reduced pressure, the resultant salt be-
tween sulfamic acid and the corresponding amine was
dissolved in toluene (50mL) and treated with phospho-
rus pentachloride (7.06g, 33.9mmol). The mixture was
refluxed for 1h and the inorganic by products removed
by filtration. The filtrate was evaporated under reduced
pressure to give the sulfamoyl chloride as an oily residue
that was used in the next synthetic step without further
purification.
2,2-dioxide (6)32 were prepared using the procedure de-
scribed above.
4.1.2. General procedure for the synthesis of 1,3-disubsti-
tuted 2,1,3-BTDs (7–19). The suitable 3-substituted 1H-
2,1,3-benzothiadiazin-4(3H)one 2,2-dioxide (4.16mmol)
and triphenylphosphine (TPP) (1.48g, 5.64mmol) were
added to a solution of the proper alcohol (2.82mmol)
in anhydrous tetrahydrofuran (THF) (10mL). Over a
period of 5min, diisopropylazodicarboxylate (DIAD)
(1.14g, 5.64mmol) was added: the orange-red colour
of DIAD disappears immediately with slight liberation
of heat. The mixture was stirred at room temperature
overnight in N2 atmosphere. The solvent was evapo-
rated under reduced pressure and the residue was puri-
fied by flash column chromatography (cyclohexane/
ethyl acetate 5/5, 7; cyclohexane/ethyl acetate 6/4, 8;
chloroform/acetone 9.5/0.5, 9, 10, 14, 15; chloroform/
acetone 9.8/0.2, 12, 13, 18; chloroform 19) and recrystal-
lized from proper solvent(s).
To a solution of suitable 2-aminobenzoate (18.32mmol)
and triethylamine (2.73g, 27mmol) in toluene (50mL)
was slowly added a solution of appropriate sulfamoyl
chloride (21.70mmol) in toluene (10mL) and the result-
ing mixture was heated at 80ꢁC for 1h. The triethylam-
ine hydrochloride was filtered and the filtrate was
evaporated under reduced pressure to give an oily resi-
due. The residue was dissolved in 75mL of a 0.5M so-
dium methoxide methanolic solution, freshly prepared.
After stirring for 2h at 40ꢁC, the solvent was evaporated
under reduced pressure and the residue dissolved in
water. After cooling and acidification with hydrochloric
acid 6N, the aqueous solution supplied 1–6 derivatives
as a solid residue.
4.1.2.1.
1-(4-Hydroxybenzyl)-3-isopropyl-1H-2,1,3-
benzothiadiazin-4(3H)-one 2,2-dioxide (7). Yield 0.20g
(21%), mp 133ꢁC (CH3OH/H2O); 1H NMR: d 9.51
(1H, s*, OH), 7.98 (1H, dd, J = 1.6, 7.8Hz, H-5), 7.78
(1H, ddd, J = 1.6, 7.5, 8.2Hz, H-7), 7.60 (1H, dd,
J = 1.1, 8.2Hz, H-8), 7.46 (1H, ddd, J = 1.1, 7.5,
7.8Hz, H-6), 6.90 (2H, m, meta phenolic), 6.64(2H,
m, ortho phenolic), 4.99 (2H, s, NCH2Ph), 4.77 (1H,
sep, J = 6.9Hz, CH), 1.38 (6H, d, J = 6.9Hz, CH3).
IR: mmax (cmꢀ1) 3284, 1651, 1379, 1199, 1031. Anal.
Calcd for C17H18N2O4S: C, 58.95; H, 5.24; N, 8.09.
Found: C, 58.96; H, 5.46; N, 8.23.
The solid was collected by filtration and recrystallized
from proper solvent(s).
4.1.1.1. 3-Propyl-6,7-dimethoxy-1H-2,1,3-benzothiad-
iazin-4(3H)-one 2,2-dioxide (2). Yield 1.38g (25%), mp
1
151–152ꢁC (DMF/H2O); H NMR: d 7.40 (1H, s, aro-
4.1.2.2. 1-(3,5-Di-tert-butyl-4-hydroxybenzyl)-3-iso-
propyl-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide
(8). Yield 0.58g (45%), mp 97–100ꢁC (CH3OH/H2O);
1H NMR: 8.01 (1H, dd, J = 1.6, 7.9Hz, H-5), 7.78
(1H, ddd, J = 1.6, 7.5, 8.2Hz, H-7), 7.65 (1H, bd,
J = 8.2Hz, H-8), 7.48 (1H, bdd, J = 7.5, 7.9Hz, H-6),
6.92 (1H, s*, OH), 6.67 (2H, s, aromatic H), 4.96 (2H,
s, NCH2Ph), 4.54 (1H, sep, J = 6.9Hz, CH), 1.20
(18H, s, CH3 tert-but.), 1.15 (6H, d, J = 6.9Hz, CH3).
IR: mmax (cmꢀ1) 3589, 1670, 1602, 1310, 1194, 1116.
