Novel potential agents for human cytomegalovirus infection: Synthesis and antiviral activity evaluation of benzothiadiazine dioxide acyclonucleosides

Article abstract of DOI:10.1021/jm980327z

The first acyclonucleosides based on the benzothiadiazine dioxide system were synthesized following the silylation procedure. Several acyclic moieties, including acetoxyethoxymethyl, benzyloxymethyl, and propargyloxymethyl groups, were introduced. Two synthetic strategies were designed to selectively obtain the N-1 or N-3 derivatives. Lipase-mediated deacylation was used for the deprotection of the acyclonucleosides. Some of the benzothiadiazine dioxide acyclonucleosides, in particular 16, proved active against human cytomegalovirus (CMV) at concentrations slightly higher than that found for ganciclovir [50% inhibitory concentration (IC₅₀) = 3.5- 3.7 μg/mL, cytotoxicity (CC₅₀) ≥ 40 μg/mL, MCC = 20 μg/mL]. Additionally, compound 16 inhibited the replication of human immunodeficiency virus type 1 (HIV-1) and HIV-2 in CEM cells at concentrations that were 5- fold lower than its cytotoxic concentration.

Full text of DOI:10.1021/jm980327z

J . Med. Chem. 1999, 42, 1145-1150
1145
Novel P oten tia l Agen ts for Hu m a n Cytom ega lovir u s In fection : Syn th esis a n d
An tivir a l Activity Eva lu a tion of Ben zoth ia d ia zin e Dioxid e Acyclon u cleosid es
Ana Martinez,*,† Ana I. Esteban,† Ana Castro,† Carmen Gil,† Santiago Conde,† Graciela Andrei,‡ Robert Snoeck,‡
J an Balzarini,‡ and Erik De Clercq‡
Instituto de Quı´mica Me´dica (CSIC), J uan de la Cierva 3, 28006 Madrid, Spain, and Rega Institute for Medical Research,
Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received May 27, 1998
The first acyclonucleosides based on the benzothiadiazine dioxide system were synthesized
following the silylation procedure. Several acyclic moieties, including acetoxyethoxymethyl,
benzyloxymethyl, and propargyloxymethyl groups, were introduced. Two synthetic strategies
were designed to selectively obtain the N-1 or N-3 derivatives. Lipase-mediated deacylation
was used for the deprotection of the acyclonucleosides. Some of the benzothiadiazine dioxide
acyclonucleosides, in particular 16, proved active against human cytomegalovirus (CMV) at
concentrations slightly higher than that found for ganciclovir [50% inhibitory concentration
(IC50) ) 3.5-3.7 µg/mL, cytotoxicity (CC50) g 40 µg/mL, MCC ) 20 µg/mL]. Additionally,
compound 16 inhibited the replication of human immunodeficiency virus type 1 (HIV-1) and
HIV-2 in CEM cells at concentrations that were 5-fold lower than its cytotoxic concentration.
Sch em e 1a
In tr od u ction
Human cytomegalovirus (CMV) is a ubiquitous hu-
man pathogen that affects 50-90% of adults. In healthy
individuals, CMV infections are usually asymptomatic.
However, in immunocompromised individuals CMV is
responsible for the most commom sight- and life-
threatening opportunistic viral infection.1 CMV can
cause severe morbidity and mortality in congenitally
infected newborns and immunocompromised patients.
It is the primary cause of death in recipients of allogenic
bone marrow transplants2 and renal transplants.3 In
addition, patients suffering from the acquired immune
deficiency syndrome (AIDS) are nearly always HCMV
seropositive and often develop symptomatic reactivation
disease as the immunodeficiency progresses, retinitis
and colitis being the most frequent manifestations.4
Few therapeutic options are available for the treat-
ment of CMV infections. Only ganciclovir,5 foscarnet,6
and cidofovir7 have been approved by the FDA for the
treatment of CMV diseases. Although treatment of CMV
infections with these drugs has produced clinical im-
provement in the majority of the patients, the com-
pounds suffer from poor oral bioavailability8 and adverse
side effects. Therefore, there still is an urgent need for
effective, nontoxic anti-CMV drugs with good oral
availability. Moreover, with the advent of virus strains
there are resistant to current drugs,9 new drugs that
act by a new mechanism of action may be highly
desirable for the treatment of CMV infections.
i
ii
iii
iv
v
a
Reagents: (i) C6H6; (ii) 6 N NaOH; (iii) HMDS/N2; (iv)
BF3‚Et2O/CH2Cl2/RCH2OCH2OAc; (v) CAL/t-BuOH/buffer pH )
7.
Ch em istr y
The benzothiadiazine dioxide ring was obtained in two
steps following the Cohen and Klarberg procedure13
starting from methyl anthranylate and sulfamoyl chlo-
ride.
