Synthesis and antiviral activity of new benzothiadiazine dioxide derivatives

Article abstract of DOI:10.1002/jhet.5570410516

A series of 2,1,3- and 1,2,4-benzothiadiazine derivatives were synthesized by alkylation via Mitsunobu reaction and evaluated for their antiviral activity against ADV, HHV-6, Cox-B5 and H-CMV. Most of them were active at micromolar level against one or more viral strains. All the molecules studied are poorly cytotoxic (maximum non toxic concentrations were >25μM), except one compound that presents a higher cytotoxicity (maximum non toxic concentration was 6 μM).

Full text of DOI:10.1002/jhet.5570410516

Sep-Oct 2004
747
Synthesis and Antiviral Activity of
New Benzothiadiazine Dioxide Derivatives
Annalisa Tait*1, Amedeo Luppi1, Claudio Cermelli2
1
2
Dipartimento di Scienze Farmaceutiche, Dipartimento di Scienze Igienistiche, Microbiologiche e Biostatistiche
Università degli Studi di Modena e Reggio Emilia, via Campi 183, 41100 Modena, Italy
Received February 20, 2004
A series of 2,1,3- and 1,2,4-benzothiadiazine derivatives were synthesized by alkylation via Mitsunobu
reaction and evaluated for their antiviral activity against ADV, HHV-6, Cox-B5 and H-CMV. Most of them
were active at micromolar level against one or more viral strains. All the molecules studied are poorly cyto-
toxic (maximum non toxic concentrations were >25µM), except one compound that presents a higher cyto-
toxicity (maximum non toxic concentration was 6 µM).
J. Heterocyclic Chem., 41, 747 (2004).
Introduction.
Chemistry.
During the last past decade, a greater understanding of
viral life cycles has resulted in the discovery and valida-
tion of several targets for therapeutic intervention, and an
increase in the number of licensed antiviral drugs from 5 in
1990 to over 30 in 2004. However, it is necessary to con-
tinue the studies in this field, as these compounds are not
always efficacious (owing to virus resistance) or well tol-
erated [1].
Martinez and co-worker have reported interesting antivi-
ral properties for 2,1,3-benzo and imidazothiadiazine
derivatives particularly against Human Cytomegalovirus
and Varicella Zoster Virus infections [2-8]. Also the 1,2,4-
benzothiadiazine and the 1,1,3-trioxo-2H, 4H-thieno[3, 4-
e][1,2,4]thiadiazine rings were investigated as antiviral
systems and found to effectively inhibit the replication of a
variety of HIV-1 strains at the reverse transcription steps,
including strains that are resistant to AZT but not against
HIV-2 (ROD) [9-10]. The benzothiadiazine derivatives
(BTDs) were described also as heterocyclic inhibitors of
PDE7 with concurrent inhibitory activity at PDE4 and
PDE3 [11-12].
The N-1-,N-3-disubstituted-2,1,3- (1 - 9) and the N-2-
monosubstituted 1,2,4-benzothiadiazine (1 0 ) were
obtained by alkylation via Mitsunobu reaction [17] with
the appropriate alcohol from the N- 3 - a r a l k y l - 1H-2,1,3-
benzothiadiazin-4(3H)-ones 2,2-dioxide (11-15) (Scheme
1) and the 6-methyl-2H-1, 2, 4-benzothiadiazin-3(4H)-one
1,1-dioxide (16) (Scheme 2). The site of alkylation of 16
was determined by means of NOESY experiments: thus
aromatic H-5 shows NOE effect with N-H of 10 but not
with CH2 of 4-OH-benzyl moiety.
Scheme 1
(i) TPP, DIAD, THF, N , r.t., overnight.
2
As a part of an ongoing search in the field of benzothia-
diazine molecules [13-16], we reported here the synthesis
and the antiviral activities against Adenovirus (ADV),
Human-Herpesvirus 6 (HHV-6), Coxsackievirus B5 (Cox-
B5) and Human Cytomegalovirus (H-CMV) of new N-
substituted 2,1,3- and 1,2,4-benzothiadiazine derivatives.
The tested viruses widely diffused in the population, are
etiological agents of important diseases and hardly
tractable with the drugs actually available, especially in
immuno-compromised subjects such as HIV and trans-
plant patients. Moreover, in the absence of effective vac-
cines to control the mentioned viral infections new active
compounds are greatly desired. It is worth mentioning that
some of these compounds are substituted with the 2,6-di-
tert-butylphenol group endowed with antioxidant and anti-
inflammatory properties and were therefore tested as free
radical scavengers by reaction with 2,2-diphenyl-1-(2,4,6-
trinitrophenyl)hydrazil (DPPH) using UV spectrometry.
Scheme 2
(i) TPP, DIAD, THF, N , r.t., overnight.
2

748
A. Tait, A. Luppi, C. Cermelli
Vol. 41
The N-3-monosubstituted 2,1,3-benzothiadiazines (11-
substitution (compound 7) improves it.
15) were obtained following the Cohen and Klarberg pro-
cedure [18] starting from the appropriate methyl 2-
aminobenzoate and sulfamoyl chloride. The use of sodium
methoxide instead of 6 N NaOH for the cyclization of the
intermediate N-sulfamoylaminobenzoates improves yields
(Scheme 3).
Biological Results.
The antiviral activity of the synthesized compounds was
evaluated on 5 different viruses, including DNA and RNA
viruses: Adenovirus (ADV), Herpes simplex virus 1
(HSV-1), Human Cytomegalovirus (H-CMV), Human
Scheme 3
(i) Toluene, Et N, 80°C, 1 hour; (ii) NaOCH , CH OH, 40 °C, 2 hours
3
3
3
The structures of new compounds were elucidated from
their analytical and spectroscopic data (1H and 13C nmr).
