Synthesis and anti-HIV activity of 1,1,3-trioxo-2H,4H-thieno[3,4-e][1,2,4]thiadiazines (TTDs): A new family of HIV-1 specific non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1016/S0968-0896(99)00221-7

The anti-HIV activity of a novel series of 1,1,3-trioxo-2H,4H-thieno[3,4-e][1,2,4]thiadiazines (TTDs) has been described. The compounds were synthesized via Curtius rearrangement of appropriate sulfamoylcarboxy azides which, in turn, were prepared from known starting materials. Several 4-substituted-2-benzyl-derivatives were found to selectively inhibit human immunodeficiency virus type 1 [HIV-1 (III(B))] replication in MT-4 and CEM cells. These TTDs were also effective against other strains of HIV-1 (RF, HE, MN, NDK), including those that are resistant to AZT, but not against HIV-2 (ROD) or simian immunodeficiency virus [SIV(MAC251)] at subtoxic concentrations. Some of the test compounds exhibited antiviral activity against L100I RT mutant virus, but significantly lost antiviral activity against K103N, V106A, E138K, Y181C and Y188H RT mutant viruses. Compounds 6dScheme 1Reagents: (i) NH₃/CH₂Cl₂ or R₁NH₂/THF; (ii) N₂H₄.H₂O/EtOH; (iii) 2 N HCl or 2 N HNO₃, NaNO₂, H₂O; (iv) Δ/toluene or CHCl₃; (v) NaH/DMF, R₂X., 6f and 6g were inhibitory to HIV-1 RT at concentrations that rank between 16.4 and 59.8μM (nevirapine: IC₅₀=4.5μM against HIV-1 RT). Inhibition of HIV-1 RT by compound 6g was purely non-competitive with respect to the natural substrate (dGTP), which is in agreement with the nature of inhibition shown by other NNRTIs such as nevirapine and delarvidine. A structure-activity relationship was established for the anti-HIV activity of these heterocyclic compounds. TTDs represent a new chemical class of non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTIs). Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00221-7

Bioorganic & Medicinal Chemistry 7 (1999) 2811±2822
Synthesis and Anti-HIV Activity of 1,1,3-Trioxo-2H,4H-
thieno[3,4-e][1,2,4]thiadiazines (TTDs): A New Family of HIV-1
Speci®c Non-Nucleoside Reverse Transcriptase Inhibitors
Ma Esther Arranz, a Juan A. Dõaz, a Simon T. Ingate, a Myriam Witvrouw, b
Christophe Pannecouque, b Jan Balzarini, b Erik De Clercq b and Salvador Vega a,*
a
Â
Â
Instituto de Quõmica Medica, CSIC, Juan de la Cierva, 3, 28006 Madrid, Spain
bRega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received 12 March 1999; accepted 14 July 1999
AbstractÐThe anti-HIV activity of a novel series of 1,1,3-trioxo-2H,4H-thieno[3,4-e][1,2,4]thiadiazines (TTDs) has been described.
The compounds were synthesized via Curtius rearrangement of appropriate sulfamoylcarboxy azides which, in turn, were prepared
from known starting materials. Several 4-substituted-2-benzyl-derivatives were found to selectively inhibit human immunode®-
ciency virus type 1 [HIV-1 (IIIB)] replication in MT-4 and CEM cells. These TTDs were also e€ective against other strains of HIV-1
(RF, HE, MN, NDK), including those that are resistant to AZT, but not against HIV-2 (ROD) or simian immunode®ciency virus
[SIV(MAC251)] at subtoxic concentrations. Some of the test compounds exhibited antiviral activity against L100I RT mutant virus,
but signi®cantly lost antiviral activity against K103N, V106A, E138K, Y181C and Y188H RT mutant viruses. Compounds 6d, 6f
and 6g were inhibitory to HIV-1 RT at concentrations that rank between 16.4 and 59.8 mM (nevirapine: IC50=4.5 mM against HIV-
1 RT). Inhibition of HIV-1 RT by compound 6g was purely non-competitive with respect to the natural substrate (dGTP), which is
in agreement with the nature of inhibition shown by other NNRTIs such as nevirapine and delarvidine. A structure±activity rela-
tionship was established for the anti-HIV activity of these heterocyclic compounds. TTDs represent a new chemical class of non-
nucleoside HIV-1 reverse transcriptase inhibitors (NNRTIs). # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
enzyme, responsible for the synthesis of double-stran-
ded viral DNA from proviral RNA for subsequent
incorporation into host cell chromosome, and therefore
essential for the replication of HIV.4
Since the early 1980s, more than 26 million people have
been infected with human immunode®ciency virus
(HIV), the causative agent of the acquired immunode®-
ciency syndrome (AIDS).1 By the year 2000, the World
Health Organization (WHO) estimates that 30±40 mil-
lion people will have been infected with HIV and that 10
million people will have developed AIDS.2 E€orts to
eradicate the disease have intensi®ed in the last 10 years
and, as a consequence, massive resources have been
focused on both the study of HIV and the development
of antiretroviral agents.3 The discovery that the virus
requires the catalytic activity of several unique enzymes
for its life cycle made them ideal therapeutic targets.
Research strategies have most particularly targeted
inhibition of reverse transcriptase (RT), a multi-purpose
Three main classes of RT inhibitors have been hitherto
discovered: the 20,30-dideoxynucleoside (ddN) and
acyclic nucleoside phosphonate (ANP) analogues which
compete with natural substrates and function as chain
terminatorsÐAZT, ddI, ddC, d4T, 3TC, PMEA,
PMPA, etc.Ðand the non-nucleoside inhibitors
(NNRTIs) which bind to an allosteric site on the
enzyme at a close distance from its polymerase active
site. These latter compounds, in contrast with nucleo-
side analogues, are highly speci®c for HIV-1 group M
isolates, do not bind to HIV-2 reverse transcriptase and
are e€ective without toxic e€ects at relatively high con-
centrations.5 As in the case of nucleoside congeners, the
rapid emergence of NNRTI-resistant viral strains has
greatly limited the clinical ecacy of these compounds
until the recent encouraging results with combination
therapy.6±9
Key words: Antivirals; enzyme inhibitors; 1,1,3-trioxo-2H,4H-
thieno[3,4-e][1,2,4]thiadiazines (TTDs); HIV-1 reverse transcriptase.
* Corresponding author. Tel.: +34-91-5622900; fax: 34-91-5644853;
e-mail: chavo@pinar1.csic.es
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00221-7

2812
Ma E. Arranz et al. / Bioorg. Med. Chem. 7 (1999) 2811±2822
The ®rst compounds of this type discovered were
the 1-(2-hydroxyethoxymethyl)-6-(phenylthio)thymine
(HEPT)10,11 and tetrahydroimidazo[4,5,1-jk]benzodi-
azepin-2(1H)-one and -thione (TIBO) derivatives.12,13
Following HEPT and TIBO, several other classes of
speci®c HIV-1 RT inhibitors were disclosed: nevirapine
(BI-RG-587),14,15 pyridinone derivatives (L-696,229 and
L-697,661),16,17 bis(heteroaryl)piperazine (BHAP),18,19
TSAO derivatives,20,21 a-anilinophenylacetamides (a-
APA),22 phenylethylthioureathiazole (PETT),23 oxa-
thiincarboxanilides (although the latter were originally
thought not to be targeted at RT)24 and quinoxaline
derivatives.25 Subsequently, more than 25 novel classes
of NNRTIs have been reported. Several overviews, with
descriptions of the discovery and development of the
majority of these NNRTIs, have appeared recently.5,6,26±28
.