The synthesis of the acyclonucleosides was achieved
following the silylation procedure.14 Thus benzothiadi-
azine 1 was first silylated using hexamethyldisilazane
(HMDS) and acetonitrile as cosolvent under nitrogen
atmosphere. Several acyclic moieties, including acetoxy-
ethoxymethyl, benzyloxymethyl, and propargyloxy-
methyl groups, were introduced in a second step using
dichloromethane and boron trifluoride as catalyst
(Scheme 1). In all conditions assayed, acycloglycosyla-
tion took place exclusively at the N-3 position yielding
derivatives 2-4. Deprotection of compound 2 was
achieved quantitatively using a lipase-mediated deacyla-
tion procedure with Candida antarctica lipase (CAL) in
a hydrolytic medium (t-BuOH/buffer pH ) 7). This
methodology has been previously described by our
group10 and has been applied efficiently to the regiose-
lective deacylation of thiadiazine dioxide diacyclonucleo-
sides.15
In our search for antivirally effective thiadiazine
acyclonucleosides,10,11 we discovered that benzothiadi-
azine dioxide acyclonucleosides could be considered as
new lead compounds in the development of anti-CMV
agents.12 In the present report we describe the synthe-
sis, molecular modeling, and further antiviral evalua-
tion of these new compounds.
† Instituto de Qu´ımica Me´dica.
‡ Katholieke Universiteit Leuven.
10.1021/jm980327z CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/12/1999

1146 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Martinez et al.
Sch em e 2a
fibroblasts. Cytotoxicity measurements were based on
the inhibition of cell growth. The results are presented
in Table 1. Two of the test compounds (7 and 8) were
active against CMV and VZV at concentrations that
were only 2.5-5-fold below the cytotoxic concentration
for the host cells, which resulted in a low selective
antiviral action. Taking into account the chemical
structure of compounds 7 and 8 that is unique because
of the nature of the heterocyclic base and the lack of
the hydroxyl group in the acyclic side chain, some
preliminar structure-activity analysis of compounds
2-14 can be done. So, it pointed to the necessity of a
double substitution in the benzothiadiazine moiety
together with the lipophilicity in the acyclic side chain.
These factors were considered when preparing the new
acyclonucleosides 15 and 16.
A lipophilic moiety was introduced in the side chain
of acyclonucleoside 13 by treatment with benzoyl chlo-
ride in refluxing pyridine yielding compound 15 (Scheme
3). Additionally, the positions of the substituents in
derivative 8 were interchanged. Thus, acyclonucleoside
3 was benzylated by reaction with benzyl bromide in
aqueous bicarbonate. Compound 16 was isolated from
the reaction medium after column chromatography
purification (Scheme 3).
ii
i
iii
iv
v
v
a
Reagents: (i) BrBn, HCO3Na; (ii) HMDS/N2; (iii) BF3‚Et2O/
CH2Cl2/RCH2OCH2OAc; (iv) [H2]/Pd; (v) CAL/t-BuOH/buffer.
The N-1-substituted acyclic nucleosides were pre-
pared using a protection-deprotection strategy from
3-benzylbenzothiadiazine 6, which was previously ob-
tained by reaction of 1 with benzyl bromide in aqueous
bicarbonate. Silylation of 6 with HMDS and catalytic
amounts of ammoniun sulfate, followed by reaction with
several acyclic moieties in dichloromethane and boron
trifluoride etherate as catalyst, yielded, after chromato-
graphic purification, the N-3-protected, N-1 acyclic
nucleosides 10-12 (Scheme 2). The N-benzyl group was
eliminated by catalytic hydrogenolysis using Pd/C as
catalyst. It is worth mentioning that in the case of
compound 7, simultaneously with the cleavage of the
N-benzyl moiety, reduction of the acyclic moiety mul-
tiple bond was accomplished. The lipase-mediated deacyl-
ation procedure was employed to obtain the hydroxy-
ethoxymethyl derivates 13 and 14 in quantitative yields.
The structures of all new compounds were elucidated
according to analytical and spectroscopic data (see
Experimental Section). Unequivocal assignment of all
chemical shifts (1H and 13C NMR) was done using
bidimensional experiments such as COSY or HMQC for
one-bond correlation. The site of glycosylation was
determined from sequences of HMBC for long distance
proton/carbon correlation experiments.
Sch em e 3
The new benzothiadizine acyclonucleosides thus pre-
pared (15 and 16) were evaluated for their activity
against CMV and VZV in HEL cells. Compound 16 was
active against CMV at a concentration that was only
slightly higher than for ganciclovir [50% inhibitory
concentration (IC50) 3.6 µg/mL for compound 16 com-
pared to 1.2-1.9 µg/mL for ganciclovir]. Although
compound 16 only inhibited cell growth after 3 days of
incubation at a concentration of g40 µg/mL, cell mor-
phology was altered at 20 µg/mL when cell cultures were
exposed to the compound for 7 days.
Additionally, the benzothiadiazine derivatives 2-16
were also evaluated for their activity against HIV-1
(strain IIIB) and HIV-2 (strain ROD) in CEM cells, and
the results are shown in Table 2. Compound 16 was
found to inhibit the replication of HIV-1 and HIV-2 in
CEM cells at an IC50 of 15-20 µg/mL. The CC50 was
around 100 µg/mL for 16 in CEM cells. All the other
compounds were inactive against the replication of
HIV-1 at subtoxic concentrations.