In the case of compound 1 unequivocal assignment of all
chemical shifts was accomplished using bi-dimensional
experiments such as COSY and Heteronuclear Multiple
Quantum Coherence (HMQC) for one bond correlation
and Heteronuclear Multiple Bond Correlation (HMBC) for
long distance proton/carbon correlations. The nmr data are
in agreement with values reported by A. Castro et al. [19]
for closely related benzothiadiazine dioxide derivatives.
Herpesvirus 6 (HHV-6), and Coxsackievirus B5 (Cox-B5)
(Table 2). Cytotoxicity measurements, based on the inhibi-
tion of cell growth, reveal all compounds to be non-toxic
until 25 µM concentration except 1 that presents a cyto-
toxic activity also at 12 µM and was tested at 6 µM.
No antiviral activity was observed against Herpes simplex
virus 1 (HSV-1). Compounds 3 and 5 show a wider activity
spectrum involving three viral strains; moreover they are
able to abolish almost completely the viral growth of HHV-6.
Introduction of the pyridyl group into the 1-position of
the 2,1,3-BTD heterocycle led to active compounds irre-
spectively of insertion point of this group (4 for compound
3 and 2 for compound 8) and elongation of the link
between the BTD heterocycle and the pyridyl group. On
the contrary, the furylmethyl (compound 1) and the N-
methylphtalimide group (compounds 2 and 9) lead to a
total loss of antiviral activity.
The bulky antioxidant 2,6-dit e rt-butylphenol moiety on
N-1 is compatible with antiviral activity against ADV (com-
pounds 4 and 7) and Cox-B5 (compound 7); however the N-
3-para-substitution (compound 6) resulted in loss of the
antiviral activity which might reflect a spatial restriction in
the target site. The 7-chloro substitution on the benzene ring
in the BTD scaffold improves activity against Cox-B5 viral
strain (compare compound 7 with compound 4).
Radical Scavenging Effect on DPPH Radical.
UV measurement of free radical scavenging activity
(S.A.%) for 1x10-4 M solutions, after storage for 6 hours at
37 °C show that compounds 4, 6 and 7 scavenge the DPPH
radical. The radical scavenging activity of each compound
was expressed by the ratio of lowering of the absorption of
DPPH relative to the absorption of DPPH solution in the
absence of compound (control) (Table 1).
Table 1
Free Radical Scavenging Activity
Compd.
ΔA [a]
S.A.% [b]
4
6
7
0.1306 ± 0.0242
0.2012 ± 0.0122
0.2015 ± 0.0164
27.77
42.78
42.84
The middle anti-ADV activity of the 1,2,4-BTD deriva-
tive 10, N-2-monosubstituted with a 4-hydroxybenzyl
group, suggests that, in the case of this heterocyclic system
and differently to 2,1,3-BTD ring, N,N double substitution
-4
[a] Absorbance decrease at 514 nm against control for 1.0 x 10 M solu-
tions after 6 hours; [b] Scavenging activity % calculated according to the
equation S.A.% = 100 x ΔA/A where A is the control absorbance.
t
t
is not indispensable for antiviral action.
The mechanism of action of these compounds and their
It is noteworthy that N-3 and also bz-substitution on het-
erocyclic system play a significant role on the parameter
under investigation. N-3-(2-Chlorobenzyl)substitution
(compound 4) reduces scavenging activity, while 7-chloro-
viral target remain unknown; it is interesting to point out
that compounds 3, 5, 7 and 8 are able to inhibit DNA and
RNA viruses and this is in agreement with the hypothesis
that they could interact to a common viral target.

Sep-Oct 2004
Synthesis and Antiviral Activity of New Benzothiadiazine Dioxide Derivatives
749
Elemental Analyzer 1106 apparatus. Reagents and solvents
were purchased from common commercial suppliers and used
as received.
In the case of 2,1,3-BTD derivatives, it could be important
to have the ability to assume a butterfly like conformation, a
structural characteristic of widely studied antiviral drugs.
Table 2
In vitro antiviral activity against ADV, HHV-6, Cox-B5 and H-CMV
Comp.
R
R'
R''
NCC
µM [b]
TCID [c]
ADV
PFU [d] % of IFA [e]
positive cells
CoxB5 H-CMV
HHV-6
3
6
6
6
5
6
5
6
5
5
6
6
C- [a]
1
2
3
4
5
6
7
8
1.2x10
9x10
1x10
1.7x10
1.2x10
1.5x10
1.2x10
1.9x10
7.3x10
1.5x10
1.4x10
1.9x10
1.6x10
1.4x10
45
48
46
17
43
41
48
45
44
47
40
56,8
59,0
51,2
1.1
50,8
1.9
61.2
58.3
66.5
59.2
49.7
2
benzyl
benzyl
benzyl
2-furylmethyl
N-methylphtalimide
4-pyridylmethyl
H
H
H
H
H
H
7-Cl
H
H
-
6
3
25
25
25
25
25
25
25
25
25
3
1.1x10
1.9x10
2.2x10
1.3x10
4.8x10
1.1x10
2
2
3
2
2
2-Cl-benzyl
2-Cl-benzyl
4-Cl-benzyl
2-Cl-benzyl
2-phenylethyl
2-phenylethyl
-
3,5-ditert-butyl-4-OH-benzyl
4-NO -benzyl
2
3,5-ditert-butyl-4-OH-benzyl
3,5-ditert-butyl-4-OH-benzyl
2-pyridylethyl
3
9
10
N-methylphtalimide
-
1x10
2
1.3x10
[a] negative control; [b] non cytotoxic concentrations; [c] Total cells infectious dose /mL: it is the virus dilution able, in theory, to infect
50
the 50% cell cultures obtained by extrapolation from different values in a 96 well cell culture plate by means of the Reed and Muench for-
mula which provides the exact exponent of the 10 fold dilution; [d] Plaque Forming Unit/mL; [e] Immunofluorescence Assay.