Scheme 1. Reagents: (i) NH3/CH2Cl2 or R1NH2/THF; (ii) N2H4 H2O/
EtOH; (iii) 2 N HCl or 2 N HNO3, NaNO2, H2O; (iv) Á/toluene or
CHCl3; (v) NaH/DMF, R2X.
thiophene-3-carboxylate 1 with ammonia or primary
amines in the presence of a base such as potassium car-
bonate (K2CO3) or pyridine. The sulfamoylesters 2a±i
were readily converted to the sulfamoylcarboxy azides
4a±i by the action of nitrous acid on carboxy hydrazides
3a±i, which, in turn, were obtained from the corre-
sponding 2 by reaction with hydrazine hydrate in etha-
nol. Sulfamoylcarboxy azides 4a±i were not puri®ed,
but they were suciently identi®ed by the presence of
Since we have had a long-time interest in the chemistry
and pharmacological properties of heterocyclic sulfon-
amides,29±35 we decided to prepare a series of 1,1,3-
trioxo-2H,4H-thieno[3,4-e][1,2,4]thiadiazines (here named
by the acronym TTDs) and assess them as inhibitors of
HIV-1 replication. From the point of view of drug
design, such thienothiadiazines can be considered as
thiophene bioisosteres of 1,2,4-benzothiadiazines. These
latter compounds have been traditionally investigated
because of their activities as diuretic, hypoglycaemic,
antihypertensive and potassium channels opener
agents36,37 but, recently, some derivatives of this type
were also found to be highly potent HIV-1 NNRTIs.38
The bioisosteric equivalence between benzene and thio-
phene has been widely demonstrated in many ther-
apeutic ®elds.39 In general, the replacement of benzene
by thiophene in pharmacologically active molecules
gives rise to new compounds with similar or improved
potency and selectivity,40 and our research group has
reported di€erent examples of the usefulness of this
already classical design approach.34,35,41±44 Further
support for the 1,1,3-trioxo-2H,4H-thieno[3,4-e]
[1,2,4]thiadiazine ring system as the basis for the design
of useful NNRTIs is its chemical novelty, since no
compound with this structure has hitherto been descri-
bed. In the present paper we report the synthesis of the
TTDs, the results obtained for their anti-HIV evalua-
tion and their structure±function relationship (SAR)
with regard to their anti-HIV activity. Further details
on their antiretroviral activity, including virus±drug
resistance pro®le and modelling in the active HIV-1 RT
drug binding site, have been described previously.45
1
the N3 group characteristic band at 2130±2160 cm in
their IR spectra.
The target thieno[1,2,4]thiadiazines 5a±i were synthe-
sized in generally good yields by spontaneous cycliza-
tion of the isocyanates generated in the Curtius thermal
rearrangement of sulfamoylcarboxy azides 4a±i in dry
toluene, benzene or chloroform. The poorer yields
obtained in the ring-closure of carboxy azides 4b
(R1=2-ClC6H4) and 4e (R1=2-BrC6H4) (26 and 28%,
respectively) could be explained by the steric hindrance
produced by the ortho substituted anilino moieties
attached to these molecules. Compounds 5j and 5k were
better prepared by N-alkylation of the 1,1,3-trioxo-
2H,4H-thieno[3,4-e][1,2,4]thiadiazine48 under classical
conditions, by treating a solution of the sodium salt of
this thiadiazine in N,N-dimethylformamide (DMF) with
one equivalent of 2-chloroacetonitrile and methyl io-
dide, respectively. Similar conditions (NaH/DMF/alkyl
or arylalkyl halide) were successfully employed for the
transformation of thiadiazines 5a±k into disubstituted
derivatives 6a±n.
Compound 7a was prepared from 5h by reaction with
benzoyl chloride in dry pyridine. Thienothiadiazines 7b
and 7c were formed by hydrolysis of their respective
cyanomethyl derivatives 6g and 6n under basic condi-
tions (Scheme 2). Thus, 7b was obtained by treating a
dimethylsulfoxide solution of 6g with an excess of 30%
hydrogen peroxide in the presence of a catalytic amount
of K2CO3.50 Compound 6n was better hydrolyzed by a
modi®cation of this procedure realized under phase
Chemistry
The 2- and 2,4-substituted 1,1,3-trioxo-2H,4H-thieno-
[3,4-e][1,2,4]thiadiazines 5 and 6 were prepared accord-
ing to the synthetic sequence outlined in Scheme 1. The
key step for the formation of the bicyclic core fragment,
common to all these derivatives, involves the intra-
molecular ring closure of the methyl sulfamoylthio-
phencarboxylates (2) through
a classical Curtius
reaction.46±48 Compounds 2a±i were prepared in good
yields following a method described previously,49
consisting of the reaction of methyl 4-chlorosulfonyl-
Scheme 2.

Ma E. Arranz et al. / Bioorg. Med. Chem. 7 (1999) 2811±2822
2813
transfer catalysis conditions.51 In this reaction, in addi-
tion to 7c, another compound was isolated in minor
proportion; on the basis of its spectroscopic data, this
compound was assigned the open structure 8.
between the aromatic ring of the benzyl group and the
HIV-1 RT. Introduction of benzyl, cyanomethyl or
other alkyl groups into the 4 position of these 2-ben-
zyl-thienothiadiazines (6e±i) also led to signi®cantly
active compounds (SI values of 49±578). In the 4-alkyl
series, the methyl and ethyl derivatives 6c and d
showed the same order of potency, the second being
less toxic and therefore more selective. However, the
increase of the methylene side chain (6e, h and j)
clearly impaired the antiviral activity while not a€ect-
ing the cytotoxicity pro®le. The 4-cyanomethyl deriva-
tive 6g proved to be the most potent inhibitor of HIV-
1 replication of the series (EC50=0.87 mM), followed by
the isostere propargyl derivative 6i (EC50=0.96 mM),
both showing scarce cytotoxicity and therefore good SI
values. In contrast, the introduction of a benzoyl or
acetamido group (7a and b), or the absence of sub-
stituents at this position (5h) resulted in loss of the
antiviral activity.
Thienothiadiazines 10a±c and 11 (Scheme 3) were pre-
pared from 5-nitro-, 5-bromo- and 5-chloro-1,1,3-
trioxo-2H,4H-thieno[3,4-e][1,2,4]thiadiazines 9a±c48 fol-
lowing the same sequence of reactions utilized for the
synthesis of 5j, 5k and 6a±n. For comparison purposes,
the 4-substituted 2-benzyl-benzo[1,2,4]thiadiazine 1,1-
dioxides 13a, b were also prepared by alkylation of
12 (Scheme 4), according to a procedure reported pre-
viously.52 Tables 1±3 summarize the physicochemical
and analytical characteristics of all the thienothiadiazine
derivatives synthesized.
Results and Discussion
The compounds synthesized were initially evaluated for
their activity as inhibitors of HIV-1 replication in vitro.
The results are summarized in Tables 4 and 5. These
biological data led to a series of considerations with
regard to the structure±activity relationships for the
compounds studied. Since the presence of aromatic
substituents on the benzothiadiazine analogues was
fundamental for their anti-HIV-1 activity, we ®rst
introduced a phenyl or substituted phenyl group at the 2
position of the thieno[3,4-e][1,2,4]thiadiazine structure
(compounds 5a±g and 6a, b). This modi®cation, how-
ever, led to products devoid of anti-HIV-1 activity,
irrespective of the nature of the R2 group present at
position 4 of the heterocycle. The lack of activity of
these derivatives is presumably due to their N-2 phenyl
group (R1), since it does not permit for the molecules to
assume the butter¯y-like conformation which seems
particularly important in other NNRTIs (TIBO, a-APA
and nevirapine).53±56
In order to improve the anti-HIV-1 activity of the new
molecules, a limited number of changes in their 2 posi-
tion were also made. These included introduction of
substituted benzyl groups (6k) or replacement of the
benzyl by methyl (6l, m), cyanomethyl (6n) or acetamido
(7c) groups. None of these alterations, however, led to
signi®cantly active products. On the other hand, mod-
i®cation of the 5 position by substitution of thiophene
proton with nitro (10a, 11), bromine (10b) or chlorine
(10c) groups did not yield active compounds at non-
cytotoxic concentrations. Moreover, neither the ben-
zothiadiazines 12±13 nor the intermediate compounds
2±3 were e€ective in the HIV-1 antiviral tests.
Compounds 6c, d, f, g and i, which rank among the
most potent inhibitors of HIV-1 in cell culture, were
evaluated against an extensive panel of mutant virus
strains that contained single mutations in their RT that
are characteristic for NNRTI resistance (Table 6). As a
rule, the compounds keep antiviral activity against
L100I RT mutant virus, but signi®cantly lose antiviral
activity against K103N, V106A, E138K, Y181C and
Y188H RT mutant viruses. Only 6f kept activity against
Y188H RT mutant virus (although it should be recog-
nized that the antiviral activity was close to its cytostatic
activity in CEM cells), 6g lost only six fold activity
against E138K RT HIV-1 whereas 6c had a ®ve to seven
fold decreased activity against E138K and Y181C RT
mutant viruses.