Anal. Calcd for C25H34N2O4S: C, 65.47; H, 7.47; N,
6.11. Found: C, 65.46; H, 7.54; N, 6.37.
matic H-5), 6.70 (1H, s, aromatic H-8), 3.85 (3H, s,
OCH3), 3.80 (3H, s, OCH3), 3.77 (2H, t, J = 7.4Hz,
NCH2), 1.67 (2H, sxt, J = 7.4Hz, CH2), 0.89 (3H, t,
J = 7.4Hz, CH3). IR: mmax (cmꢀ1) 3130, 1731, 1022,
985, 791. Anal. Calcd for C12H16N2O5S: C, 47.99; H,
5.37; N, 9.33. Found: C, 48.13; H, 5.45; N, 9.61.
4.1.1.2. 3-Benzyl-7-chloro-1H-2,1,3-benzothiadiazin-
4(3H)-one 2,2-dioxide (4). Yield 0.89g (15%), mp 192–
193ꢁC (CH3OH/H2O); 1H NMR: d 7.90 (1H, d,
J = 8.5Hz, H-5), 7.26 (7H, m, aromatic H), 4.97 (2H,
s, NCH2Ph). IR: mmax (cmꢀ1) 3172, 1650, 1351, 1176,
720, 700. Anal. Calcd for C14H11ClN2O3S: C, 52.10;
H, 3.44; N, 8.68. Found: C, 52.49; H, 3.83; N, 8.92.
4.1.2.3. 1-(3-Nitrobenzyl)-3-isopropyl-1H-2,1,3-benzo-
thiadiazin-4(3H)-one 2,2-dioxide (9). Yield 0.87g (82%),
mp 137–140ꢁC (trituration with petrol ether 40–60ꢁC);
1H NMR: d 8.16 (1H, m, aromatic H), 8.05 (1H, pseudo
s, aromatic H), 8.01 (1H, dd, J = 1.7, 7.9Hz, H-5), 7.76
(1H, ddd, J = 1.7, 7.5, 8.1Hz, H-7), 7.55 (3H, m, aro-
matic H), 7.45 (1H, ddd, J = 1.1, 7.5, 7.9Hz, H-6),
5.28 (2H, s, NCH2Ph), 4.82 (1H, sep, J = 6.9Hz, CH),
1.38 (6H, d, J = 6.9Hz, CH3). IR: mmax (cmꢀ1) 1681,
1531, 1195, 1174, 977. Anal. Calcd for C17H17N2O5S:
C, 54.39; H, 4.56; N, 11.19. Found: C, 54.11; H, 4.18;
N, 11.27.
4.1.1.3. 3-Benzyl-6,7-dimethoxy-1H-2,1,3-benzothiadi-
azin-4(3H)-one 2,2-dioxide (5). Yield 5.74g (90%), mp
160–161ꢁC (CH3OH/H2O); 1H NMR: d 7.40 (1H, s,
H-5) 7.32 (5H, m, aromatic H), 6.72 (1H, s, H-8), 5.00
(2H, s, NCH2Ph), 3.86 (3H, s, OCH3), 3.80 (3H, s,
OCH3). IR: mmax (cmꢀ1) 3170, 1673, 1277, 1165, 1004,
722. Anal. Calcd for C16H16N2O5S: C, 55.16; H, 4.63;
N, 8.04. Found: C, 54.88; H, 4.63; N, 8.43.
The
known
3-isopropyl-1H-2,1,3-benzothiadiazin-
4(3H)-one 2,2-dioxide (Bentazon)30 (1), 3-benzyl-1H-
2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (3)31 and
3-(2-phenylethyl)-1H-2,1,3-benzothiadiazin-4(3H)-one
4.1.2.4. 1-Cyclohexyl-3-isopropyl-1H-2,1,3-benzothi-
adiazin-4(3H)-one 2,2-dioxide (10). Yield 0.11g (12%),
mp 80–84ꢁC (trituration with petrol ether 40–60ꢁC);

A. Tait et al. / Bioorg. Med. Chem. 13 (2005) 1393–1402
1399
1H NMR: d 8.02 (1H, dd, J = 1.7, 7.9Hz, H-5), 7.76
(1H, ddd, J = 1.7, 7.5, 8.1Hz, H-7), 7.55 (1H, ddd,
J = 1.2, 7.5, 7.9Hz, H-6), 7.52 (1H, dd, J = 1.2, 8.1Hz,
H-8), 4.88 (1H, sep, J = 6.9Hz, CH), 3.98 (1H, m, CH
cyclohexyl), 1.51 (10H, m, cyclohexyl), 1.46 (6H, d,
J = 6.9Hz, CH3). IR: mmax (cmꢀ1) 1668, 1596, 1298,
1193, 985. Anal. Calcd for C16H22N2O3S: C, 59.60; H,
6.88; N, 8.69. Found: C, 60.00; H, 6.55; N, 8.95.
7.59 (1H, dd, J = 8.5Hz, J = 2.0Hz, aromatic H-6),
7.33 (5H, m, aromatic H), 7.04(1H, s*, OH), 6.78
(2H, s, aromatic H), 5.03 (2H, s, NCH2Ph), 4.70 (2H,
s, NCH2Ph), 1.27 (18H, s, CH3). IR: mmax (cmꢀ1) 3575,
1683, 1597, 1292, 1174, 1115. Anal. Calcd for
C29H33ClN2O4S: C, 64.37; H, 6.15; N, 5.18. Found: C,
64.51; H, 6.35; N, 5.54.