Biologica l Resu lts a n d Discu ssion
All acyclonucleosides derived from benzothiadiazine
dioxide system were evaluated for their antiviral activ-
ity in a wide variety of assay systems:16 herpes simplex
virus type 1 (strains KOS, F. McIntyre), herpes simplex
virus type 2 (strains G, 196, Lyons), thymidine kinase-
deficient (TK-) herpes simplex virus type 1 (strains B
2006, VMW 1837), vaccinia virus, and vesicular stoma-
titis virus in E6SM cells; vesicular stomatitis virus,
poliovirus type 1, and Coxsackie B4 virus in HeLa cells;
parainfluenza virus type 3, reovirus type 1, Sindbis
virus, Coxsackie B4 virus, and Semliki forest virus in
Vero cells. However, no antiviral activity was noted in
any of these antiviral assay systems (at compound
concentrations up to 400 µg/mL).
The conformation of 16 was studied with ab initio
Hartree-Fock methods to asses its structural determi-
nants. The geometry was optimized with a 3-21G* basic
set to determine the optimum position of the two
aromatic systems. Molecular modeling, using the Gauss-
ian 94 program,19 revealed that the benzothiadiazine
In addition, antiviral activity against CMV, strains
AD-169 and Davis, and varicella-zoster virus (VZV),
strains OKA, YS, 07/1, and YS/R, was determined by
plaque reduction (VZV)17 or CPE reduction (CMV)18
assays in confluent human embryonic lung (HEL)

Novel Potential Agents for Human CMV Infection
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1147
Ta ble 1. Antiviral Activity and Cytotoxicity of Benzothiadiazine Dioxide Acyclonucleosides 2-16 against Human Cytomegalovirus
(CMV) and Varicella-Zoster Virus (VZV) in Human Embryonic Lung (HEL) Cells
antiviral activity IC50 (µg/mL)a
cytotoxicity (µg/mL)
CMV
TK+ VZV
TK- VZV
cell growth
cell morphology
compd no.
AD-169
Davis
OKA
YS
07/1
YS/R
CC50
MCCc
b
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
>50
>50
>50
>50
>20
12
>50
>50
>50
>50
>20
12
>50
>50
>50
38
>50
>50
48
11
>5
44
>50
>50
>50
55
>50
31
>50
>50
>50
13
>200
148
169
>50
>50
>50
>50
50
50
g50
50
>50
>50
>50
>50
>50
50
34
>50
>50
>50
21
46
>50
>50
>50
24
160
>50
>50
>50
100
>50
>50
>200
125
112
17
g40
>200
>50
8
11
20
42
20
>5
>20
>50
>50
>50
>20
>50
13
>50
>50
>50
>50
>50
>50
g20
3.7
>50
>50
>50
>50
>50
>50
>20
>5
>20
>50
>50
>50
>20
>50
12
>50
>50
>50
43
>50
>20
>5
>50
>20
>5
3.5
3.0
26
20
>50
>50
acyclovir
ganciclovir
0.73
0.78
24
1.2
1.9
a
50% inhibitory concentration, or concentration required to reduce virus plaque formation by 50%. Virus input was 100 PFU in CMV
b
and 20 PFU in VZV. Assays were performed in duplicate. 50% cytotoxic concentration, or concentration required to reduce cell growth
by 50%. Assays were performed in duplicate. c Minimum cytotoxic concentration that causes a microscopically detectable alteration of
cell morphology. Assays were performed in duplicate.
Ta ble 2. Anti-HIV-1 and Anti-HIV-2 Activity and Cytotoxic
Properties of Benzothiadiazine Dioxide Acyclonucleosides 2-16
in Human T-Lymphocyte (CEM) Cells
the antiviral compounds, as it is the first to show
activity against both CMV and HIV, at a yet to be
identified target site.
antiviral activity EC50 (µg/mL)a
compd
no.
cytotoxicity CC50
(µg/mL)b
Exp er im en ta l Section
HIV-1(IIIB)
HIV-2(ROD)
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
>200
>40
>200
>40
>200
>8
>200
>40
>200
>40
>200
>8
>200
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.
NOE difference spectra were measured under the same
conditions, using a presaturation time of 3 s. 13C NMR
experiments were carried out on the Varian Gemini-200
spectrometer operating at 50 MHz. The acquisiton parameters
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 performed by the
analytical departement at CNQO (CSIC), and the results
obtained were within (0.4% of the theoretical values. The C.
antarctica lipase used was Novo Nordisk’s immobilized prepa-
ration Novozym 435.
91 ( 0.71
>200
>200
>200
23 ( 0.71
25 ( 1.4
103 ( 3.5
120 ( 28
>200
>8
>8
>40
>40
>40
>200
>40
>40
g40
15
>40
>40
>40
>200
>40
>40
g40
20
>200
89 ( 1.4
15 ( 4.9
100
100
a
50% effective concentration, or concentration required to
protect CEM cells against the cytopathogenicity of HIV by 50%.
Assays were performed in triplicate. b 50% cytotoxic concentration,
or concentration required to reduce CEM cell viability by 50%.
Assays were performed in triplicate.