The ®rst compounds of this series found to inhibit the
HIV-1 replication were 6c and d, with selective indexes
(SI) of 196 and >316, respectively. Compounds 6c and
d bear at the 2 position a benzyl group which seems to
provide a conformation that permits a certain interaction
Four TTD derivatives that rank among the most potent
inhibitors of HIV-1 in cell culture were chosen to mea-
sure their inhibitory activity against HIV-1 RT (Table
7). Compounds 6d, f and g, which were inhibitory to
HIV-1 replication in cell culture within the 1.2±1.8 mM
range for CEM and 0.87±2.4 mM range for MT-4, were
inhibitory to HIV-1 RT at concentrations that rank
between 16.4 and 59.8 mM. Nevirapine, that is inhibi-
tory to HIV-1 replication at 0.11 mM (CEM) ± 0.03 mM
(MT-4), had an IC50 of 4.5 mM against HIV-1 RT.
Scheme 3. Reagents: (ii) NaH/DMF, C6H5CH2Br; (iii) NaH/DMF,
R2X.
Compound 6g was evaluated in more detail on its
inhibitory activity against HIV-1 RT in the presence of
Scheme 4.

2814
Ma E. Arranz et al. / Bioorg. Med. Chem. 7 (1999) 2811±2822
Table 1. 4-N-Substituted sulfamoylthiophene-3-methyl carboxylates 2 and -3-carbohydrazides 3 prepared
Compound no.
R1
R2
Yield (%)
mp (ꢀC)
Solvent
Formula
Anal.
2a
2b
2c
2d
2e
2f
2g
2h
2i
3a
3b
3c
3d
3e
3f
3g
3h
3i
Ph
2-Cl-Ph
3-Cl-Ph
4-Cl-Ph
2-Br-Ph
4-F-Ph
4-OMe-Ph
Bn
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
NHNH2
NHNH2
NHNH2
NHNH2
NHNH2
NHNH2
NHNH2
NHNH2
NHNH2
88
84
79
95
70
95
89
89
50
89
65
94
81
87
70
86
91
87
130±132
127±129
161±163
157±159
130±132
129±131
115±116
73±75
125±127
222±224
226±228
196±198
255±257
215±217
217±219
212±214
152±154
175±176
EtOH
EtOH
MeOH
MeOH
MeOH
EtOH
MeOH
EtOH
EtOH
C
12H11NO4S2
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C12H10ClNO4S2
12H10ClNO4S2
C12H10ClNO4S2
C12H10BrNO4S2
C
C12H10FNO4S2
C13H13NO5S2
C13H13NO4S2
CH2(2,4-diOMe-Ph)
Ph
2-Cl-Ph
C15H17NO6S2
C11H11N3O3S2
C11H10ClN3O3S2
C11H10ClN3O3S2
C11H10ClN3O3S2
C11H10BrN3O3S2
C11H10FN3O3S2
C12H13N3O4S2
C12H13N3O3S2
C14H17N3O5S2
MeOH
n-PrOH±H2O
EtOH
3-Cl-Ph
4-Cl-Ph
2-Br-Ph
4-F-Ph
MeOH
MeCN
EtOH
MeCN
EtOH
EtOH
4-OMe-PH
Bn
CH2(2,4-diOMe-Ph)
Table 2. 2-Substituted 1,1,3-trioxo-2H,4H-thieno[3,4-e][1,2,4]thiadiazines 5 synthesized
1
IR (KBr) cm
Compound no.
R1
Yield (%)
mp (ꢀC)
Solvent
CˆO
SO2
Formula
Anal.
5a
5b
5c
5d
5e
5f
5g
5h
5i
Ph
2-Cl-Ph
3-Cl-Ph
4-Cl-Ph
2-Br-Ph
4-F-Ph
80
26
67
54
28
54
82
82
56
75
60
244±245
>250 (d)
245±247
252±254 (d)
263±265
260±262
182±183
160±162
163±164
178±180
185±187
EtOH
EtOH±H2O
MeOH
MeOH±H2O
MeOH
1690
1690
1695
1700
1695
1695
1695
1690
1675
1690
1710
1340,1160
1340,1170
1340,1170
1330,1170
1335,1170
1335,1170
1335,1165
1335,1175
1330,1180
1310,1160
1330,1195
C
11H8N2O3S2
C11H7ClN2O3S2
11H7ClN2O3S2
C,H,N,S
C,H,N,S
C,N,H,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C
C11H7ClN2O3S2
C11H7BrN2O3S2
C11H7FN2O3S2
4-OMe-Ph
Bn
CH2(2,4-diOMe-Ph)
Me
CH2CN
Toluene
EtOH±H2O
Toluene
EtOH±H2O
EtOH
C12H10N2O4S2
C12H10N2O3S2
C14H14N2O5S2
C6H6N2O3S2
C17H5N3O3S2
5j
5k
six di€erent concentrations of [2,8-3H]dGTP (i.e. 20, 10,
5, 3, 2 and 1.5 mM) at 30, 15 and 0 (control) mM. A
dose-dependent inhibition was observed. When plotted
as a Lineweaver±Burk diagram (1/velocity versus 1/
substrate), the inhibition was purely non-competitive
with respect to the natural substrate (dGTP). This
kinetic behaviour is in agreement with the nature of
inhibition of HIV-1 RT by other NNRTIs such as
nevirapine and delarvidine (data not shown).
substituent in the 4 position displayed any anti-HIV
activity. These TTDs were also e€ective against other
strains of HIV-1 (RF, HE, MN, NDK), including those
that are resistant to AZT, but not against HIV-2 (ROD)
or simian immunode®ciency virus [SIV(MAC251)] at
subtoxic concentrations. Some of the test compounds
kept antiviral activity against L100I RT mutant virus,
but signi®cantly lose antiviral activity against K103N,
V106A, E138K, Y181C and Y188H RT mutant viruses.
Compounds 6d, f and g were inhibitory to HIV-1 RT at
concentrations that rank between 16.4 and 59.8 mM
(nevirapine: IC50=4.5 mM against HIV-1 RT). Inhibi-
tion of HIV-1 RT by compound 6g was purely non-
competitive with respect to the natural substrate
(dGTP), which is in agreement with the nature of inhi-
bition shown by other NNRTIs such as nevirapine and
delarvidine. The 2-benzyl-4-cyanomethyl and 2-benzyl-
4-propargyl derivatives 6g and i were the most active of
In summary, based on the Curtius rearrangement of the
4-(N-substituted-sulfamoyl)thiophene-3-carboxy azides
4, we have described the synthesis of the novel 1,1,3-
trioxo-2H,4H-thieno[3,4-e][1,2,4]thiadiazines (TTDs) 5,
6, 7, 10 and 11. Several 4-substituted-2-benzyl-deriva-
tives were found to selectively inhibit human immuno-
de®ciency virus type 1 [HIV-1 (IIIB)] replication in MT-4
and CEM cells. None of the derivatives without a

Ma E. Arranz et al. / Bioorg. Med. Chem. 7 (1999) 2811±2822
2815
Table 3. 2,4-Disubstituted 1,1,3-trioxo-2H,4H-thieno[3,4-e][1,2,4]thiadiazines 6 and 7 synthesized
Compound no.
R1
R2
Yield (%)
mp (ꢀC)
Solvent
EI-MS (m/z)
Formula
Anal.
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6na
7ab
7b
7cc
3-Cl-Ph
3-Cl-Ph
Bn
Bn
Bn
Bn
Bn
Bn
Me
Et
Me
Et
n-Pr
Bn
CH2CN
Allyl
CH2CꢁCH
CH2CH2CN
Et
Bn
CH2(2-Cl-Ph)
Bn
COPh
CH2CONH2
Bn
87
93
90
91
74
93
89
90
90
45
91
95
90
89
45
46
43
177±178
125±127
158±160
140±142
134±136
128±130
182±184
134±136
161±163
137±139
167±169
97±99
132±134
119±120
131±133
228±230
238±240
MeOH
MeOH
EtOH
EtOH
MeOH
MeOH
EtOH
EtOH
EtOH
EtOH
MeOH
MeOH
EtOH
Ð
328 (M+
342 (M+
308 (M+
322 (M+
336 (M+
384 (M+
333 (M+
334 (M+
332 (M+
347 (M+
382 (M+
308 (M+
342 (M+
333 (M+
398 (M+
351 (M+
351 (M+
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
C12H9ClN2O3S2
C13H11ClN2O3S2
C13H12N2O3S2
C14H14N2O3S2
C15H16N2O3S2
C19H16N2O3S2
C14H11N3O3S2
C15H14N2O3S2
C15H12N2O3S2
C15H13N3O3S2
C16H18N2O5S2
C13H12N2O3S2
C13H11ClN2O3S2
C14H11N2O3S2
C19H14N2O4S2
C14H13N3O4S2
C14H13N3O4S2
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
Bn
Bn
CH2(2,4-diOMe-PH)
Me
Me
CH2CN
Bn
Bn
Ð
MeCN
Ð
CH2CONH2
a
Puri®ed by column chromatography (hexane:ethyl acetate 3:1).