4.1.2.10.
1-(2-Nitrobenzyl)-3-(2-phenylethyl)-1H-
4.1.2.5. 1-[2-(3-Isopropyl-2,2-dioxido-4-oxo-3,4-dihy-
dro-1H-2,1,3-benzothiadiazin-1-yl)ethyl]pyrrolydine-2,5-
dione (12). Yield 0.20g (20%), mp 135ꢁC (trituration
2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (19). Yield
0.91g (74%), mp 112–115ꢁC (trituration with petroleum
ether 40–60ꢁC); 1H NMR: d 8.09 (2H, dt, J = 1.5,
8.2Hz, aromatic H), 7.76 (1H, dt, J = 1.5, 7.6Hz, aro-
matic H), 7.65 (2H, m, aromatic H), 7.47 (2H, m, aro-
matic H), 7.26 (6H, m, aromatic H), 5.33 (2H, s,
NCH2Ph), 4.06 (2H, m, NCH2CH2Ph), 2.93 (2H, m,
NCH2CH2Ph). IR: mmax (cmꢀ1) 1682, 1601, 1537, 1262,
1147. Anal. Calcd for C22H19N3O5S: C, 60.40; H, 4.38;
N, 9.61. Found: C, 60.78; H, 4.40; N, 9.99.
1
with petrol ether 40–60ꢁC); H NMR: d 8.05 (1H, dd,
J = 1.5, 8.0Hz, H-5), 7.78 (1H, dt, J = 1.5, 8.1Hz, H-
7), 7.54(1H, bd,
J = 8.1Hz, H-8), 7.43 (1H, bt,
J = 8.1Hz, H-6), 4.86 (1H, sep, J = 7.0Hz, CH), 4.07
(2H, m, NCH2–CH2N), 3.66 (2H, m, NCH2CH2N),
2.46 (4H, m, CH2), 1.47 (6H, d, J = 7.0Hz, CH3). IR:
mmax (cmꢀ1) 1704, 1681, 1604, 1168, 757. Anal. Calcd
for C16H19N3O5S: C, 52.59; H, 5.24; N, 11.50. Found:
C, 52.73; H, 5.24; N, 11.46.
The known 2-[(3-isopropyl-2,2-dioxido-4-oxo-3,4-dihy-
dro-1H-2,1,3-benzothiadiazin-1-yl)methyl]-1H-isoindo-
le-1,3(2H)dione (11),33 3-benzyl-1-(2-furylmethyl)-1H-
2,1,3-benzothidiazin-4(3H)-one 2,2-dioxide (16),32 2-
[(3-benzyl-2,2-dioxido-4-oxo-3,4-dihydro-1H-2,1,3-benz-
othiadiazin-1-yl)methyl]-1H-isoindole-1,3(2H)dione (17)32
were prepared using the procedure described above.
4.1.2.6. 1-(3,5-Di-tert-butyl-4-hydroxybenzyl)-3-prop-
yl-6,7-dimetoxy-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-
dioxide (13). Yield 0.83g (57%), mp 163–165ꢁC
1
(CH3OH/H2O); H NMR: d 7.34(1H, s, aromatic H-
5), 7.17 (1H, s, aromatic H-8), 6.94(1H, s*, OH), 6.68
(2H, s, aromatic H), 4.87 (2H, s, NCH2Ph), 3.91 (3H,
s, OCH3), 3.83 (3H, s, OCH3), 3.46 (2H, t, J = 7.3Hz,
NCH2), 1.49 (2H, sxt, J = 7.3Hz, CH2), 1.24(18H, s,
tert-butyl), 0.82 (3H, t, J = 7.3Hz, CH3). IR: mmax
(cmꢀ1) 3615, 1672, 1606, 1263, 1181, 1011. Anal. Calcd
for C27H38N2O6S: C, 62.52; H, 7.38; N, 5.40. Found: C,
62.47; H, 7.46; N, 5.80.
4.1.2.11.
6-Chloro-3-chloromethyl-3,4-dihydro-2H-
1,2,4-benzothiadiazin 1,1-dioxide (26). To a solution of
2-amino-4-chlorobenzensulfonamide (10g, 48mmol) in
DMF (50mL) was added a solution of 45% aqueous
chloroacetaldehyde (16.9g, 96mmol) and NH4Cl (3.0g,
58mmol) in water (10mL). The reaction mixture was
heated for 1h at 100ꢁC, cooled and poured into water
to obtain 26 as a beige solid. Yield 12.18g (95%) mp
173ꢁC (DMF/water); 1H NMR: d 7.83 (1H, d*,
J = 11.2Hz, NH-4), 7.52 (1H, s*, NH-2), 7.50 (1H, d,
J = 8.4Hz, H-8), 6.92 (1H, d, J = 2.0Hz, H-5), 6.77
(1H, dd, J = 2.0, 8.4Hz, H-7), 4.92 (1H, m, H-3), 3.81
(2H, d, J = 7.6Hz, CH2Cl). IR: mmax (cmꢀ1) 3359,
3218, 1603, 1166, 765. Anal. Calcd for C8H8Cl2N2O2S:
C, 35.97; H, 3.02; N, 10.49. Found: C, 36.00; H, 3.12;
N, 10.56.