3-(2-chlorobenzyl)-1 H-2, 1, 3-benzothiadiazin-4(3H)-one 2,2-
Dioxide (12).
EXPERIMENTAL
Melting points determined in open capillary tubes (Büchi
13
General Procedure.
1
510 capillary apparatus) were uncorrected. Both H and
C
nmr spectra were recorded with a Brucker DPX 200 spectrom-
eter; chemical shifts (δ) are reported as ppm downfield from
To a stirred and cooled (0 °C) solution of 2-chlorobenzylamine
(5.00 g, 35 mmol) in dichloromethane (50 mL), chlorosulfonic
acid (1.36 g, 12 mmol) was added drop by drop and the reaction
mixture was stirred for an additional hour after being warmed to
room temperature gradually. The resultant white salt between
2-chlorobenzylsulfamic acid and 2-chlorobenzylamine was col-
lected, dissolved in toluene (50 mL), and treated with phosphorus
pentachloride (3.00 g, 14.4 mmol). The mixture was refluxed for
1 hour and the inorganic by-products removed by filtration. The
filtrate was evaporated in vacuo to give the N-2-chlorobenzylsul-
famoyl chloride as an oily residue that was used in the next syn-
thetic step without further purification.
To a solution of methyl 2-aminobenzoate (1.81 g, 12.5 mmol)
and triethylamine (1.92 g, 19 mmol) in toluene (50 mL) was slowly
added a solution of N-2-chlorobenzylsulfamoyl chloride (2.89 g,
12 mmol) in toluene (10 mL) and the resulting mixture was heated
at 80 °C for 1 hour. The resultant triethylamine hydrochloride was
filtered and the filtrate was evaporated in vacuo to give an oily
residue. The residue was dissolved in 50 mL of a freshly prepared
0.5 M sodium methoxide methanolic solution. After stirring for 2
13
tetramethylsilane as external standard, and the C chemical
shifts were referenced to the solvent peaks: dimethylsulfoxide-
d (39.6 ppm). Coupling constants (J) are in Hertz (Hz), multi-
6
plicities are abbreviated as follows: s, singlet; d, doublet; t,
triplet; dd, double doublet; ddd, double double doublet; dt,
double triplet; m, a more complex multiplet or overlapping
multiplets; b, indicates a broadening of the signal. Ir spectra
were recorded on a Perkin-Elmer 1600 FT-IR spectrometer as
Nujol mull. The spectra were in agreement with the proposed
structures. The purity of compounds was checked by thin layer
chromatography performed on aluminium sheets silica gel 60
F
, 0.2 mm thick. Flash chromatography was performed on
254
Silica gel Merck (230-400 mesh). UV spectra were acquired on
a Cary 50 Bio UV-VISIBLE Spectrophotometer Va r i a n .
Elemental analyses for C, H and N were performed in
Microanalysis Laboratory of Dipartimento di Scienze
Farmaceutiche of Modena University on a Carlo Erba

750
A. Tait, A. Luppi, C. Cermelli
Vol. 41
hours at 40 °C the solvent was evaporated under reduced pressure
and the residue dissolved in water. After cooling the aqueous solu-
tion gave compound 12 on acidification with hydrochloric acid as a
H), 7.17 (dd, 1H, J=0.7, 8.1 Hz, H-8), 4.04 (m, 2H, NCH ) 2.97
13
2
(m, 2H, CH Ph); C nmr (dimethylsulfoxide-d ): δ 162.3 (C),
2
6
138.7 (C), 138.4 (C), 135.8 (CH), 129.8 (CH), 129.2 (2xCH),
129.0 (2xCH), 127.0 (CH), 124.6 (CH), 120.2 (CH), 118.1 (C),
1
white solid (2.55 g, 66%); mp 142-144 °C (DMF/water); H nmr
(dimethylsulfoxide-d ): δ 10.00 (bs, 1H, NH deuterium oxide
-1
43.1 (CH ), 35.0 (CH ); ir (nujol) 3127, 1638, 1606, 1176 cm .
2
2
6
exchangeable), 7.97 (dd, 1H, J=1.5, 7.8 Hz, H-5), 7.65 (dt, 1H,
Anal. Calcd. for C H N O S: C, 59.59%; H, 4.67%; N,
15 14 2 3
9.27%. Found: C, 58.86%; H, 4.80%; N, 9.21%.
J=1.5, 7.6 Hz, H-7), 7.47 (m, 1H, aromatic), 7.28 (m, 4H, aro-
13
matic), 7.13 (dd, 1H, J=1.0, 7.6 Hz, H-8), 5.08 (s, 2H, CH Ph); C
nmr (dimethylsulfoxide-d ): δ 162.5 (C), 138.7 (C), 136.1 (CH),
2
3-Benzyl-1-(2-furylmethyl)-1H-2,1,3-benzothiadiazin-4(3H)-
one 2,2-Dioxide (1).
6
133.6 (C), 132.0 (C), 130.1 (CH), 129.8 (CH), 129.5 (CH), 128.4
(CH), 127.8 (CH), 125.0 (CH), 120.7 (CH), 118.3 (C), 42.8 (CH );
2
General Procedure.
-1
(ir (nujol) 3172, 1640, 1361, 1175, 1049, 750 cm .