3-Ben zyl-2,1,3-ben zoth ia d ia zin -4(1H)-on e 2,2-Dioxid e
(6). Benzyl bromine (0.17 g, 1 mmol) was added to a solution
of benzothiadiazine 113 (0.198 g, 1 mmol) in a sodium bicar-
bonate aqueous solution (25 mL). The reaction mixture was
refluxed for 1 h. After the mixture cooled to room temperature,
the aqueous phase was extracted with AcOEt (5 × 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:
3) as eluent. Compound 6 was obtained (0.10 g, 34%) as a white
crystalline solid: mp 250-251 °C; 1H NMR (DMSO-d6) δ 4.93
(s, 2H, N-CH2), 6.72 (d, 1H, J ) 8.2 Hz, H-8), 6.85 (t, 1H, J
) 6.7 Hz, H-6), 7.19-7.42 (m, 6H, Ar-H and H-7), 7.85 (d,
1H, J ) 7.8 Hz, H-5); 13C NMR (DMSO-d6) δ 46.45 (CH2),
113.66 (C-4a), 119.09 (Cp), 119.73 (C-8), 126.59 (Cm), 126.65
(C-7), 128.31 (Co), 128.75 (Ci), 131.72 (C-5), 137.68 (C-6),
142.21 (C-8a), 165.67 (C-4). Anal. (C14H12N2O3S) C, H, N, S.
Gen er a l P r oced u r e for th e Syn th esis of Ben zoth ia d i-
a zin e Acyclon u cleosid es. To a solution in CH2Cl2 (25 mL)
of the silyl derivative of the benzothiadiazine dioxide (1 mmol)
F igu r e 1. Two different views of the 3-21G*-optimized
structure of benzothiadiazine 16.
derivative 16 adopts a butterfly-like conformation, the
valence angle R between the two rings being 114.10°
(Figure 1).
In conclusion, the benzothiadiazine dioxide acyclo-
nucleoside 16 can be considered as a new lead among

1148 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Martinez et al.
prepared by refluxing the base in hexamethyldisilazane (3 mL)
under nitrogen using suitable catalyst and cosolvents, the
dissolved acyclic moiety precursor in CH2Cl2 (25 mL) was
added. The mixture was cooled, and BF3‚Et2O (1.40 mmol) was
added under vigorous stirring and exclusion of moisture. The
resulting mixture was stirred at room temperature for 1-3 h
and was then shaken with saturated sodium hydrogencarbon-
ate solution (50 mL). The organic phase was separated, dried
over sodium sulfate, and evaporated under reduced pressure.
The residue was chromatographed on a silica gel column, using
as eluent mixtures of solvents in the proportions indicated for
each particular case.
3-[(2-Acet oxyet h oxy)m et h yl]-2,1,3-b en zot h ia d ia zin -
4(1H)-on e 2,2-Dioxid e (2). Reagents: (1) benzothiadiazine
dioxide 1 (0.39 g, 2 mmol), HMDS (6 mL), SO4(NH4)2 (catalytic
amounts), CH3CN (1 mL); (2) acetoxyethyl acetoxymethyl
ether20 (0.35 g, 2 mmol), BF3‚Et2O (2.8 mmol). Purification:
compound 2 was obtained as white needles from the acidified
aqueous phase (0.34 g, 54%); mp 122-123 °C; 1H NMR
(DMSO-d6) δ 1.97 (s, 3H, CH3CO), 3.77 (m, 2H, CH2O), 4.12
(m, 2H, AcOCH2), 5.35 (s, 2H, NCH2O), 7.20 (d, 1H, J ) 7.8
Hz, H-8), 7.33 (t, 1H, H-6), 7.71 (t, 1H, H-7), 8.04 (d, 1H, H-5);
13C NMR (DMSO-d6) δ 20.63 (CH3), 62.79 (CH2), 67.03 (CH2),
71.66 (N-CH2-O), 117.64 (C-4a), 119.97 (C-8), 124.47 (C-7),
129.77 (C-5), 135.79 (C-6), 138.53 (C-8a), 162.40 (C-4), 170.74
(CdO). Anal. (C12H14N2O6S) C, H, N, S.
H-7), 8.04 (d, 1H, H-5); 13C NMR (DMSO-d6) δ 55.12 (CH2Ph),
70.51 (CH2), 72.19 (N-CH2-O), 121.82 (C-4a), 122.91 (C-8),
126.70 (C-7), 127.39, 128.27 (Cp), 128.33, 128.54 (Cm), 128.80,
129.01 (Co), 129.87 (C-5), 134.01, 137.22 (Ci), 135.81 (C-6),
139.67 (C-8a), 161.86 (C-4). Anal. (C22H20N2O4S) C, H, N, S.
1-[(2-Acetoxyeth oxy)m eth yl]-3-ben zyl-2,1,3-ben zoth ia -
d ia zin -4-on e 2,2-Dioxid e (9). Reagents: (1) benzothiadiazine
dioxide 6 (0.57 g, 2 mmol), HMDS (6 mL), SO4(NH4)2 (catalytic
amounts); (2) acetoxyethyl acetoxymethyl ether20 (0.35 g, 2
mmol), BF3‚Et2O (2.8 mmol). Purification: hexane:AcOEt (10:
3); yield 0.39 g (50%); 1H NMR (DMSO-d6) δ 1.99 (s, 3H, CH3-
CO), 3.71 (m, 2H, CH2O), 4.12 (m, 2H, AcOCH2), 5.10 (s, 2H,
CH2Ph), 5.23 (s, 2H, NCH2O), 7.23-7.28 (m, 5H, Ar-H), 7.47
(t, 1H, H-6), 7.53 (d, 1H, H-8), 7.80 (t, 1H, J ) 7.8 Hz, H-7),
8.00 (d, 1H, H-5); 13C NMR (DMSO-d6) δ 20.63 (CH3), 32.79
(CH2), 67.03 (CH2), 71.66 (N-CH2-O), 122.02(C-4a), 123.17
(C-8), 126.93 (C-7), 130.04 (C-5), 136.05 (C-6), 139.91 (C-8a),
162.00 (C-4), 170.74 (CdO). Anal. (C19H20N2O6S) C, H, N, S.