4.1.2.7. 3-Benzyl-1-(4-hydroxybenzyl)-1H-2,1,3-ben-
zothiadiazin-4(3H)-one 2,2-dioxide (14). Yield 0.38g
1
(34%), mp 98ꢁC; H NMR: d 9.44 (1H, s*, OH), 7.97
(1H, dd, J = 1.7, 7.7Hz, H-5), 7.78 (1H, dt, J = 1.7,
7.7Hz, H-7), 7.61 (1H, dd, J = 1.2, 7.7Hz, H-8), 7.45
(1H, dt, J = 1.2, 7.7Hz, H-6), 7.33 (5H, m, aromatic
H), 6.82 (2H, m, meta phenolic), 6.56 (2H, m, ortho phen-
olic), 4.97 (2H, s, NCH2), 4.93 (2H, s, NCH2). IR: mmax
(cmꢀ1) 3296, 1650, 1461, 1183, 1024, 629. Anal. Calcd
for C21H18N2O4S: C, 63.95; H, 4.60; N, 7.10. Found:
C, 64.05; H, 4.88; N, 7.41.
4.1.3. General procedure for the synthesis of 2-substituted
1,2,4-BTDs (20–25). The title compounds 20–25 were
obtained following the same procedure described for
7–19 by reaction of 6-chloro-3-chloromethyl-3,4-dihy-
dro-2H-1,2,4-benzothiadiazine 1,1-dioxide 26 with the
appropriate alcohol. The residue was purified by flash
column chromatography (cyclohexane/ethyl acetate
6.5/3.5, 20; chloroform/acetone 8/2, 21; chloroform, 22,
23; chloroform/acetone 9.8/0.2, 24; cyclohexane/ethyl
acetate 9/1, 25).
4.1.2.8. 3-Benzyl-1-(3-nitrobenzyl)-1H-2,1,3-benzothi-
adiazin-4(3H)-one 2,2-dioxide (15). Yield 0.76g (64%),
mp 108–109ꢁC (CH3OH); 1H NMR: d 8.14(1H, m, aro-
matic H), 8.04(2H, dd, J = 1.6, 7.8Hz, aromatic H),
7.80 (1H, ddd, J = 1.6, 7.4, 8.0Hz), 7.58 (3H, m, aro-
matic H), 7.49 (1H, ddd, J = 1.0, 7.6, 8.0Hz), 7.33
(5H, m, aromatic H), 5.28 (2H, s, NCH2Ph), 4.99 (2H,
s, NCH2Ph). IR: mmax (cmꢀ1) 1688, 1601, 1526, 1180.
Anal. Calcd for C21H17N3O5S: C, 59.57; H, 4.05; N,
9.92. Found: C, 60.19; H, 3.79; N, 10.15.
4.1.3.1. 6-Chloro-3-chloromethyl-2-(4-hydroxybenzyl)-
3,4-dihydro-2H-1,2,4-benzothiadiazin 1,1-dioxide (20).
1
4.1.2.9. 3-Benzyl-1-(3,5-di-tert-butyl-4-hydroxybenz-
yl)-7-chloro-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-di-
oxide (18). Yield 0.55g (36%), mp 115–119ꢁC
(CH3OH/H2O); 1H NMR: d 7.98 (1H, d, J = 8.5Hz,
aromatic H-5), 7.91 (1H, d, J = 2.0Hz, aromatic H-8),
Yield 0.91g (86%), mp 156–158ꢁC; H NMR: d 9,34
(1H, s*, OH), 7,68 (1H, d*, J = 2,0Hz, NH), 7.57 (1H,
d, J = 8,5Hz, aromatic H-8), 7.16 (2H, m, meta phen-
olic), 6.97 (1H, d, J = 2.0Hz, aromatic H-5), 6.84(1H,
dd, J = 8.5, 2.0Hz, aromatic H-7), 6.70 (2H, m, ortho

1400
A. Tait et al. / Bioorg. Med. Chem. 13 (2005) 1393–1402
phenolic), 5.10 (1H, bt, J = 6.8Hz, H-3), 4.17 (1H, d,
J = 17.5Hz, NCHHPh), 3.86 (3H, m, CH2Cl and
NCHHPh). IR: mmax (cmꢀ1) 3249, 1737, 1688, 1599,
1526, 1260, 1110, 1051, 926. Anal. Calcd for
C15H14Cl2N2O3S: C, 48.27; H, 3.78; N, 7.51. Found:
C, 48.14; H, 3.69; N, 7.39.
8.5Hz, aromatic H-7), 5.35 (1H, ddd, J = 3.5, 6.7,
7.5Hz, H-3), 5.10 (1H, d, J = 14.4Hz, NCHHN), 4.96
(1H, d, J = 14.4Hz, NCHHN), 4.05 (1H, dd, J = 7.5,
11.2Hz, CHHCl), 3.87 (1H, dd, J = 6.7, 11.2Hz,
CHHCl). IR: mmax (cmꢀ1) 3381, 1777, 1715, 1325,
1130, 881, 718. Anal. Calcd for C17H13Cl2N3O4S: C,
47.90; H, 3.07; N, 9.86. Found: C, 48.30; H, 2.91; N,
10.12.