Puri®ed by column chromatography in dichloromethane.
Puri®ed by column chromatography (chloroform:ethanol 30:1).
b
c
Table 4. Anti-HIV-l activity of 2-substituted 1,1,3-trioxo-2H,4H-thieno[3,4-c][1,2,4]thiadiazines 5 and 10 in MT-4 and CEM cell linesa
Compound no.
R1
X
HIV-1 (IIIB)
EC50 (mM)
CC50 (mM)
SI
MT-4
CEM
(MT-4)
(MT-4)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
Ph
o-Cl-Ph
m-Cl-Ph
p-Cl-Ph
o-Br-Ph
p-F-Ph
p-MeO-Ph
Bn
H
H
H
H
H
H
H
H
H
H
H
NO2
Br
Cl
>517.7
Ð
>110.7
>266.5
>695
>435.8
>507.2
>78.8
>705.4
>411.1
>99.8
>5.9
>418.2
>416.7
0.03
53.5
Ð
517.7
Ð
<1
Ð
<1
<1
1
<1
<1
<1
1
<1
<1
1
>63.5
>12.7
>55.7
>67.0
>64.4
ꢂ67.9
>282.2
Ð
110.7
266.5
>695
435.8
507.2
78.8
705.4
410.3
>99.8
<5.9
418.2
416.7
683
CH2(2,4-diMeO-Ph)
CH2CN
Me
Ð
10a
10b
10c
Nevirapine
AZT
Bn
Bn
Bn
>2.3
ꢂ53.5
48.6
<1
<1
22,767
50,857
0.11
0.015
0.0007
35.6
a
All data represent mean values for at least two separate experiments.
the series and were therefore chosen as lead compounds
for the design of more potent molecules.
analyses were performed with a Heraeus CHN-RAPID
instrument at the Centro Nacional de Quõmica Orga-
nica, CSIC, Madrid. Analytical results which are only
indicated by symbols were found within ‹0.4% of the
1
theoretical values. H NMR spectra were recorded on
Experimental
Varian Gemini 200, Bruker AM-200 and Varian XL-
300 spectrometers operating at 200 and 300 MHz in the
indicated solvent. Chemical shifts are expressed in d
Melting points were determined on a Gallenkamp
capillary apparatus and are uncorrected. Elemental

2816
Ma E. Arranz et al. / Bioorg. Med. Chem. 7 (1999) 2811±2822
Table 5. Anti-HIV-l activity of TTDs 6, 7 and 11 in MT-4 and CEM cell linesa
Compound no.
R1
R2
X
HIV-1 (IIIB)
CC50 (mM)
EC50 (mM)
SI
MT-4
CEM
(MT-4)
(MT-4)
6a
6b
6c
6d
6e
6f
6g
6h
6
6j
6k
6l
6m
6n
7a
m-Cl-Ph
m-Cl-Ph
Bn
Bn
Bn
Bn
Bn
Bn
Me
Et
Me
Et
n-Pr
Bn
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NO2
>483.6
61.3
2.7
Ð
Ð
484.5
62.4
<1
<1
196
>316
>12.1
>298
578
2.3
1.5
14.9
1.8
1.2
Ð
527.9
775.4
>743.1
>650.3
502.7
303.8
>376.0
130.4
255.2
79.1
2.4
6.1
2.2
0.87
6.3
0.96
4.9
CH2CN
CH2CHˆCH2
CH2CꢁCH
CH2CN2CN
Et
49
Bn
Bn
5.1
Ð
>387
27
<1
<1
<1
<1
<1
<1
1
<1
22,767
50,857
CH2(2,4-diMeO-Ph)
>255.2
>77.8
>70
>291
>133
>401.3
>355.7
>368.8
0.03
>261.5
>64.8
>11.7
Ð
Me
Me
CH2CN
Bn
Bn
Bn
CH2(o-Cl-Ph)
Bn
COPh
CH2CONH2
Bn
Me
68.8
291.3
132.3
402.7
>355.7
37.1
Ð
7b
7c
136.5
>284.5
Ð
CH2CONH2
Bn
11
Nevirapine
AZT
0.11
0.015
683
35.6
0.0007
a
All data represent mean values for at least two separate experiments.
Table 6. Inhibitory activity of TTD derivatives against a series of mutant HIV-1 strains in CEM cell cultures
Compound no.
EC50 (mM)a
HIV-1 (WT)
L100I
K103N
V106A
E138K
Y181C
Y188H
6c
6d
6f
6g
2.3
1.5
1.8
1.2
5.1
0.11
12.9
10.2
ꢂ10
7.8
>324
>310
>10
>300
>60
>324
>310
ꢂ10
Ð
ꢂ324
ꢂ310
ꢂ10
7.2
>324
>310
ꢂ10
ꢂ324
>310
7.8
>300
36.1
864
ꢂ60
6i
Nevirapine
6.0
0.07
Ð
2.44
30.1
0.03
Ð
>37
1.54
a
50% e€ective concentration or compound concentration required to inhibit virus-induced giant cell formation by 50%. All data represent mean
values for at least two separate experiments.
Table 7. Inhibitory activity of TTD derivatives against recombinant
HIV-1 reverse transcriptase
cator 254 nm) were used for thin layer chromatography
(TLC). Developed plates were visualized by UV light.
Flash column chromatography was performed on col-
umns packed with silica gel 60 (230±400 mesh) (Merck).
Compound no.
IC50 (mM)a
6d
6f
6g
31.0
59.8
16.4
4.5
Chemistry
Nevirapine
General procedure for the preparation of derivatives
2a±i. Example: 4-(N-phenylsulfamoyl)thiophene-3-methyl
carboxylate (2a). To a solution of 1 (2.40 g, 0.01 mol) in
THF (10 mL) was added dropwise a solution of aniline
(1.82 mL, 0.02 mol) in the same solvent whilst main-
taining the temperature below 10ꢀC. The reaction mix-
ture was stirred at 5ꢀC for 1 h and at room temperature
for 1 h more. The solvent was evaporated and the resi-
a
50% inhibitory concentration or compound concentration required
to inhibit [2,8-3H]dGTP incorporation into the poly rC.oligo dG tem-
plate by 50%. 1 mM [2,8-3H]dGTP served as the natural substrate of
the enzyme reaction.
units from tetramethylsilane (TMS) as an internal
standard. IR spectra were measured with a Shimadzu
IR-435 spectrometer. Silica gel/TLC cards (Fluka, silica
gel-precoated aluminium cards with ¯uorescent indi-
due was treated with water to give a solid which was
1
puri®ed by recrystallization to give 2a. IR (KBr, cm
)
3250 (NH); 1695 (CˆO); 1345, 1155 (SO2); 1265 (C O).

Ma E. Arranz et al. / Bioorg. Med. Chem. 7 (1999) 2811±2822
2817
1H NMR (DMSO-d6) d 9.70 (bs, 1H, NH); 8.40 (d, 1H,
J=3.3 Hz, thiophene); 8.31 (d, 1H, J=3.3 Hz, thio-
phene); 7.22±7.01 (m, 5H, benzene); 3.84 (s, 3H, CH3).
General procedure for the preparation of sulfamoylcarbo-
hydrazides 3a±i. Example: 4-(N-phenylsulfamoyl)thio-
phene-3-carbohydrazide (3a). A mixture of compound
2a (1.49 g, 5 mmol) and hydrazine hydrate (98%)
(0.72 mL, 0.015 mol) in ethanol (5 mL) was re¯uxed for
1 h. Upon cooling, the precipitated solid was ®ltered o€,
washed with ice cold ethanol and recrystallized to yield
3a. IR (KBr, cm 1) 3360 (NH); 1660 (CˆO); 1335, 1140
(SO2). 1H NMR (DMSO-d6) d 10.20±8.50 (bs, 2H, NH);
8.07 (d, 2H, thiophene); 7.28±7.04 (m, 5H, benzene);
5.10±4.00 (bs, 2H, NH2).