Gen er a l P r oced u r e for th e En zym a tic Clea va ge of
Acetyl Gr ou p . A solution of the acylated acyclonucleoside (2
mmol) in t-BuOH:buffer pH ) 7 (90:10) was incubated with
10 mg‚mL-1 C. antarctica lipase (CAL) at 45 °C and 250 rpm
in an orbital shaker for 4 h. When all the starting material
had disappeared, the enzyme was removed by filtration and
washed with methanol. The filtrate was evaporated in vacuo,
and the deprotected compound was obtained as a syrup. The
compounds thus obtained were as follows.
3-[(2-H yd r oxyet h oxy)m et h yl]-2,1,3-b en zot h ia d ia zin -
4(1H)-on e 2,2-d ioxid e (5): yield 0.08 g (92%); 1H NMR
(DMSO-d6) δ 3.54 (m, 4H, CH2-CH2O), 5.22 (s, 2H, NCH2O),
6.83 (m, 2H, H-6 and H-8), 7.37 (t, 1H, J ) 7.8 Hz, H-7), 7.82
(t, 1H, H-5); 13C NMR (DMSO-d6) δ 60.11 (CH2), 70.53 (CH2),
71.02 (N-CH2-O), 115.87 (C-4a), 118.16 (C-8), 121.75 (C-7),
128.80 (C-5), 133.88 (C-6), 149.13 (C-8a), 164.48 (C-4). Anal.
(C10H12N2O5S) C, H, N, S.
1-[(2-Hyd r oxyeth oxy)m eth yl]-3-ben zyl-2,1,3-ben zoth i-
a d ia zin -4-on e 2,2-d ioxid e (13): yield 0.08 g (98%); 1H NMR
(DMSO-d6) δ 3.52 (m, 4H, CH2-CH2O), 5.12 (s, 2H, CH2Ph),
5.21 (s, 2H, NCH2O), 7.13-7.27 (m, 5H, Ar-H), 7.57 (d, 1H, J
) 7.8 Hz, H-8), 7.45 (t, 1H, H-6), 7.78 (t, 1H, H-7), 8.01 (d,
1H, H-5); 13C NMR (DMSO-d6) δ 55.10 (CH2Ph), 59.88 (CH2),
71.21 (CH2), 72.96 (N-CH2-O), 121.87 (C-4a), 122.89 (C-8),
126.68 (C-7), 128.29 (Cp), 128.31 (Cm), 128.54 (Co), 129.77 (C-
5), 134.01 (Ci), 135.76 (C-6), 139.67 (C-8a), 161.81 (C-4). Anal.
(C17H18N2O5S) C, H, N, S.
3-(Be n zyloxym e t h yl)-2,1,3-b e n zot h ia d ia zin -4(1H )-
on e 2,2-Dioxid e (3). Reagents: (1) benzothiadiazine dioxide
1 (0.59 g, 3 mmol), HMDS (9 mL), SO4(NH4)2 (catalytic
amounts), CH3CN (1 mL); (2) acetoxymethyl benzyl ether21
(0.54 g, 3 mmol), BF3‚Et2O (4.2 mmol). Purification: CH2Cl2:
1
MeOH (50:1); yield 0.60 g (62%); H NMR (DMSO-d6) δ 4.59
(s, 2H, CH2O), 5.25 (s, 2H, NCH2O), 6.72 (t, 1H, H-7), 6.74 (d,
1H, J ) 8.0 Hz, H-8), 7.25-7.34 (m, 6H, Ar-H, H-6), 7.78 (d,
1H, H-5); 13C NMR (DMSO-d6) δ 69.80 (CH2), 70.38 (N-CH2-
O), 115.45 (C-4a), 116.94 (C-8), 122.07 (C-7), 127.29 (Cp),
127.55 (Cm), 128.11 (Co), 128.60 (C-5), 133.51 (C-6), 138.41
(Ci), 149.92 (C-8a), 164.89 (C-4). Anal. (C15H14N2O4S) C, H,
N, S.
3-(P r op a r gyloxym eth yl)-2,1,3-ben zoth ia d ia zin -4(1H)-
on e 2,2-Dioxid e (4). Reagents: (1) benzothiadiazine dioxide
1 (0.396 g, 2 mmol), HMDS (6 mL), SO4(NH4)2 (catalytic
amounts), CH3CN (1 mL); (2) (propargyloxy)methyl chloride22
(0.21 g, 2 mmol), BF3‚Et2O (2.8 mmol). Purification: CH2Cl2:
1
MeOH (50:1); yield 0.34 g (62%); H NMR (DMSO-d6) δ 3.47
(t, 1H, J ) 1.5 Hz, HCt), 4.25 (d, 2H, J ) 1.5 Hz, tCCH),
5.36 (s, 2H, NCH2O), 7.19 (d, 1H, J ) 6.4 Hz, H-8), 7.34 (t,
1H, H-6), 7.71 (t, 1H, H-7), 8.04 (d, 1H, H-5); 13C NMR (DMSO-
d6) δ 56.60 (CHO), 70.72 (N-CH2-O), 78.02 (CHt), 79.88
(Ct), 118.14 (C-4a), 120.56 (C-8), 125.01 (C-7), 130.24 (C-5),
136.28 (C-6), 138.96 (C-8a), 162.78 (C-4). Anal. (C11H10N2O4S)
C, H, N, S.