4.1.3.2. 6-Chloro-3-chloromethyl-2-(pyridin-4-ylme-
thyl)-3,4-dihydro-2H-1,2,4-benzothiadiazin 1,1-dioxide
1
(21). Yield 0.66g (65%), mp 121–123ꢁC; H NMR: d
4.1.3.6. 2-{[6-Chloro-3-(chloromethyl)-1,1-dioxido-3,4-
dihydro-2H-1,2,4-benzothiadiazin-2-yl]ethyl}-pyrrolidine-
2,5-dione (25). Yield 0.4g (36%), mp 139–142 ꢁC; 1H
NMR: d 7.74(1H, d*, J = 2.1Hz, NH), 7.55 (1H, d,
J = 8.5Hz, aromatic H-8), 6.96 (1H, d, J = 2.0Hz, aro-
matic H-5), 6.84(1H, dd, J = 2.0, 8.5Hz, aromatic H-
7), 5.23 (1H, ddd, J = 2.1, 6.5, 6.7Hz, H-3), 3.94(2H,
m, CH2Cl), 3.65–2.75 (4H, m, NCH2CH2N), 2.58 (4H,
s, pyrrolidine). IR: mmax (cmꢀ1) 3330, 1693, 1594, 1164,
721. Anal. Calcd for C14H15Cl2N3O4S: C, 42.87; H,
3.85; N, 10.71. Found: C, 43.16; H, 3.96; N, 10.02.
8.51 (2H, bd, J = 6.0Hz, pyridine H-2 and H-6), 7.80
(1H, d*, J = 2.0Hz, NH), 7.58 (1H, d, J = 8.5Hz, aro-
matic H-8), 7.42 (2H, bd, J = 6.0Hz, pyridine H-3 and
H-5), 7.02 (1H, d, J = 2.0Hz, aromatic H-5), 6.87 (1H,
dd, J = 2.0, 8.5Hz, aromatic H-7), 5.32 (1H, m, H-3),
4.39 (1H, d, J = 17.0Hz, NCHHPh), 3.96 (3H, m,
CH2Cl and NCHHPh). IR: mmax (cmꢀ1) 3370, 1704,
1697, 1599, 1337, 1167, 1080, 722. Anal. Calcd for
C14H13Cl2N3O2S: C, 46.94; H, 3.66; N, 11.73. Found:
C, 47.05; H, 3.99; N, 12.00.
4.1.3.3. 6-Chloro-3-chloromethyl-2-(2-nitrobenzyl)-
3,4-dihydro-2H-1,2,4-benzothiadiazin 1,1-dioxide (22).
Yield 0.42g (37%), mp 144–145ꢁC; H NMR: d 8.06
4.2. Biological assays
1
4.2.1. Chemicals. Benzamidine, bovine serum albumine
(BSA, fraction V powder), cAMP, EGTA (ethylenegly-
col-bis-[b-amino-ethylether]-N,N,N0,N0-tetraacetic acid),
3-isobutyl-1-methyl-xanthine (IBMX), leupeptin, b-
mercaptoethanol, pepstatin A and trysin inhibitor were
purchased from Sigma Chemie (Deisenhofen, Ger-
many). [50,8-3H]cAMP and phosphodiesterase SPA
assay beads were obtained from Amersham Biosciences
(Freiburg, Germany). PefablockR SC was purchased
from Boehringer Mannheim (Germany). Dimethyl sulf-
oxide (DMSO) and tris(hydroxymethyl)-aminoethan
(Tris) were obtained either from Merck (Darmstadt,
Germany) or from Sigma Chemie. All other chemicals
were of analytical grade and were obtained from Merck.
(H, d, J = 8.1Hz, nitrobenzyl aromatic H-3), 8.02 (H,
d, J = 7.8Hz, nitrobenzyl aromatic H-6), 7.86 (1H, d*,
J = 2.0Hz, NH), 7.82 (1H, dd, J = 7.8, 8.6Hz, nitro-
benzyl aromatic H-5), 7.59 (1H, d, J = 8.5Hz, aromatic
H-8), 7.57 (1H, dd, J = 8.1, 8.6Hz, nitrobenzyl aromatic
H-4), 7.06 (1H, d, J = 2.0Hz, aromatic H-5), 6.89 (1H,
dd, J = 2.0, 8.5Hz, aromatic H-7), 5.34(1H, ddd,
J = 2.0, 5.7, 7.9Hz, H-3), 4.70 (1H, d, J = 17.5Hz,
NCHHPh), 4.36 (1H, d, J = 17.5Hz, NCHHPh), 4.06
(1H, dd, J = 7.9, 11.7Hz, CHHCl), 3.87 (1H, dd,
J = 5.7, 11.7Hz, CHHCl). IR: mmax (cmꢀ1) 3371, 1595,
1561, 1321, 1160, 1093, 859, 727. Anal. Calcd for
C15H13Cl2N3O4S: C, 44.79; H, 3.26; N, 10.45. Found:
C, 45.11; H, 3.35; N, 10.57.