4-[N-(2-Chlorophenyl)sulfamoyl]thiophene-3-methyl car-
boxylate (2b). IR (KBr, cm 1) 3230 (NH); 1710 (CˆO);
1
1335, 1160 (SO2); 1250 (C±O). H NMR (DMSO-d6) d
9.03 (s, 1H, exchange with D2O, NH); 8.49 (d, 1H,
J=3.3 Hz, thiophene); 8.27 (d, 1H, J=3.3 Hz, thio-
phene); 7.44±7.17 (m, 4H, benzene); 3.84 (s, 3H, CH3).
4-[N-(3-Chlorophenyl)sulfamoyl]thiophene-3-methyl car-
boxylate (2c). IR (KBr, cm 1) 3250 (NH), 1705 (CˆO),
4-[N-(2-Chlorophenyl)sulfamoyl]thiophene-3-carbohydra-
zide (3b). IR (KBr, cm 1) 3400, 3320 (NH); 1660
1
1315, 1160 (SO2); 1250 (C±O). H NMR (DMSO-d6) d
10.13 (bs, 1H, NH); 8.41 (d, 2H, thiophene); 7.29±7.03
(m, 4H, benzene); 3.84 (s, 3H, CH3).
1
(CˆO); 1150 (SO2). H NMR (DMSO-d6) d 10.00 (bs,
1H, NH); 8.24 (d, 1H, J=3.2 Hz, thiophene); 8.11 (d,
1H, J=3.2 Hz, thiophene); 7.44±7.13 (m, 5H, benzene
and NH); 6.00±4.00 (bs, 2H, NH2).
4-[N-(4-Chlorophenyl)sulfamoyl]thiophene-3-methyl car-
boxylate (2d). IR (KBr, cm 1) 3180 (NH); 1695 (CˆO);
1
1270, 1155 (SO2). H NMR (DMSO-d6) d 9.96 (bs, 1H,
4-[N-(3-Chlorophenyl)sulfamoyl]thiophene-3-carbohydra-
zide (3c). IR (KBr, cm 1) 3300 (NH); 1610 (CˆO);
exchange with D2O, NH); 8.41 (d, 1H, J=3.3 Hz, thio-
phene); 8.34 (d, 1H, J=3.3 Hz, thiophene); 7.31±7.09
(m, 4H, benzene); 3.84 (s, 3H, CH3).
1
1250, 1110 (SO2). H NMR (DMSO-d6) d 9.95 (bs, 1H,
NH); 8.23 (d, 1H, J=3.2 Hz, thiophene); 8.02 (d, 1H,
J=3.2 Hz, thiophene); 7.31±7.06 (m, 5H, benzene and
NH); 5.70±4.20 (bs, 2H, NH2).
4-[N-(2-Bromophenyl)sulfamoyl]thiophene-3-methyl car-
boxylate (2e). IR (KBr, cm 1) 3250 (NH); 1710 (CˆO);
1
1260, 1150 (SO2). H NMR (DMSO-d6) d 8.42 (s, 1H,
4-[N-(4-Chlorophenyl)sulfamoyl]thiophene-3-carbohydra-
zide (3d). IR (KBr, cm 1) 3300 (NH); 1620 (CˆO);
1240, 1130 (SO2). 1H NMR (DMSO-d6) d 11.62 (bs, 1H,
NH); 7.95 (d, 2H, thiophene); 7.03±6.90 (m, 5H, ben-
zene and NH); 5.70±3.80 (bs, 2H, NH2).
NH); 8.16 (d, 1H, J=3.3 Hz, thiophene); 8.06 (d, 1H,
J=3.3 Hz, thiophene); 7.66 (dd, 1H, J=8.1 Hz, J=
1.5 Hz, benzene); 7.45 (dd, 1H, J=8.1 Hz, J=1.5 Hz,
benzene); 7.25 (m, 1H, benzene); 6.97 (ddd, 1H, J=
8.1 Hz, J=7.5 Hz, J=1.5 Hz, benzene); 3.96 (s, 3H,
CH3).
4-[N-(2-Bromophenyl)sulfamoyl]thiophene-3-carbohydra-
zide (3e). IR (KBr, cm 1) 3400, 3300, 3200 (NH); 1650
(CˆO); 1340, 1160 (SO2). 1H NMR (DMSO-d6) d 10.07
(bs, 1H, NH); 8.28 (d, 1H, J=3.3 Hz, thiophene); 8.11
(d, 1H, J=3.3 Hz, thiophene); 7.59 (dd, 1H, J=7.9 Hz,
J=1.4 Hz, benzene); 7.35 (m, 2H, benzene); 7.06 (m,
1H, benzene); 5.80±4.20 (bs, 3H, NH and NH2).
4-[N-(4-Fluorophenyl)sulfamoyl]thiophene-3-methyl car-
boxylate (2f). IR (KBr, cm 1) 3300 (NH); 1695 (CˆO);
1
1150 (SO2). H NMR (DMSO-d6) d 9.50 (bs, 1H, NH);
8.38 (d, 1H, J=3.4 Hz, thiophene); 8.23 (d, 1H, J=
3.4 Hz, thiophene); 7.09±7.05 (m, 4H, benzene); 3.84 (s,
3H, CH3).
4-[N-(4-Fluorophenyl)sulfamoyl]thiophene-3-carbohydra-
zide (3f). IR (KBr, cm 1) 3380, 3200 (NH); 1645
(CˆO); 1330, 1145 (SO2). 1H NMR (DMSO-d6) d 10.00
(bs, 1H, NH); 8.06 (d, 2H, thiophene); 7.12±7.09 (m,
4H, benzene); 5.30±3.70 (bs, 2H, NH2).
4-[N-(4-Methoxyphenyl)sulfamoyl]thiophene-3-methyl
car-boxylate (2g). IR (KBr, cm 1): 3300 (NH); 1705
1
(CˆO); 1155 (SO2); 1245 (C±O). H NMR (DMSO-d6)
d 8.32 (d, 1H, J=3.4 Hz, thiophene); 8.09 (d, 1H,
J=3.4 Hz, thiophene); 6.94 (dd, 2H, J=8.9 Hz, J=
2.3 Hz, benzene); 6.75 (dd, 2H, J=8.9 Hz, J=2.3 Hz,
benzene); 3.85 (s, 3H, CH3); 3.65 (s, 3H, CH3).
4-[N-(4-Methoxyphenyl)sulfamoyl]thiophene-3-carbohydra-
zide (3g). IR (KBr, cm 1) 3340 (NH); 1617 (CˆO); 1150
(SO2). 1H NMR (DMSO-d6) d 12.00±9.00 (bs, 2H, NH);
7.95 (d, 2H, thiophene); 6.91±6.87 (dd, 2H, J=8.9 Hz, J=
2.2 Hz, benzene); 6.70±6.66 (dd, 2H, J=8.9 Hz, J=2.2Hz,
benzene); 6.50±4.20 (bs, 2H, NH2); 3.62 (s, 3H, CH3).
4-(N-Benzylsulfamoyl)thiophene-3-methyl carboxylate
(2h). IR (KBr, cm 1): 3300 (NH); 1717 (CˆO); 1330,
1
1150 (SO2); 1210 (C±O). H NMR (DMSO-d6) d 8.05
(d, 2H, thiophene); 7.20 (s, 5H, benzene); 6.56 (t, J=
6.9 Hz, NH); 4.16 (d, 2H, J=6.9 Hz, CH2).
4-(N-Benzylsulfamoyl)thiophene-3-carbohydrazide (3h).
IR (KBr, cm 1) 3300 (NH); 1660 (CˆO); 1320, 1140
1
4-[N-(2,4-Dimethoxybenzyl)sulfamoyl]thiophene-3-methyl
carboxylate (2i). IR (KBr, cm 1) 3250 (NH); 1705
(SO2). H NMR (DMSO-d6) d 8.12 (d, 1H, J=3.0 Hz,
thiophene); 7.95 (d, 1H, J=3.0 Hz, thiophene); 6.00±
4.10 (bs, 4H, NH); 4.03 (s, 2H, CH2).
1
(CˆO); 1320, 1155 (SO2). H NMR (DMSO-d6) d 8.34
(d, 1H, J=3.3 Hz, thiophene); 8.08 (d, 1H, J=3.3 Hz,
thiophene); 6.91±6.83 (m, 2H, benzene and NH);
6.34±6.28 (m, 2H, benzene); 4.00 (d, 2H, J=5.1 Hz,
CH2N); 3.79 (s, 3H, CH3); 3.68 (s, 3H, CH3); 3.64 (s,
3H, CH3).