Anal. Calcd for C H ClN O S: C, 52.10%; H, 3.44%; N,
All of these reactions were carried out under a nitrogen atmos-
phere. To a stirred and cooled (0 °C) solution of 3-benzyl-1H-
2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (11) (1.00 g, 3.2
mmol), 2-furylmethanol (0.21 g, 2.1 mmol) and triphenyl-
phosphine (1.10 g, 4.2 mmol) in dry THF (20 mL), diisopropyla-
zodicarboxylate (0.85 g, 4.2 mmol) was added dropwise in five
minutes and the reaction mixture was allowed to stand at that
temperature for an additional 30 minutes. The orange-red colour
of diisopropylazodicarboxylate disappears immediately. After
being gradually warmed to room temperature, the mixture was
stirred overnight. Removal of the solvent in vacuo gave a residue,
which was washed with petroleum ether 40-60 °C, then purified
by silica gel flash column chromatography using
cyclohexane/ethyl acetate (6:4, v/v) as eluent to afford 1 (0.38 g,
14 11
8.68%. Found: C, 52.18%; H, 3.58%; N, 8.94%.
2 3
The already known 3-benzyl-1H-2,1,3-benzothiadiazin-4(3H)-
one 2,2-dioxide (11) [20] and compounds 13-15 were prepared
using the procedure described above. The physical and spectral
data for 13-15 are listed below.
3-(4-Chlorobenzyl)-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-
Dioxide (13).
This compound was obtained starting from methyl 2-
aminobenzoate by reaction with 4-chlorobenzylsulfamoyl chlo-
ride as a white solid in 26% yield; mp 167-170 °C
1
(methanol/water); H nmr (dimethylsulfoxide-d ): δ 10.30 (bs,
6
1H, NH deuterium oxide exchangeable), 7.98 (ddd, 1H, J=0.4,
1.6, 7.9 Hz, H-5), 7.65 (dt, 1H, J=1.6, 7.4 Hz, H-7), 7.39 (m, 4H,
aromatic), 7.26 (dt, 1H, J=1.1, 7.4 Hz, H-6), 7.14 (1H, ddd, J=
1
yield 49%); mp 115°C (acetone/petroleum ether 40-60 °C); H
nmr (dimethylsulfoxide-d ): δ 7.99 (dd, 1H, J=1.6, 7.9Hz, H-5),
6
7.81 (ddd, 1H, J=1.6, 7.4, 8.0 Hz, H-7), 7.65 (dd, 1H, J=1.2, 8.0
Hz, H-8), 7.50 (dd, 1H, J=0.9, 1.9 Hz, H-5 furyl), 7.49 (ddd, 1H,
J=1.2, 7.4, 7.9 Hz, H-6), 7.39-7.25 (m, 5H, benzyl), 6.30 (dd, 1H,
J=1.9, 3.2Hz, H-4 furyl), 6.17 (dd, 1H, J=0.9, 3.2 Hz, H-3 furyl),
13
0.4, 1.1, 7.4 Hz, H-8), 5.00 (2H, s, CH Ph); C nmr (dimethyl-
2
sulfoxide-d ): δ 162.4 (C), 138.4 (C), 136.0 (CH), 135.8 (C),
6
132.7 (C), 130.2 (2xCH), 130.0 (CH), 128.9 (2xCH), 125.1 (CH),
120.5 (CH), 118.3 (C), 44.4 (CH ); (ir (nujol) 3165, 1655, 1610,
2
13
5.08 (s, 2H, CH furyl), 4.95 (s, 2H, CH benzyl); C nmr
-1
1329, 1175, 1017, 756 cm .
A n a l. Calcd for C H ClN O S: C, 52.10%; H, 3.44%; N,
2
(dimethylsulfoxide-d ): δ 162.0 (C-4), 148.0 (C-2' furyl), 144.3
2
6
14 11
8.68%. Found: C, 52.39%; H, 3.15%; N, 8.96%.
2 3
(C-5' furyl), 140.0 (C-8a), 136.4 (C-1' benzyl), 135.8 (C-7), 129.3
(C-5), 129.0 (C-3' and C-5' benzyl), 128.4 (C-2' and C-6' benzyl),
128.2 (C-4' benzyl), 127.6 (C-6), 124.2 (C-8), 123.0 (C-4a),
7-Chloro-3-(2-chlorobenzyl)-1H-2, 1, 3-benzothiadiazin-4(3H)-
one 2,2-Dioxide (14).
111.2 (C-3' furyl), 111.1 (C-4' furyl), 49.1 (CH benzyl), 46.8
-1
2
(CH furyl); ir (nujol): 1673, 1379, 1180, 1010, 603 cm .
2
Anal. Calcd. for C
This compound was obtained starting from methyl 2-amino-4-
chlorobenzoate by reaction with 2-chlorobenzylsulfamoyl chlo-
ride as a white solid in 20% yield; mp 128-130 °C
H N O S: C, 61.94%; H, 4.38%; N,
19 16 2 4
7.60%. Found: C, 62.18%; H, 4.15%; N, 7.81%.
The compounds 2-10 were prepared using the procedure
described above. Their physical and spectral data are listed
below.
1
(methanol/water); H nmr (dimethylsulfoxide-d ): δ 8.98 (bs,
6
1H, NH deuterium oxide exchangeable), 7.86 (dd, 1H, J=1.2, 7.8
Hz, aromatic), 7.43 (m, 1H, aromatic), 7.26 (m, 3H, aromatic),
7.03 (dd, 1H, J=1.9, 7.8 Hz, aromatic), 7.00 (m, 1H, aromatic),
2-[(3-Benzyl-2,2-dioxido-4-oxo-3,4-dihydro-1H-2,1,3-benzothia-
diazin-1-yl)methyl]-1H-isoindole-1, 3(2H)dione (2).