For the inhibition experiments (see below), stock solu-
tions (10mM) of the compounds were prepared in
DMSO, which were serially diluted 1:10 (v/v) in DMSO
to achieve the desired final concentrations of the com-
pounds in the assays after pipetting of 1lL of these dilu-
tions into the 100lL-assays (representing a final dilution
step of 1:100 v/v).
4.1.3.4. 6-Chloro-3-chloromethyl-2-(3-nitrobenzyl)-
3,4-dihydro-2H-1,2,4-benzothiadiazin 1,1-dioxide (23).
Yield 0.79g (70%), mp 163–165ꢁC; H NMR: d 8.30
1
(1H, bs, nitrobenzyl aromatic H-2), 8.12 (1H, bd, J =
8.3Hz, nitrobenzyl aromatic H-4), 7.86 (1H, bd,
J = 7.8Hz, nitrobenzyl aromatic H-6), 7.79 (1H, d*,
J = 2.0Hz NH), 7.66 (1H, dd, J = 7.8, 8.3Hz, nitro-
benzyl aromatic H-5), 7.59 (1H, d, J = 8.5Hz, aromatic
H-8), 6.98 (1H, d, J = 2.0Hz, aromatic H-5), 6.86 (1H,
dd, J = 2.0, 8.5Hz, aromatic H-7), 5.34(1H, ddd,
J = 2.0, 5.7, 8.1Hz, H-3), 4.50 (1H, d, J = 16,6Hz,
NCHHPh), 4.16 (1H, d, J = 16.6Hz, NCHHPh), 4.05
(1H, dd, J = 8.1, 11.7Hz, CHHCl), 3.89 (1H, dd,
J = 5.7, 11.7Hz, CHHCl). IR: mmax (cmꢀ1) 3385, 1594,
1561, 1526, 1332, 1165, 1088, 726. Anal. Calcd for
C15H13Cl2N3O4S: C, 44.79; H, 3.26; N, 10.45. Found:
C, 44.48; H, 3.37; N, 10.41.
4.2.2. PDE enzymes. PDE3 was analyzed using cytosol
from human platelets; the homogenate was prepared
in analogy to the method described below for insect
cells. The human PDE4D3 (GB no. U50159) was a gift
of Prof. Marco Conti (Stanford University, USA); the
ORF (GB no. U50159) was cut from the original
pCMV5 vector with the restriction enzymes EcoRI
and XbaI and subcloned in the expression vector
pBP9. The human PDE7A1 (GB no. L12052) was iso-
lated using RT-PCR, from total cellular RNA derived
from the T-cell line CCRF-CEM using the primers
4.1.3.5. 2-{[6-Chloro-3-(chloromethyl)-1,1-dioxido-3,4-
dihydro-2H-1,2,4-benzothiadiazin-2-yl]methyl}-1H-isoin-
dole-1,3-(2H)-dione (24). Yield 0.38g (32%), mp 228–
CP2PD7S
(50-GGGCGGGCGGATCCAATGGAA-
GTG-30) and CP3PD7A (50-CTGGTTCTGGGGGT-
TATGATAACCG-30) and cloned into the Baculovirus
expression vector pCRBac. Recombinant PDEs were ex-
pressed in a Baculovirus/Sf21 insect cell system. After
infection (usually for 48h) the Sf21 cells were resus-
1
230ꢁC dec; H NMR: d 7.87 (5H, m, aromatic H and
NH), 7.52 (1H, d, J = 8.5Hz, aromatic H-8), 6.85 (1H,
d, J = 2.0Hz, aromatic H-5), 6.77 (1H, dd, J = 2.0,

A. Tait et al. / Bioorg. Med. Chem. 13 (2005) 1393–1402
1401
pended in ice-cold (4ꢁC) homogenization buffer (20mM
Tris, pH8.2, containing the following additions: 140mM
NaCl, 3.8mM KCl, 1mM EGTA, 1mM MgCl2, 10mM
b-mercaptoethanol, 2mM benzamidine, 0.4mM Pefa-
block, 10lM leupeptin, 10lM pepstatin A and 5lM
trypsin inhibitor) at a concentration of approximately
107 cells/mL, and disrupted by ultrasonication on ice.
The homogenate was then centrifuged for 10min at
1.000g (4ꢁC) and the supernatant was stored at ꢀ80ꢁC
until subsequent use.
The three-dimensional structure co-ordinate file of
PDE4D catalytic domain (PDB entry 1MKD) in com-
plex with zardaverine was obtained from the Protein
Data Bank.36 A two-step docking protocol was em-
ployed. In a first phase, each inhibitor was docked into
the active site by means of the FlexX module, as imple-
mented in Sybyl v6.825 with the macromolecule and the
ligands being flexible. Preparation of the protein for
FlexX requires definition of the binding pocket in terms
of Ôinteraction pointsÕ. In this work the active site was
˚
defined as all atoms within a distance of 10 A from
zardaverine. The specific distance was determined in
order to ensure a significative portion of the active site
for the docking experiments.