4-[N-(2,4-Dimethoxybenzyl)sulfamoyl]thiophene-3-carbo-
hydrazide (3i). IR (KBr, cm 1) 3340, 3280, 3200 (NH);
1
1670 (CˆO); 1285, 1150 (SO2). H NMR (DMSO-d6) d
9.90 (bs, 1H, NH); 8.10±7.90 (d, 2H, thiophene); 7.30

2818
Ma E. Arranz et al. / Bioorg. Med. Chem. 7 (1999) 2811±2822
(bs, 1H, NH); 7.00 (d, 1H, J=8.0 Hz, benzene); 6.50±
6.30 (m, 2H, benzene); 4.60 (bs, 2H, NH2); 3.92 (s, 2H,
CH2); 3.70 (s, 3H, CH3); 3.64 (s, 3H, CH3).
J=3.2 Hz, thiophene); 7.65±7.53 (m, 4H, benzene); 7.16
(d, 1H, J=3.2 Hz, thiophene).
2-(m-Chlorophenyl)-1,1,3-trioxo-2H,4H-thieno[3,4-e]-
[1,2,4]thiadiazine (5c). Compound 4c (2.30 g, 7 mmol) in
dry chloroform (70 mL) was re¯uxed for 6 h to give 5c.
1H NMR (DMSO-d6) d 11.56 (s, 1H, NH); 8.68 (d, 1H,
J=3.2 Hz, thiophene); 7.60±7.50 (m, 3H, benzene); 7.40
(dd, 1H, J=7.4 Hz, J=1.7 Hz, benzene); 7.11 (d, 1H,
J=3.2 Hz, thiophene).
General procedure for the preparation of sulfamoylcar-
boxy azides 4a±i. Example: 4-(N-phenylsulfamoyl)thio-
phene-3-carboxy azide (4a). To a solution of compound
3a (1.19 g, 4 mmol) in 2 N hydrochloric acid (20 mL)
was added dropwise a solution of sodium nitrite (0.56 g)
in water (2 mL), maintaining the reaction temperature
below 10ꢀC. The mixture was stirred at this temperature
for 2 h and the insoluble product was ®ltered, washed
with water and dried to yield 4a, 1.17 g (95%). The
compound was pure enough to be used as such in the
following step. IR (KBr, cm 1) 3300 (NH); 2150, 1220
(N3); 1670 (CˆO); 1150 (SO2).
2-(p-Chlorophenyl)-1,1,3-trioxo-2H,4H-thieno[3,4-e]-
[1,2,4]thiadiazine (5d). Compound 4d (2.85 g, 8 mmol)
in dry chloroform (94 mL) was re¯uxed for 2 h to give
1
5d. H NMR (DMSO-d6) d 11.50 (s, 1H, NH); 8.87 (d,
1H, J=3.2 Hz, thiophene); 7.59 (AB, 2H, J=8.6 Hz,
benzene); 7.30 (AB, 2H, J=8.6 Hz, benzene); 7.13 (d,
1H, J=3.2 Hz, thiophene).
4-[N-(2-Chlorophenyl)sulfamoyl]thiophene-3-carboxy azide
(4b). Yield: 95%. IR (KBr, cm 1) 3250 (NH); 2150,
1215 (N3); 1680 (CˆO); 1340, 1155 (SO2).
2-(o-Bromophenyl)-1,1,3-trioxo-2H,4H-thieno[3,4-e]-
[1,2,4]thiadiazine (5e). Compound 4e (3.0 g, 8 mmol) in
dry chloroform (85 mL) was re¯uxed for 6 h to give 5e.
1H NMR (DMSO-d6) d 11.59 (s, 1H, NH); 8.67 (d, 1H,
J=3.1 Hz, thiophene); 7.83±7.44 (m, 4H, benzene); 7.13
(d, 1H, J=3.1 Hz, thiophene).
4-[N-(3-Chlorophenyl)sulfamoyl]thiophene-3-carboxy azide
(4c). Yield: 96%. IR (KBr, cm 1) 3250 (NH); 2150,
1220 (N3); 1680 (CˆO); 1330, 1160 (SO2).
4-[N-(4-Chlorophenyl)sulfamoyl]thiophene-3-carboxy azide
(4d). Yield: 81%. IR (KBr, cm 1) 3300 (NH); 2150,
1220 (N3); 1670 (CˆO); 1380, 1155 (SO2).
2-(p-Fluorophenyl)-1,1,3-trioxo-2H,4H-thieno[3,4-e]-
[1,2,4]thiadiazine (5f). Compound 4f (2.96 g, 9 mmol) in
dry chloroform (95 mL) was re¯uxed for 3 h to give 5f.
1H NMR (DMSO-d6) d 11.50 (bs, 1H, NH); 8.66 (d,
1H, J=3.2 Hz, thiophene); 7.43±7.33 (m, 4H, benzene);
7.13 (d, 1H, J=3.2 Hz, thiophene).
4-[N-(2-Bromophenyl)sulfamoyl]thiophene-3-carboxy azide
(4e). Yield: 87%. IR (KBr, cm 1) 2150 (N3); 1680
(CˆO).
4-[N-(4-Fluorophenyl)sulfamoyl]thiophene-3-carboxy azide
(4f). Yield: 95%. IR (KBr, cm 1) 3300 (NH); 2130,
1220 (N3); 1675 (CˆO); 1155 (SO2).
2-(p-Methoxyphenyl)-1,1,3-trioxo-2H,4H-thieno[3,4-e]-
[1,2,4]thiadiazine (5g). Compound 4g (1.50 g, 4 mmol)
in dry xylene (50 mL) was re¯uxed for 2 h to give 5g. 1H
NMR (DMSO-d6) d 11.35 (bs, 1H, NH); 8.64 (d, 1H,
J=3.3 Hz, thiophene); 7.28 (AB, 2H, J=8.9 Hz, ben-
zene); 7.07 (d, 1H, J=3.3 Hz, thiophene); 7.04 (AB, 2H,
J=8.9 Hz, benzene); 3.80 (s, 3H, CH3).
4-[N-(4-Methoxyphenyl)sulfamoyl]thiophene-3-carboxy
azide (4g). Yield: 86%. IR (KBr, cm 1) 3300 (NH);
2140, 1210 (N3); 1675 (CˆO); 1155 (SO2).
4-(N-Benzylsulfamoyl)thiophene-3-carboxy azide (4h).
Yield: 85%. IR (KBr, cm 1) 3300 (NH); 2150, 1220
(N3); 1685 (CˆO); 1330, 1150 (SO2).
2-Benzyl-1,1,3-trioxo-2H,4H-thieno[3,4-e][1,2,4]thiadi-
azine (5h). Compound 4h (0.5 g, 1 mmol) in dry ben-
zene (13 mL) was re¯uxed for 7 h to give 5h. H NMR
(DMSO-d6) d 11.36 (s, 1H, NH); 8.61 (d, 1H, J=3.2 Hz,
thiophene); 7.30±7.20 (m, 5H, benzene); 7.04 (d, 1H,
J=3.2 Hz, thiophene); 4.92 (s, 2H, CH2).
1
4-[N-(2,4-Dimethoxybenzyl)sulfamoyl]thiophene-3-carboxy
azide (4i). Yield: 90%. IR (KBr, cm 1) 3270 (NH); 2150,
1210 (N3); 1670 (CˆO); 1160 (SO2).
2-(2,4-Dimethoxybenzyl)-1,1,3-trioxo-2H,4H-thieno[3,4-
e][1,2,4]thiadiazine (5i). Compound 4i (1.0 g, 3 mmol) in
General procedure for the preparation of thienothiadi-
azines 5a±i. Example: 2-phenyl-1,1,3-trioxo-2H,4H-
thieno[3,4-e][1,2,4]thiadiazine (5a). A suspension of the
carboxy azide 4a (4.62 g, 0.01 mol) in dry benzene
(90 mL) was re¯uxed for 3 h. The precipitate was ®ltered
o€ and recrystallized to give 5a. 1H NMR (DMSO-d6) d
11.45 (s, 1H, NH); 8.66 (d, 1H, J=3.2 Hz, thiophene);
7.52±7.36 (m, 5H, benzene); 7.10 (d, 1H, J=3.2 Hz,
thiophene).
1
dry benzene (50 mL) was re¯uxed for 6 h to give 5i. H
NMR (DMSO-d6) d 11.30 (bs, 1H, NH); 8.57 (d, 1H,
J=3.1 Hz, thiophene); 7.04 (d, 1H, J=3.1 Hz, thio-
phene); 6.92 (d, 1H, J=8.4 Hz, benzene); 6.53 (d, 1H,
J=2.2 Hz, benzene); 6.44 (dd, 1H, J=8.4 Hz, J=
2.2 Hz, benzene); 4.82 (s, 2H, CH2); 3.77 (s, 3H, CH3);
3.71 (s, 3H, CH3).