13
5.03 (s, 2H, CH Ph); C nmr (dimethylsulfoxide-d ): δ 162.2
6
2
(C), 142.5 (C), 139.9 (C), 133.9 (C), 131.9 (C), 131.6 (CH),
129.7 (CH), 129.4 (CH), 128.5 (CH), 127.8 (CH), 123.4 (CH),
120.3 (CH), 116.3 (C), 42.8 (CH ); ir (nujol) 3117, 1666, 1602,
This compound was obtained starting from 3-benzyl-1H-2,1,3-
benzothiadiazin-4(3H)-one 2,2-dioxide (11) by reaction with 2-
(hydroxymethyl)-1H-isoindole-1, 3(2H)-dione. Flash column
chromatography: eluent chloroform/acetone (9.5:0.5, v/v). Yield
2
-1
1325, 1161, 752 cm .
Anal. Calcd. for C H Cl N O S: C, 47.07%; H, 2.82%; N,
14 10 2 2 3
7.84%. Found: C, 47.36%; N, 8.11%.
1
82%; mp 148-150 °C (acetone/petrol ether 40-60 °C); H nmr
(dimethylsulfoxide-d ): δ 7.96 (m, 1H, aromatic ), 7.84 (m, 5H,
6
3 - ( 2 - P h e n y l e t h y l ) - 1H-2, 1, 3-benzothiadiazin-4(3 H)-one 2,2-
Dioxide (15).
aromatic ), 7.79 (dd, 1H, J=1.5, 8.2 Hz, aromatic ), 7.51 (ddd, 1H,
J=2.2,6.3, 6.5 Hz, aromatic), 7.29 (m, 5H, aromatic H), 5.64 (s,
13
2H, NCH N), 5.00 (s, 2H, NCH Ph); C nmr (dimethylsulfox-
This compound was obtained starting from methyl 2-
aminobenzoate by reaction with 2-phenylethylsulfamoyl chloride
as a white solid in 76% yield; mp138-140 °C (acetone/petroleum
2
ide-d ): δ 167.1 (2xC), 162.1 (C), 138.5 (C), 136.3 (C), 135.7
2
6
(CH), 135.5 (2xCH), 131.3 (2xC), 129.8 (CH), 128.9 (2xCH),
128.3 (2xCH), 128.2 (CH), 128.1 (CH), 125.3 (CH), 124.1
(2xCH), 123.6 (C), 54.2 (CH ), 47.0 (CH ); ir (nujol): 1782,
1
ether 40-60 °C); H nmr (dimethylsulfoxide-d ): δ 10.80 (bs, 1H,
6
NH deuterium oxide exchangeable), 7.99 (dd, 1H, J=1.6, 7.6 Hz,
H-5), 7.67 (dt, 1H, J=1.6, 7.6 Hz, H-6/7), 7.28 (m, 6H, aromatic
2
2
-1
1737, 1688, 1025, 891cm .

Sep-Oct 2004
Synthesis and Antiviral Activity of New Benzothiadiazine Dioxide Derivatives
751
A n a l. Calcd. for C
9.39%. Found: C, 62.14%; H, 3.52%; N, 9.58%.
H
N O S: C, 61.74%; H, 3.83%; N,
Anal. Calcd. for C H ClN O S: C, 55.09%; H, 3.52%; N,
21 16 3 5
9.18%. Found: C, 55.45%; H, 3.90%; N, 9.53%.
23 17
3
5
3-Benzyl-1-(4-pyridiylmethyl)-1H-2,1,3-benzothiadiazin-4(3H)-
one 2,2-Dioxide (3).
3-(4-Chlorobenzyl)-1-(3,5-di-ter t-butyl-4-hydroxybenzyl)-1H-
2,1,3-benzothiadiazin-4(3H)-one 2,2-Dioxide (6).
This compound was obtained starting from 3-benzyl-1H-2,1,3-
benzothiadiazin-4(3H)-one 2,2-dioxide (11) by reaction with 4-
(hydroxymethyl)pyridine. Flash column chromatography: eluent
chloroform/acetone (8:2, v/v). Yield 69%; mp 100 °C
This compound was obtained starting from 3-(4-chloroben-
zyl)-1H-2, 1, 3-benzothiadiazin-4(3H)-one 2,2-dioxide (13) by
reaction with 2,6-di-t e rt-butyl-4-(hydroxymethyl)phenol. Flash
column chromatography: eluent cyclohexane/ethyl acetate (1:1,
1
1
(acetone/petroleum ether 40-60 °C); H nmr (dimethylsulfoxide-
d ): δ 8.46 (bd, 2H, J=6.0 Hz, pyridine H-2 and H-6), 8.06 (dd,
v/v). Yield 53%; mp 142-145 °C (methanol/water); H nmr
(dimethylsulfoxide-d ): δ 8.00 (dd, 1H, J=1.4, 7.9 Hz, H-5), 7.88
6
1H, J=1.6, 7.8 Hz, H-5), 7.78 (ddd, 1H, J=1.6, 7.5, 8.0 Hz, H-7),
7.43 (m, 7H, aromatic), 7.15 (bd, 2H, J=6.0 Hz, pyridine H-3 and
6
(ddd, 1H, J=1.4, 7.9, 8.1 Hz, H-7), 7.82 (dd, 1H, J=1.0, 8.1 Hz,
H-8), 7.54 (ddd, 1H, J=1.0, 7.9, 8.1 Hz, H-6), 7.40 (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, 1H, OH, deuterium oxide
13
H-5), 5.18 (s, 2H, CH Py), 5.03 (s, 2H, CH Ph); C nmr
2
(dimethylsulfoxide-d ): δ 162.0 (C), 150.1 (2xCH), 144.2 (C),
2
6
139.6 (C), 136.2 (C and CH), 130.2 (CH), 129.0 (2xCH), 128.6
(2xCH), 128.3 (CH), 126.9 (CH), 123.0 (2xCH), 122.1 (CH),
121.6 (C), 53.5 (CH ), 46.6 (CH ); ir (nujol) 1694, 1603, 1313,
exchangeable), 6.71 (s, 2H, H aromatic), 4.98 (s, 2H, CH Ph),
1 3
2
4.64 (s, 2H, CH Ph), 1.26 (s, 18H, t e rt-butyl); C nmr
2
(dimethylsulfoxide-d ): δ 161.8 (C), 154.7 (C), 140.1 (C), 139.3
2
2
6
-1
1173, 703 cm .