1-[(2-H yd r oxyet h oxy)m et h yl]-2,1,3-b en zot h ia d ia zin -
4(3H)-on e 2,2-d ioxid e (14): yield 0.12 g (90%); 1H NMR
(DMSO-d6) δ 3.59 (m, 4H, CH2-CH2O), 5.21 (s, 2H, NCH2O),
6.78 (d, 1H, J ) 8 Hz, H-8), 6.76 (t, 1H, H-6), 7.32 (t, 1H, H-7),
7.79 (t, 1H, H-5); 13C NMR (DMSO-d6) δ 60.49 (CH2), 70.78
(CH2), 71.30 (N-CH2-O), 116.00 (C-4a), 117.95 (C-8), 122.35
(C-7), 129.80 (C-5), 134.14 (C-6), 149.55 (C-8a), 165.17 (C-4).
Anal. (C10H12N2O5S) C, H, N, S.
1-(P r op yloxym e t h yl)-2,1,3-b e n zot h ia d ia zin -4(3H )-
on e 2,2-Dioxid e (10). A solution of acyclonucleoside 7 (0.09
g, 0.3 mmol) in MeOH (15 mL) was hydrogenated with 10 psi
of hydrogen in the presence of 20% palladium/charcoal catalyst
(0.018 g) at room temperature. After 30 min, the catalyst was
filtered off and the solvent removed in vacuo. The residue was
dissolved in AcOEt and washed with water. The organic phase
was dried over sodium sulfate and the solvent eliminated
under reduced pressure yielding 0.06 g (90%) of derivative
10: 1H NMR (DMSO-d6) δ 0.83 (t, 3H, CH3), 1.49 (q, 2H, CH2),
3.50 (m, 2H, CH2O), 5.30 (s, 2H, NCH2O), 7.17 (d, 1H, J ) 7.8
Hz, H-8), 7.29 (t, 1H, H-6), 7.68 (t, 1H, H-7), 8.02 (d, 1H, H-5);
13C NMR (DMSO-d6) δ 10.68 (CH3), 22.48 (CH2), 70.71 (CH2O),
71.89 (N-CH2-O), 117.88 (C-4a), 120.32 (C-8), 124.29 (C-7),
129.95 (C-5), 135.84 (C-6), 139.44 (C-8a), 162.78 (C-4). Anal.
(C11H14N2O4S) C, H, N, S.
3-Ben zyl-1-(pr opar gyloxym eth yl)-2,1,3-ben zoth iadiazin -
4-on e 2,2-Dioxid e (7). Reagents: (1) benzothiadiazine dioxide
6 (0.29 g, 1 mmol), HMDS (3 mL), SO4(NH4)2 (catalytic
amounts); (2) (propargyloxy)methyl chloride22 (0.11 g, 1 mmol),
BF3‚Et2O (1.4 mmol). Purification: hexane:AcOEt (4:1); yield
1
0.27 g (77%); H NMR (DMSO-d6) δ 3.54 (t, 1H, J ) 1.5 Hz,
HCt), 4.21 (d, 2H, J ) 1.5 Hz, tCCH), 5.10 (s, 2H, CH2Ph),
5.25 (s, 2H, NCH2O), 7.33 (m, 5H, Ar-H), 7.49 (d, 1H, J ) 7.8
Hz, H-6), 7.59 (d, 1H, H-8), 7.82 (t, 1H, H-7), 8.01 (d, 1H, H-5);
13C NMR (DMSO-d6) δ 55.61 (CH2), 56.57 (CHO), 71.44 (N-
CH2-O), 78.09 (CHt), 79.58 (Ct), 122.10 (C-4a), 123.32 (C-
8), 127.09 (C-7), 128.75 (Co, Cp), 128.85 (Cm), 130.12 (C-5),
134.23 (Ci), 136.16 (C-6), 139.93 (C-8a), 162.04 (C-4). Anal.
(C18H16N2O4S) C, H, N, S.