4.2.3. Measurement of PDE activity. PDE activity was
measured by a modified SPA (scintillation proximity
assay) test from Amersham Biosciences (see procedural
instructions Ôphosphodiesterase [3H]cAMP SPA enzyme
assay, code TRKQ 7090Õ), carried out in 96 well micro-
plates (MTPs). The test volume is 100lL and contains
20mM Tris buffer (pH7.4), 0.1mg/mL BSA, 5mM
Mg2+, 0.5lM of the substrate cAMP (including about
50.000cpm of the corresponding [3H]-labelled cyclic
nucleotide), 1lL of the respective substance dilution in
DMSO and sufficient enzyme to ensure that 10–20% of
the substrate is converted. The final concentration of
DMSO in the assays (1% v/v) does not substantially af-
fect any of the PDEs investigated.
Starting from the best-docked geometries, as obtained
with FlexX, the second step consisted in a further refine-
ment of the complex performed with QXP.37 Also the
algorithm implemented in the QXP program allows
for fully flexibility of the inhibitors and simultaneous
flexibility of the active site side chains. Each docking
run included 15,000 steps of Monte Carlo perturbation,
subsequent fast searching and final energy minimization.
The results were evaluated in terms of total estimated
binding energy, internal strain energy of the ligand,
van der Waals and electrostatic interaction energies.
After a preincubation of 5min at 37ꢁC in the presence of
the compounds, the reaction is started by adding the
substrate and the assays are incubated for a further
15min; after that, they are stopped by adding SPA
beads (50lL). In accordance with the manufacturerÕs
instructions, the SPA beads had previously been resus-
pended in water, but were then further diluted 1:3
(v/v) in water; the diluted solution also contains 3mM
IBMX to ensure a complete PDE activity stop. After
the beads have been sedimented (>30min), the MTPs
are analyzed in commercially available luminescence
detection devices. IC50 values were calculated from the
concentration–inhibition-curves by nonlinear regression
analysis using GraphPad Prism.
For verifying the variability of amino acids residues in
S1, S2 and S3 sub-pockets of PDE4D catalytic site,
amino acids sequences of PDE family members were re-
trieved from the SWISSPROT database.27 Multiple se-
quence alignment of PDE isozymes was performed
employing CLUSTALW.28 All calculations were carried
out on a SGI O2 workstations and on a standard per-
sonal computer running under Linux.
Acknowledgements
Thanks are due to Prof. Marco Conti (Stanford Univer-
sity, USA) for the gift of the human PDE4D3 (GB no.
U50159), Mrs. Rossella Gallesi who performed elemen-
tal analyses of synthesized compounds and Mr. Daniele
Montanini, Mr. Dennis Cattabriga, Mrs. Elena Balla-
beni and Mrs. Eleonora Di Iorio for their valuable
assistance.
4.3. Radical scavenging effect on DPPH radical
Two millilitres of an ethanolic solution (1 · 10ꢀ4 M) of
the tested compound was added to 2mL of a DPPH
solution (1 · 10ꢀ4 M), and the reaction mixture was sha-
ken vigorously and kept at 37ꢁC 0.02 (Haake F 3C
Thermocriostat) in air. DPPH absorption was measured
at 514nm every 15min. The mean values were obtained
from quadruplicate experiments.
References and notes
1. Essayan, D. M. Biochem. Pharmacol. 1999, 57, 965.
2. Conti, M.; Richter, W.; Mehats, C.; Livera, G.; Park
J.-Y.; Jin, C. J. Biol. Chem. 2003, 278, 5493.
4.4. Molecular modelling
3. Dent, G.; Giembyez, M. A. In Phosphodiesterase Inhibi-
tors; Schudt, C., Dent, G., Rabe, K. F., Eds.; Academic:
San Diego, CA, 1997.
4. Schmidt, D.; Dent, G.; Rabe, K. F. Clin. Exp. Allergy
1999, 29, 99.
5. Teixeira, M. M.; Gristwood, R. W.; Cooper, N.; Helle-
well, P. G. Trends Pharmacol. Sci. 1997, 18, 164.
6. Li, L.; Yee, C.; Beavo, J. A. Science 1999, 283, 848.
7. Barnes, M. J.; Cooper, N.; Davenport, R. J.; Dyke, H. J.;
Galleway, F. P.; Galvin, F. C. A.; Gowers, L.; Haughan,
A. F.; Lowe, C.; Meissner, J. W. G.; Montana, J. G.;
Molecular structures of ligands 8, 13, 18 were built and
energy minimized within MacroModel.34 Conforma-
tional analysis was carried out using the AMBER* force
field, as included in MacroModel. For all compounds,
the resulting geometries of the lower energy conformers
were re-optimized with semi-empirical quantum me-
chanic calculations, using the Hamiltonian AM1 as
implemented in Spartan35 and atomic charges were
calculated.

1402
A. Tait et al. / Bioorg. Med. Chem. 13 (2005) 1393–1402
Morgan, T.; Picken, C. L.; Watson, R. J. Bioorg. Med.
Chem. Lett. 2001, 11, 1081.
8. Hatzelmann, A.; Schudt, C. J. Pharmacol. Exp. Ther.
2001, 297, 267.