General procedure for the preparation of thienothiadi-
azines 5j±k. Example: 2-methyl-1,1,3-trioxo-2H,4H-
thieno[3,4-e][1,2,4]thiadiazine (5j). To a solution of
1,1,3-trioxo-2H,4H-thieno[3,4-e][1,2,4]thiadiazine48 (1.0 g,
5 mmol) in dry DMF (25 mL), under N2, was added
2-(o-Chlorophenyl)-1,1,3-trioxo-2H,4H-thieno[3,4-e]-
[1,2,4]thiadiazine (5b). Compound 4b (3.0 g, 9 mmol) in
dry chloroform (85 mL) was re¯uxed for 6 h to give 5b.
1H NMR (DMSO-d6) d 11.67 (s, 1H, NH); 8.67 (d, 1H,

Ma E. Arranz et al. / Bioorg. Med. Chem. 7 (1999) 2811±2822
2819
portionwise sodium hydride (60% dispersion in mineral
oil) (0.2 g, 5 mmol) maintaining the temperature below
10ꢀC. After 15 min, methyl iodide (0.31 mL, 5 mmol)
was added and the reaction mixture was stirred at room
temperature for 3 h. The DMF was evaporated in vacuo
2,4-Dibenzyl-1,1,3-trioxo-2H,4H-thieno[3,4-e][1,2,4]thi-
adiazine (6f). From 5h and benzyl bromide. IR (KBr,
cm 1) 1695 (CˆO); 1318, 1165 (SO2). 1H NMR
(DMSO-d6) d 8.69 (d, 1H, J=3.2 Hz, thiophene); 7.34±
7.22 (m, 11H, benzene and thiophene); 5.19 (s, 2H,
CH2); 5.01 (s, 2H, CH2).
1
and the crude residue was recrystallized to yield 5j. H
NMR (DMSO-d6) d 11.25 (s, 1H, NH); 8.60 (d, 1H,
J=3.2 Hz, thiophene); 7.01 (d, 1H, J=3.2 Hz, thio-
phene); 3.13 (s, 3H, CH3).
2-Benzyl-4-cyanomethyl-1,1,3-trioxo-2H,4H-thieno[3,4-
e][1,2,4]thiadiazine (6g). From 5h and chloroacetoni-
trile. IR (KBr, cm 1) 1707 (CˆO); 1340, 1172 (SO2). 1H
NMR (DMSO-d6) d 8.78 (d, 1H, J=3.1 Hz, thiophene);
7.62 (d, 1H, J=3.1 Hz, thiophene); 7.33±7.29 (m, 5H,
benzene); 5.13 (s, 2H, CH2); 4.98 (s, 2H, CH2).
2-Cyanomethyl-1,1,3-trioxo-2H,4H-thieno[3,4-e][1,2,4]-
1
thiadiazine (5k). H NMR (DMSO-d6) d 11.50 (bs, 1H,
NH); 8.70 (d, 1H, J=3.1 Hz, thiophene); 7.11 (d, 1H,
J=3.1 Hz, thiophene); 4.84 (s, 2H, CH2).
4-Allyl-2-benzyl-1,1,3-trioxo-2H,4H-thieno[3,4-e][1,2,4]-
thiadiazine (6h). From 5h and allyl bromide. IR (KBr,
cm 1) 1692 (CˆO); 1333, 1180 (SO2). 1H NMR
(DMSO-d6) d 8.71 (d, 1H, J=3.2 Hz, thiophene); 7.40
(d, 1H, J=3.2 Hz, thiophene); 7.33±7.27 (m, 5H, ben-
zene); 5.90±5.81 (m, 1H, CH); 5.19 (dd, 1H, J=5.7 Hz,
J= 1.4 Hz, ˆCH); 5.14 (dd, 1H, J=12.3 Hz, J=
1.4 Hz, ˆCH); 4.97 (s, 2H, CH2); 4.58 (d, 2H, J=
4.9 Hz, CH2).
General procedure for the preparation of thienothiadia-
zines 6a±n. To a solution of the thiadiazine 5 (1 mmol)
in dry DMF (15 mL), under Ar, was added slowly
sodium hydride (60% dispersion in mineral oil, 0.06 g,
1.5 mmol) maintaining the temperature below 10ꢀC.
After 15 min, the alkyl halide (1.5 mmol) was added and
the reaction mixture was stirred at 50±70ꢀC for 15 h.
The solvent was evaporated to dryness and the crude
solid was ®ltered, washed with water, dried and recrys-
tallized from the appropriate solvent.
2-Benzyl-4-propargyl-1,1,3-trioxo-2H,4H-thieno[3,4-e]-
[1,2,4]thiadiazine (6i). From 5h and propargyl chloride.
IR (KBr, cm 1) 3280 (CꢁCH); 1675 (CˆO); 1320, 1165
Example: 2-(m-chlorophenyl)-4-methyl-1,1,3-trioxo-2H,
4H-thieno[3,4-e][1,2,4]thiadiazine (6a). By reaction of 5c
with methyl iodide at room temperature. IR (KBr,
cm 1) 1700 (CˆO); 1335, 1180 (SO2). 1H NMR
(DMSO-d6) d 8.77 (d, 1H, J=3.2 Hz, thiophene); 7.61±
7.37 (m, 5H, benzene and thiophene); 3.43 (s, 3H, CH3).
1
(SO2). H NMR (DMSO-d6) d 8.74 (d, 1H, J=3.2 Hz,
thiophene); 7.47 (d, 1H, J=3.2 Hz, thiophene); 7.31 (s,
5H, benzene); 4.96 (s, 2H, CH2); 4.76 (d, 2H, J=2.4 Hz,
CH2 C); 3.36 (t, 1H, J=2.4 Hz, CH).
2-Benzyl-4-cyanoethyl-1,1,3-trioxo-2H,4H-thieno[3,4-e]-
[1,2,4]thiadiazine (6j). From 5h and 3-chloropropioni-
trile. Compound 6j was isolated from the crude solid by
column chromatography using CH2Cl2 as eluent. IR
(KBr, cm 1) 2260 (CN); 1670 (CˆO); 1355, 1165 (SO2).
1H NMR (DMSO-d6) d 8.73 (d, 1H, J=3.1 Hz, thio-
phene); 7.64 (d, 1H, J=3.1 Hz, thiophene); 7.34±7.27
(m, 5H, benzene); 4.97 (s, 2H, CH2); 4.25 (t, 2H, J=
6.7 Hz, CH2); 2.94 (t, 2H, J=6.7 Hz, CH2).
2-(m-Chlorophenyl)-4-ethyl-1,1,3-trioxo-2H,4H-thieno-
[3,4-e][1,2,4]thiadiazine (6b). From thiadiazine 5c and
ethyl iodide at room temperature. IR (KBr, cm 1) 1697
1
(CˆO); 1340, 1175 (SO2). H NMR (DMSO-d6) d 8.77
(d, 1H, J=3.1 Hz, thiophene); 7.65±7.37 (m, 5H, ben-
zene and thiophene); 3.98 (c, 2H, J=7.0 Hz, CH2); 1.24
(t, 3H, J=7.0 Hz, CH3).
2-Benzyl-4-methyl-1,1,3-trioxo-2H,4H-thieno[3,4-e][1,2,4]-
thiadiazine (6c). From 5h and methyl iodide at room
temperature. IR (KBr, cm 1): 1680 (CˆO); 1330, 1160
2-(2,4-Dimethoxybenzyl)-4-ethyl-1,1,3-trioxo-2H,4H-thi-
eno[3,4-e][1,2,4]thiadiazine (6k). From 5i and ethyl io-
dide. IR (KBr, cm 1) 1683 (CˆO); 1340, 1172 (SO2). 1H
NMR (DMSO-d6) d 8.64 (d, 1H, J=3.2 Hz, thiophene);
7.50 (d, 1H, J=3.2 Hz, thiophene); 6.92 (d, 1H, J=
8.3 Hz, benzene); 6.52 (d, 1H, J=2.4 Hz, benzene); 6.43
(dd, 1H, J=8.3 Hz, J=2.4 Hz, benzene); 4.85 (s, 2H,
CH2); 3.85 (c, 2H, J=7.0 Hz, CH2); 3.74 (s, 3H, CH3O);
3.71 (s, 3H, CH3O); 1.19 (t, 3H, J=7.0 Hz, CH3).