3-Ben zyl-1-(b en zyloxym et h yl)-2,1,3-b en zot h ia d ia zin -
4-on e 2,2-Dioxid e (8). Reagents: (1) benzothiadiazine dioxide
6 (0.57 g, 2 mmol), HMDS (6 mL), SO4(NH4)2 (catalytic
amounts); (2) acetoxymethyl benzyl ether21 (0.36 g, 2 mmol),
BF3‚Et2O (2.8 mmol). Purification: hexane:AcOEt (3:1); yield
1-(Be n zyloxym e t h yl)-2,1,3-b e n zot h ia d ia zin -4(3H )-
on e 2,2-Dioxid e (11). Following the above procedure, benzyl
acyclonucleoside 8 (0.11 g, 0.3 mmol) was hydrogenated in the
presence of palladium/charcoal (0.02 g). After workup, com-
pound 11 was obtained (0.08 g, 97%) as a syrup: 1H NMR
1
0.56 g (70%); H NMR (DMSO-d6) δ 4.59 (s, 2H, CH2O), 5.11
(s, 2H, CH2Ph), 5.30 (s, 2H, NCH2O), 7.58 (d, 1H, J ) 6.4 Hz,
H-8), 7.12-7.33 (m, 5H, Ar-H), 7.47 (t, 1H, H-6), 7.76 (t, 1H,

Novel Potential Agents for Human CMV Infection
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1149
(DMSO-d6) δ 4.66 (s, 2H, CH2O), 5.45 (s, 2H, NCH2O), 7.20 (t,
1H, J ) 8.0 Hz, H-6), 7.31-7.35 (m, 5H, Ar-H), 7.30 (d, 1H,
H-8), 7.68 (t, 1H, H-7), 8.04 (d, 1H, H-5); 13C NMR (DMSO-d6)
δ 70.44 (CH2), 71.32 (N-CH2-O), 117.67 (C-4a), 120.04 (C-8),
124.36 (C-7), 127.71 (Cp), 128.28 (Cm), 128.39 (Co), 129.79 (C-
5), 137.41 (Ci), 135.72 (C-6), 138.76 (C-8a), 162.49 (C-4). Anal.
(C15H14N2O4S) C, H, N, S.
1-[(2-Acet oxyet h oxy)m et h yl]-2,1,3-b en zot h ia d ia zin -
4(3H)-on e 2,2-Dioxid e (12). According to the method de-
scribed for derivative 10, the benzothiadiazine acyclonucleoside
9 (0.20 g, 0.4 mmol) was hydrogenated with 50 psi of hydrogen
using 20% palladium/charcoal (0.04 g) as catalyst. After 2 h,
the reaction mixture was worked up yielding 0.13 g (90%) of
compound 12: 1H NMR (DMSO-d6) δ 1.98 (s, 3H, CH3CO), 3.76
(m, 2H, CH2O), 4.13 (m, 2H, AcOCH2), 5.35 (s, 2H, NCH2O),
7.20 (d, 1H, J ) 7.8 Hz, H-8), 7.32 (t, 1H, H-6), 7.70 (t, 1H,
H-7), 8.04 (d, 1H, H-5); 13C NMR (DMSO-d6) δ 21.02 (CH3),
63.19 (CH2), 67.40 (CH2), 72.06 (N-CH2-O), 117.93 (C-4a),
120.45 (C-8), 124.56 (C-7), 130.01 (C-5), 136.08 (C-6), 139.42
(C-8a), 162.90 (C-4), 170.74 (CdO). Anal. (C12H14N2O6S) C, H,
N, S.
1-[(2-Ben zoyloxyet h oxy)m et h yl]-3-b en zyl-2,1,3-b en -
zoth ia d ia zin -4-on e 2,2-Dioxid e (15). A solution of acyclo-
nucleoside 13 (0.08 g, 0.2 mmol) in CH2Cl2:pyridine (10:1) (10
mL) was cooled at -20 °C for 15 min, and benzoyl chloride
(107 µL, 0.9 mmol) dissolved in CH2Cl2 (3 mL) was slowly
added. The reaction mixture was stirred at room temperature
for 24 h. The solvent was evaporated under reduced pressure,
and the residue was purified by silica gel column chromatog-
raphy eluting with hexane:AcOEt (1:3) and yielding 0.14 g
(75%) of derivative 15 as a syrup: 1H NMR (CDCl3) δ 3.91
(m, 2H, CH2O), 4.42 (m, 2H, AcOCH2), 4.89 (s, 2H, CH2Ph),
5.29 (s, 2H, NCH2O), 7.04-8.10 (m, 14H, Ar-H, H-5, H-6, H-7,
H-8); 13C NMR (CDCl3) δ 56.56 (CH2Ph), 63.62 (CH2), 67.69
(CH2), 72.84 (N-CH2-O), 122.71 (C-4a), 122.61 (C-8), 126.65
(C-7), 130.67, 130.17 (Cp), 128.45, 128.27 (Cm), 128.73, 129.70
(Co), 132.93 (C-5), 133.72, 133.56 (Ci), 135.13 (C-6), 140.32 (C-
8a), 162.47 (C-4), 166.48 (CdO). Anal. (C24H22N2O6S) C, H, N,
S.
had been clarified by low-speed centrifugation. The virus
stocks were stored at -80 °C.
An tivir a l Assa ys. Confluent HEL cells grown in 96-well
microtiter plates were infected with the different strains at
20 (VZV) or 100 (CMV) plaque forming units (PFU). After a
2-h incubation period, residual virus was removed and the
infected cells were further incubated with MEM supplemented
with 2% inactivated FCS, 1% L-glutamine, and 0.3% sodium
bicarbonate containing serial dilutions of the test compounds
(in duplicate). After 5 (VZV) or 7 (CMV) days of incubation at
37 °C in 5% CO2 atmosphere, the cells were fixed with ethanol
and strained with 2.5% Giemsa solution. Virus plaque forma-
tion (virus input: 20 PFU, VZV) or viral cytopathic effect (virus
input: 100 PFU, CMV) was monitored microscopically. The
antiviral activity is expressed as IC50 which represents the
compound concentration required to reduce virus plaque
formation or cytopathicity by 50%. IC50 values were estimated
from graphic plots of the number of plaques (percentage of
control) or percentage of cytopathicity as a function of the
concentration of the test compounds.