Anal. Calcd. for C
11.07%. Found: C, 63.44%; H, 4.57%; N, 10.98%.
(2xC), 139.2 (C), 136.0 (CH), 135.2 (C), 132.9 (C), 130.1
(2xCH), 129.8 (CH), 129.0 (2xCH), 125.5 (2xCH), 124.8 (CH),
124.2 (C), 123.9 (C), 57.8 (CH ), 46.0 (CH ), 34.7 (2xC), 30.5
H
N O S: C, 63.31%; H, 4.52%; N,
3 3
20 17
2
2
-1
(6xCH ); ir (nujol) 3577, 1677, 1605, 1326, 1176 cm .
H ClN O S: C, 64.37%; H, 6.15%; N,
29 33 2 4
3
Anal. Calcd. for C
3-(2-Chlorobenzyl)-1-(3,5-di-ter t-butyl-4-hydroxybenzyl)-1H-
2,1,3-benzothiadiazin-4(3H)-one 2,2-Dioxide (4).
5.18%. Found: C, 64.62%; H, 6.24%; N, 5.48%.
This compound was obtained starting from 3-(2-chloroben-
zyl)-1H-2, 1, 3-benzothiadiazin-4(3H)-one 2,2-dioxide (12) by
reaction with 2,6-di-t e rt-butyl-4-(hydroxymethyl)phenol. Flash
column chromatography: eluent cyclohexane/ethyl acetate (6:4,
v/v). Yield 17%; mp183-185 °C (acetone/petroleum ether 40-60
7-Chloro-3-(2-chlorobenzyl)-1-(3,5-di-tert-butyl-4-hydroxyben-
zyl)-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (7).
This compound was obtained starting from 7-chloro-3-(2-
chlorobenzyl)-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide
(14) by reaction with 2,6-di-tert-butyl-4-(hydroxymethyl)phenol.
Flash column chromatography: eluent cyclohexane/ethyl acetate
1
°C; H nmr (dimethylsulfoxide-d ): δ 8.00 (dd, 1H, J=1.5, 7.9
6
Hz, H-5), 7.92 (dd, 1H, J=1.5, 7.3 Hz, aromatic), 7.82 (bd, 1H,
J=8.4 Hz, aromatic), 7.60 (dt, 1H, J=1.3, 7.5 Hz, aromatic), 7.50
(m, 1H, aromatic), 7.38-7.25 (m, 2H, aromatic), 7.13 (m, 1H, aro-
matic), 7.02 (s, 1H, OH, deuterium oxide exchangeable), 6.68 (s,
1
(9:1, v/v). Yield 40%; mp 165-168 °C (methanol/water); H nmr
(dimethylsulfoxide-d ): δ 8.00 (dd, 1H, J=1.5,7.9 Hz, H-5), 7.66
6
(dd, 1H, J=2.0, 8.5 Hz, H-6), 7.50 (m, 1H, aromatic), 7.32 (m,
2H, aromatic), 7.15-7.11 (m, 1H, aromatic), 7.06 (s, 1H, OH,
deuterium oxide exchangeable), 6.74 (s, 2H, aromatic), 5.04 (s,
2H, CH Ph), 4.74 (s, 2H, CH Ph), 1.28 (s, 18H, tert-butyl); ir
2H, aromatic), 4.98 (s, 2H, CH Ph), 4.67 (s, 2H, CH Ph), 1.26 (s,
13
2
18H, tert-butyl); C nmr (dimethylsulfoxide-d ): δ 161.7 (C),
2
6
154.7 (C), 140.1(C), 139.3 (2xC), 136.2 (CH), 133.1 (C), 131.8
(C), 129.9 (CH), 129.8 (CH), 129.7 (CH), 128.3 (CH), 127.9
(CH), 125.7 (2xCH), 125.5 (CH), 124.6 (CH), 123.6 (C), 58.7
2
2
-1
(nujol) 3575, 1684, 1596, 1326, 1321, 1185 cm .
Anal. Calcd. for C Cl N O S: C, 60.42%; H, 5.60%; N,
4.87%. Found: C, 60.54%; H, 5.74%; N, 4.84%.
H
29 32
2 2 4
(CH ), 44.7 (CH ), 34.7 (2xC), 30.4 (6xCH ); ir (nujol): 3585,
2
2
3
-1
1677, 1603, 1604, 1326, 1188, 1052 cm .
Anal. Calcd. for C ClN O S : C, 64.37%; N, 5.18%.
Found: C, 64.63%; N, 5.41%.
3-(2-Phenylethyl)-1-(2-pyridylethyl)-1H-2,1,3-benzothiadiazin-
4(3H)-one 2,2-Dioxide (8).
H
29 33
2 4
This compound was obtained starting from 3-(2-phenylethyl)-
1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (1 5) by reac-
tion with 2-(2-hydroxyethyl)pyridine as a yellow oil (yield 86%).