9. Bundschuh, D. S.; Eltze, M.; Barsig, J.; Wollin, L.;
Hatzelmann, A.; Beume, R. J. Pharmacol. Exp. Ther.
2001, 297, 280.
21. Mitsunobu, O. Synthesis 1981, 1.
22. Close, W. J.; Swett, L. R.; Brady, L. E.; Short, J. H.;
Vernsten, M. J. Am. Chem. Soc. 1960, 82, 1132.
23. Lee, M. E.; Markowitz, J.; Lee, J.-O.; Lee, H. FEBS Lett.
2002, 530, 53.
24. Rarey, M.; Kramer, B.; Lengauer, T.; Klebe, G. J. Mol.
Biol. 1996, 261, 470.
10. Donnelly, L. E.; Rogers, D. F. Drugs 2003, 63, 1973.
11. Spina, D. Drugs 2003, 63, 2575.
12. Martinez, A.; Castro, A.; Gil, C.; Miralpeix, M.; Segarra,
25. Sybyl 6.8, Tripos Inc. 1699 South Hanley Road, St. Louis,
Missouri 63144, USA.
26. Erickson, J. A.; Jalaie, M.; Robertson, D. H.; Lewis, R.
A.; Vieth, M. J. Med. Chem. 2004, 47, 4 5.
27. Bairoch, A.; Apweiler, R. Nucleic Acids Res. 2000, 28,
45.
´
V.; Domenech, T.; Beleta, J.; Palacios, J. M.; Ryder, H.;
Miro, X.; Bonet, C.; Casacuberta, J. M.; Azorın, F.; Pin˜a,
`
`
`
B.; Puigdomenech, P. J. Med. Chem. 2000, 43, 683.
13. Castro, A.; Abasolo, M. I.; Gil, C.; Segarra, V.; Martinez,
A. Eur. J. Med. Chem. 2001, 36, 333.
14. Burnouf, C.; Pruniaux, M. P. Curr. Pharm. Des. 2002, 8,
1255.
28. Thompson, J. D.; Higgins, D. G.; Gibson, T. J. Nucleic
Acids Res. 1994, 22, 4673, Clamp, M. Jalview, http://
www.ebi.ac.uk/~michele/jalview/, 1998.
29. Tait, A.; Ganzerli, S.; Di Bella, M. Tetrahedron 1996, 38,
12587.
30. Damjan, J.; Benczik, J. M.; Soptei, C.; Barcza, I. M.;
Kolonics, Z.; Pelyve, J.; Karacsonyi, B.; Dioszegi Eich-
hardt, E. HU Patent 56837, October 1991.
31. Dewynter, G.; Aouf, N.; Regainia, Z.; Montero, J.-L.
Tetrahedron 1996, 52, 993.
32. Tait, A.; Luppi, A.; Cermelli, C. J. Het. Chem. 2004, 41,
747.
33. Hamprecht, G.; Stubenrauch, G.; Urbach, H.; Wuerzer,
B. DE Patent 2656290, June 1978.
34. MacroModel 7.0, Schro¨dinger LLC Mohamadi, F.;
Richards, N. G. J.; Guida, W. C.; Liskamp, R.; Lipton,
M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C.
J. Comput. Chem. 1990, 11, 440.
35. Spartan ÕO2, Wavefunction Inc., Irvine, CA.
36. Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.;
Bhat, T. N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E.
Nucleic Acids Res. 2000, 28, 235.
15. Song, Y.; Connor, D. T.; Doubleday, R.; Sorensen, R. J.;
Sercel, A. D.; Unangst, P. C.; Roth, B. D.; Gilbertsen, R.
B.; Chan, K.; Schrier, D. J.; Guglietta, A.; Bornemeier, D.
A.; Dyer, R. D. J. Med. Chem. 1999, 42, 1151.
16. Inagaki, M.; Tsuri, T.; Jyoyama, H.; Ono, T.; Yamada,
K.; Kobayashi, M.; Hori, Y.; Arimura, A.; Yasui, K.;
Ohno, K.; Kakudo, S.; Koizami, K.; Suzuki, R.; Kato,
M.; Kawai, S.; Matsumoto, S. J. Med. Chem. 2000, 43,
2040.
17. Janusz, J. M.; Young, P. A.; Ridgeway, J. M.; Scherz, M.
W.; Enzweiler, K.; Wu, L. I.; Gan, L.; Chen, J.; Kellstein,
D. E.; Green, S. A.; Tulich, J. L.; Rosario-Jansen, T.;
Magrisso, I. J.; Wehmeyer, K. R.; Kuhlenbeck, D. L.;
Eichhold, T. H.; Dobson, R. L. M. J. Med. Chem. 1998,
41, 3515.
18. De Leval, X.; Julemont, F.; Delarge, J.; Pirotte, B.;
Dogne, J. M. Curr. Med. Chem. 2002, 9, 941.
19. Cohen, E.; Klarberg, B. J. Am. Chem. Soc. 1962, 84, 1994.
20. Kloek, J. A.; Leschinsky, K. L. J. Org. Chem. 1976,
25(41), 4028.
37. McMartin, C.; Bohacek, R. J. Comput.-Aided Mol. Des.
1997, 11, 333.