Cytotoxicity Assa ys. Cytotoxicity measurements were
based on the inhibition of HEL cell growth. HEL fibroblasts
were seeded at a rate of 5 × 103 cells/well microtiter plates
and allowed to proliferate for 24 h. Different concentrations
of the test compounds were then added (in duplicate), and after
3 days of incubation at 37 °C in 5% CO2 atmosphere, the cell
number was determined with a Coulter counter. Cytotoxicity
is expressed as CC50, which represents the compound concen-
tration required to reduce cell growth by 50%. As a second
parameter of cytotoxicity, the minimum toxic concentration
(MTC) to cause a microscopically detectable change in mor-
phology of normal cells treated with the compounds was
evaluated.
An tir etr ovir a l Eva lu a tion . Human immunodeficiency
virus type 1 [HIV-1 (HTLV-IIIb)] was kindly provided by Dr.
R. C. Gallo (when at the National Institutes of Health,
Bethesda, MD). Virus stocks were prepared from supernatants
of HIV-1-infected MT-4 cells. HIV-2 (strain ROD) was provided
by Dr. L. Montagnier (Pasteur Institute, Paris, France), and
virus stocks were prepared from the supernatants of HIV-2-
infected MT-4 cells. CEM cells were obtained from the
American Tissue Culture Collection (Rockville, MD). CEM cells
were infected as follows: 4 × 105 cells/mL were infected with
HIV-1 or HIV-2 at ∼100 CCID50 (50% cell culture infective
dose)/mL of cell suspension. Then 100 µL of the infected cell
suspension was transferred to 96-well microtiter plate wells
and mixed with 100 µL of the appropiate dilutions of the test
compounds. After 4 days giant cell formation was recorded
microscopically in the HIV-infected cell cultures.
1-Ben zyl-3-(b en zyloxym et h yl)-2,1,3-b en zot h ia d ia zin -
4-on e 2,2-Dioxid e (16). Benzyl bromine (0.8 µL, 0.8 mmol)
was added to a solution of acyclonucleoside 3 (0.05 g, 0.2 mmol)
in
a sodium bicarbonate aqueous solution (10 mL). The
reaction mixture was refluxed for 4 h. After cooling to room
temperature, the aqueous phase was extracted with AcOEt (5
× 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
hexane:AcOEt (4:1) as eluent. Compound 16 was obtained
(0.07 g, 95%) as a syrup: 1H NMR (CDCl3) δ 4.60 (s, 2H,
CH2O), 4.89 (s, 2H, CH2Ph), 5.26 (s, 2H, NCH2O), 7.06-7.36
(m, 12H, Ar-H, H-6, H-8), 7.54 (t, 1H, J ) 7.3 Hz, H-7), 8.09
(d, 1H, H-5); 13C NMR (CDCl3) δ 56.44 (CH2Ph), 71.44 (CH2),
72.21 (N-CH2-O), 122.49 (C-8), 122.68 (C-4a), 126.58 (C-7),
127.87, 128.64 (Cp), 128.37, 128.75 (Cm), 128.02, 128.23 (Co),
130.69 (C-5), 135.04 (C-6), 133.71, 137.14 (Ci), 140.34 (C-8a),
162.48 (C-4). Anal. (C22H20N2O4S) C, H, N, S.
An tivir a l Eva lu a tion . The compounds were evaluated for
antiviral activity following established procedures, as reviewed
in ref 16.
Cells. Human embryonic lung (HEL) fibroblasts were
propagated in Eagle’s minimum essential medium (MEM)
supplemented with 10% inactivated fetal calf serum, 1%
L-glutamine, and 0.3% sodium bicarbonate.
Vir u ses. Two reference strains of VZV expressing viral
thymidine kinase (TK+) (YS and OKA) and two reference
strains of VZV lacking the viral thymidine kinase (TK-) (07/1
and YS/R) were included in the study. Virus stocks were
prepared in HEL cells as infected cells. When 70% cytopathic
effect was obtained, the cells were trypsinized, resuspended
in medium containing 10% DMSO, and stored in aliquots at
-80 °C. The Davis and AD-169 strains of human cytomega-
lovirus were used. Virus stocks consisted of cell-free virus
obtained from the supernatant of infected cell cultures that
Ack n ow led gm en t . This investigation was sup-
ported by Fondo de Investigaciones Sanitarias (Project
No. FIS 98/253), the Biomedical Research Programme
of the European Commission, the Fonds voor Weten-
schappelijk Onderzoek (FWO) Vlaanderen, and the
Geconcerteerde Onderzoeksacties (GOA) Vlaanderen.
We thank Ann Absillis, Anita Camps, Frieda De Meyer,
Katrin Saelen, Ria Van Berwaer, and Anita Van Lierde
for excellent technical assistance. One of us (C. Gil)
acknowledges a grant from Comunidad de Madrid.
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