Flash column chromatography: eluent chloroform/acetone
3-(2-Chlorobenzyl)-1-(4-nitrobenzyl)-1H-2,1,3-benzothiadiazin-
4(3H)-one 2,2-Dioxide (5).
This compound was obtained starting from 3-(2-chloroben-
zyl)-1H-2, 1, 3-benzothiadiazin-4(3H)-one 2,2-dioxide (12) by
reaction with 4-nitrobenzyl alcohol. Flash column chromatogra-
1
(9.8:0.2, v/v). H nmr (dimethylsulfoxide-d ): δ 8.41 (m, 1H,
6
pyridine-H), 8.00 (dd, 1H, J=1.6, 7.8 Hz, H-5), 7.74 (dt, 1H,
J=1.6, 7.8 Hz, H-7), 7.61 (dt, 1H, J=1.9, 7.8 Hz, pyridine H-4/5),
7.54 (bd, 1H, J=7.8 Hz, H-8), 7.46 (bt, 1H, J=7.8, H-6), 7.29-7.12
(m, 7H, H aromatic), 4.24 (t, 2H, J=7.1 Hz, NCH CH Py), 3.97
phy: eluent cyclohexane/ethyl acetate (7:3, v/v). Yield 67%; mp
1
108-110 °C (methanol); H nmr (dimethylsulfoxide-d ): δ 8.20
6
(bd, 2H, J=8.8 Hz, 4-nitrobenzyl H-3 and H-5), 8.08 (dd, 1H,
J=1.6, 7.8 Hz, H-5), 7.83 (1H, ddd, J=1.6, 7.4, 8.0 Hz, aromatic),
7.53 (m, 3H, H aromatic), 7.50 (bd, 2H, J=8.8 Hz, 4-nitrobenzyl
2
2
(m, 2H, NCH C H Ph), 2.95 (m, 4H, NCH CH P h ,
2
2
NCH CH Py); C nmr (dimethylsulfoxide-d ): δ 161.9 (C),
2
2
13
2
2
6
157.6 (C), 149.4 (CH), 139.9 (C), 138.1 (C), 137.2 (CH), 135.8
(CH), 129.9 (CH), 129.2 (2xCH), 129.0 (2xCH), 127.1 (CH),
126.7 (CH), 124.1 (CH), 122.8 (CH), 122.4 (CH), 122.1 (C), 51.7
(CH ), 44.2 (CH ), 35.9 (CH ), 34.8 (CH ); ir (nujol) 1711,
H-2 and H-6) 7.30 (m, 3H, H aromatic), 5.30 (s, 2H, CH Ph),
13
2
5.11 (s, 2H, CH Ph); C nmr (dimethylsulfoxide-d ): δ 162.0
2
6
(C), 147.8 (C), 143.0 (CH), 139.8 (C), 136.4 (C), 133.2 (CH),
132.2 (C), 130.4 (CH), 129.9 (CH), 129.8 (CH), 129.5 (2xCH),
129.0 (CH), 127.8 (CH), 127.2 (C), 124.3 (2xCH), 122.4 (CH),
121.7 (C), 54.2 (CH ), 44.4 (CH ); ir (nujol) 1681, 1603, 1518,
2
2
2
2
-1
1604, 1461, 1378, 1261, 1171 cm .
Anal. Calcd. for C N O S: C, 64.85%; H, 5.19%; N,
10.31%. Found: C, 65.11%; H, 5.41%; N, 10.50%.
H
22 21
3 3
2
2
-1
1175, 1050, 759 cm .

752
A. Tait, A. Luppi, C. Cermelli
Vol. 41
2-{[2,2-Dioxido-4-oxo-3-(2-phenylethyl)-3,4-dihydro-1H-2,1,3-
benzothiadiazin-1-yl]methyl}-1H-isoindole-1,3-(2H)-dione (9).
Cytotoxicity Assays.
The non-toxic concentrations of each compound evaluated in
the antiviral assays were assessed by means of the MTT test [24].
Briefly, 24 hours growth VERO cells in a 24-well cell culture
plate were placed in contact with different concentrations of the
compounds under study dissolved in maintenance medium. After
48 hours incubation, the MTT stain was added to each well at a
0.5 mg/mL final concentration. MTT is a tetrazolium salt that is
converted to an insoluble purple formazan by cleavage of the
tetrazolium ring by mitochondrial dehydrogenase of live cells, so
the colour formation is proportional to the number of live cells.
After 3 hours incubation, adding 0.1 N HCl in absolute iso-
propanol the insoluble formazan developed in viable cells was
solubilized. Absorbance of converted dye was measured at a
wavelength of 570 nm with background subtraction at 630 nm.
Each dilution was always tested in triplicate and in each set of
experiments three control wells without drug were included.
This compound was obtained starting from 3-(2-phenylethyl)-
1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (5) by reaction
with N-(hydroxymethyl)phtalimide as a white powder. Flash col-
umn chromatography: eluent chloroform. Yield 62%; mp 128-
1
130 °C (acetone/petroleum ether 40-60 °C); H nmr (dimethyl-
sulfoxide-d ): δ 7.96 (bd, 1H, J=7.8 Hz, H-5), 7.87-7.76 (m, 6H,
6
aromatic), 7.51 (dt, 1H, J=2.1,7.0, aromatic), 7.37-7.19 (m, 5H,
aromatic), 5.60 (s, 2H, NCH N), 4.00 (t, 2H, J=8.0 Hz,
13
2
N CH CH Ph), 2.89 (t, 2H, J=8.0 Hz, NCH CH Ph); C nmr
2
(dimethylsulfoxide-d ): δ 167.1 (2xC), 161.8 (C), 138.4 (C),
2
2
2
6
138.1 (C and CH), 135.6 (2xCH), 131.2 (2xC), 129.6 (CH), 129.1
(2xCH), 129.0 (2xCH), 128.3 (CH), 127.1 (CH), 125.5 (CH),
124.1 (2xCH), 123.7 (C), 54.3 (CH ), 44.7 (CH ), 35.0 (CH ); ir
2
2
2
-1
(nujol) 1776, 1720, 1606, 1302, 1160, 978 cm .
Anal. Calcd. for C N O S: C, 62.46%; H, 4.15%; N,
9.11%. Found: C, 62.71%; H, 4.09%; N, 9.35%.
H
24 19
3 5
Antiviral Assays.
2-(4-Hydroxybenzyl)-6-methyl-2H-1, 2, 4-benzothiadiazin-
3(4H)-one 1,1-Dioxide (10).
Antiviral activity was ascertained by means of plaque assays
for HSV-1 and H-CMV and by viral yield assays for ADV, Cox-
B5 and HHV-6. Plaque assays were carried out as follows. For
HSV-1, 24 hours growth VERO in 24-well tissue culture plates
and 48 hours MRC-5 cells for H-CMV were infected with 50
PFU/well. After 1 hour adsorption, virus inoculum was removed,
the plates washed with PBS, and the maintenance medium with
or without (control cultures) the compounds under study. Then
0.6% human γ-globulin was added. After 48 hours incubation,
the plates were fixed with methanol, stained with Giemsa and the
plaques counted. Each compound was assayed in triplicate.
For ADV and Cox-B5, 24 hours growth VERO cells were
This compound was obtained starting from 6-methyl-2H-
1,2,4-benzothiadiazin-3(4H)-one 1,1-dioxide (6) [21] by reaction
with 4-(hydroxymethyl)phenol. Flash column chromatography:
eluent cyclohexane/ethyl acetate (5:5, v/v). Yield 64%; mp 200-
1
204 °C (acetone/petroleum ether 40-60 °C); H nmr (dimethyl-
sulfoxide-d ): δ 11.30 (s, 1H, NH, deuterium oxide exchange-
6
able), 9.35 (s, 1H, OH, deuterium oxide exchangeable), 7.72 (d,
1H, J=8.0 Hz, H-8), 7.14 (m, 3H, H-7 and 4-hydroxybenzyl H-2
and H-6), 7.05 (bs, 1H, H-5), 6.68 (m, 2H, 4-hydroxybenzyl H-3
13
and H-5), 4.82 (s, 2H, CH Ph), 2.37 (s, 3H, CH ); C nmr
2
(dimethylsulfoxide-d ): δ 157.4 (C), 150.2 (C), 146.0 (C), 134.9
3
infected at a multiplicity of infection (MOI) of 0.01 TCID /cell.
50
6
(C), 130.0 (2xCH), 127.2 (C), 124.7 (CH), 122.4 (CH), 120.2
After 1 hour adsorption at 37 °C, the inoculum was removed, the
plates washed with PBS and the compounds added to the mainte-
nance medium. After 2 days incubation, the plates were frozen
and thawed three times and the viral yield was titred by the end-
point titration. In this case, 10-fold dilutions of each cell lysate
were seeded onto 24 hours growth VERO cells in a 96-well cul-
(C), 117.3 (CH), 115.5 (2xCH), 43.5 (CH ), 21.8 (CH ); ir
3
2
1
(nujol): 3455, 3250, 1680, 1610, 1593, 1517, 1272, 1152 cm .
Anal. Calcd. for C N O S: C, 56.59%; H, 4.43%; N,
8.80%. Found: C, 56.93%; H, 4.30%; N, 8.95%.
H
15 14
2 4
Radical Scavenging Effect on DPPH Radical.
ture plate and after 2 days the titre, expressed as TCID /mL, was
50
-4
An ethanolic solution (2 mL, 1x10 M) of the tested com-
-4
pound was added to 2 mL of a DPPH solution (1 x 10 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 fifteen minutes. The mean values were
obtained from quadruplicate experiments.
read, taking account of the final dilution which afforded the typi-
cal viral cytopathic effect and the results were elaborated using
the Reed and Muench formula [25].
5
For HHV-6, 10 SupT-1 cells in 24-well plates were infected at
a MOI of 0.01 TCID /cell and incubated for 2 hours and then the
50
medium with the drugs added. After 6 days incubation, the virus
growth was ascertained determining the percentage of infected
cells by immunofluorescence assays as described elsewhere [26].
Cells and Virus.
The green monkey kidney cell line VERO was used to grow
HSV-1, Cox-B5 and ADV, while HHV-6 was grown in SupT-1
cells (a human T cell line) and H-CMV in MRC-5 (human fibrob-
lasts). VERO and MRC-5 cells were cultured in Minimum
Essential Medium (MEM) added with 10% (growth medium) or
5% (maintenance medium) fetal calf serum (FCS), penicillin
(100U/mL), streptomycin (100µg/mL) and incubated at 37 °C with
Acknowledgements.
Thanks are due to Mrs. Rossella Gallesi who performed ele-
mental analyses of synthesized compounds and Mr. Daniele
Montanini, Mr. Dennis Cattabriga and Mrs. Elena Ballabeni for
their valuable assistance.
5% CO . SupT-1 cells were grown with RPMI1640 medium added
2
REFERENCES AND NOTES
with 10 % heat inactivated FCS and antibiotics as for MEM.
HSV-1, ADV and Cox-B5 were clinical strains adapted to
grow in cells with several in vitro passages. For H-CMV and
HHV-6 the references strain Towne [22] and Z-29 [23] were
used, respectively. A same stock of each virus kept frozen at –80
°C was used in all the experiments.
*
To whom correspondence should be addressed; E-mail:
tait.annalisa@unimore.it
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