1
(SO2). H NMR (DMSO-d6) d 8.69 (d, 1H, J=3.2 Hz,
thiophene); 7.39 (d, 1H, J=3.2 Hz, thiophene); 4.94 (s,
2H, CH2); 3.39 (s, 3H, CH3).
2-Benzyl-4-ethyl-1,1,3-trioxo-2H,4H-thieno[3,4-e][1,2,4]-
thiadiazine (6d). From 5h and ethyl bromide. IR (KBr,
cm 1): 1677 (CˆO); 1330, 1180 (SO2). 1H NMR
(DMSO-d6) d 8.70 (d, 1H, J=3.2 Hz, thiophene); 7.50
(d, 1H, J=3.2 Hz, thiophene); 7.30±7.20 (m, 5H, ben-
zene); 4.95 (s, 2H, CH2); 3.95 (c, 2H, J=7.1 Hz, CH2);
1.19 (t, 3H, J=7.1 Hz, CH3).
4-Benzyl-2-methyl-1,1,3-trioxo-2H,4H-thieno[3,4-e][1,2,4]-
thiadiazine (6l). From 5j and benzyl bromide. IR (KBr,
cm 1) 1685 (CˆO); 1320, 1165 (SO2). 1H NMR
(DMSO-d6) d 8.67 (d, 1H, J=2.7 Hz, thiophene); 7.33±
7.25 (m, 6H, benzene and thiophene); 5.19 (s, 2H, CH2);
3.22 (s, 3H, CH3).
2-Benzyl-4-propyl-1,1,3-trioxo-2H,4H-thieno[3,4-e][1,2,4]-
thiadiazine (6e). From 5h and 1-bromopropane. IR
1
(KBr, cm 1): 1680 (CˆO); 1325, 1190 (SO2). H NMR
(DMSO-d6) d 8.66 (d, 1H, J=3.2 Hz, thiophene); 7.49
(d, 1H, J=3.2 Hz, thiophene); 7.30±7.20 (m, 5H, ben-
zene); 4.94 (s, 2H, CH2); 3.90 (t, 2H, J=7.0 Hz, CH2);
1.62 (m, 2H, CH2); 0.85 (t, 3H, J=7.0 Hz, CH3).
4-(o-Chlorobenzyl)-2-methyl-1,1,3-trioxo-2H,4H-thieno-
[3,4-e][1,2,4]thiadiazine (6m). From 5j and o-chloro-
benzyl bromide. IR (KBr, cm 1) 1690 (CˆO); 1335,
1165 (SO2). 1H NMR (DMSO-d6) d 8.71 (d, 1H,

2820
Ma E. Arranz et al. / Bioorg. Med. Chem. 7 (1999) 2811±2822
J=3.0 Hz, thiophene); 7.54±7.49 (dd, 1H, J=7.0 Hz,
J=1.8 Hz, benzene); 7.32±7.25 (m, 3H, benzene and
thiophene); 7.04 (d, 1H, J=6.4 Hz, benzene); 5.21 (s,
2H, CH2); 3.21 (s, 3H, CH3).
7c as a white solid. IR (KBr, cm 1) 3455 (NH2); 1705,
1
1687 (CˆO); 1335, 1165 (SO2). H NMR (DMSO-d6)
d 8.62 (d, 1H, J=3.1Hz, thiophene); 7.60 (bs, 1H,
exchange with D2O, NH); 7.32±7.23 (m, 7H, benzene,
thiophene and NH); 5.18 (s, 2H, CH2); 4.34 (s, 2H, CH2).
4-Benzyl-2-cyanomethyl-1,1,3-trioxo-2H,4H-thieno[3,4-e]-
[1,2,4]thiadiazine (6n). From 5k and benzyl bromide.
Compound 6n was isolated from the crude solid by col-
umn chromatography using hexane:ethyl acetate 3:1 as
eluent. IR (KBr, cm 1) 1695 (CˆO); 1330, 1170 (SO2).
1H NMR (DMSO-d6) d 8.77 (d, 1H, J=3.1 Hz, thio-
phene); 7.41 (d, 1H, J=3.1 Hz, thiophene); 7.39±7.26
(m, 5H, benzene); 5.23 (s, 2H, CH2); 4.93 (s, 2H, CH2).
General procedure for the preparation of thienothiadi-
azines 10a±c. Example: 2-benzyl-5-nitro-1,1,3-trioxo-
2H,4H-thieno[3,4-e][1,2,4]thiadiazine (10a). To a solu-
tion of 5-nitro-1,1,3-trioxo-2H,4H-thieno[3,4-e][1,2,4]-
thiadiazine48 (9a) (1.0 g, 4 mmol) in dry DMF, under
N2, was added slowly sodium hydride (60% dispersion
in mineral oil) (0.17 g, 4 mmol), maintaining the tem-
perature below 10ꢀC. After 15 min, benzyl bromide
(0.49 mL, 4 mmol) was added and the reaction mixture
was stirred at 50ꢀC for 23 h. The DMF was evaporated
in vacuo and the crude residue was recrystallized to give
10a, 1.24 g (60%). Mp 187±188ꢀC (EtOH). IR (KBr,
cm 1): 3325 (NH); 1705 (CˆO); 1585, 1290 (NO2);
1350, 1170 (SO2). 1H NMR (DMSO-d6) d 11.43 (bs, 1H,
NH); 9.01 (s, 1H, thiophene); 7.34 (m, 5H, benzene);
4.97 (s, 2H, CH2). Anal. (C12H9N3O5S2) C, H, N, S.
4-Benzoyl-2-benzyl-1,1,3-trioxo-2H,4H-thieno[3,4-e]-
[1,2,4]thiadiazine (7a). To a suspension of 5h (0.15 g,
0.5 mmol) in dry pyridine was added dropwise benzoyl
chloride (0.077 mL, 0.66 mmol), maintaining the tem-
perature below 10ꢀC. The reaction mixture was stirred
at room temperature for 24 h. The solvent was evapo-
rated to dryness and the crude solid was puri®ed by
column chromatography using CH2Cl2 as eluent. IR
1
(KBr, cm 1) 1707 (CˆO); 1360, 1190 (SO2). H NMR
(DMSO-d6) d 8.77 (d, 1H, J=3.2 Hz, thiophene); 8.06
(d, 1H, J=3.2 Hz, thiophene); 7.78±7.54 (m, 5H, ben-
zene); 7.34±7.27 (m, 5H, benzene); 4.92 (s, 2H, CH2).
Anal. (C19H14N2O4S2) C, H, N, S.
2-Benzyl-5-bromo-1,1,3-trioxo-2H,4H-thieno[3,4-e][1,2,4]-
thiadiazine (10b). This compound was obtained in an
89% yield from reaction of 5-bromo-1,1,3-trioxo-
2H,4H-thieno[3,4-e][1,2,4]thiadiazine48 (9b) with benzyl
bromide; mp 202±203ꢀC (MeOH). IR (KBr, cm 1) 3150
(NH); 1687 (CˆO); 1335, 1165 (SO2). 1H NMR
(DMSO-d6) d 11.37 (bs, 1H, NH); 8.69 (s, 1H, thio-
phene); 7.31 (s, 5H, benzene); 4.92 (s, 2H, CH2). Anal.
(C12H9BrN2O3S2) C, H, N, S.
2-Benzyl-4-(carbamoylmethyl)-1,1,3-trioxo-2H,4H-thi-
eno[3,4-e][1,2,4]thiadiazine (7b). To a solution of 6g
(0.14 g, 0.4 mmol) in dimethyl sulfoxide (5 mL) contain-
ing potassium carbonate (8 mg, 0.06 mmol) was added
dropwise a 30% hydrogen peroxide solution (0.06 mL),
maintaining the temperature below 10ꢀC. The reaction
mixture was stirred at room temperature for 20 h. Ice-
water was added and the solid formed was ®ltered,
washed with water, dried and recrystallized. IR (KBr,
cm 1) 3480, 3360 (NH); 1697 (CˆO); 1680 (CˆO);
2-Benzyl-5-chloro-1,1,3-trioxo-2H,4H-thieno[3,4-e][1,2,4]-
thiadiazine (10c). This compound was obtained in a
90% yield from the reaction of 5-chloro-1,1,3-trioxo-
2H,4H-thieno[3,4-e][1,2,4]thiadiazine48 (9c) with benzyl
bromide; mp 192±194ꢀC (EtOH). IR (KBr, cm 1) 3190
(NH); 1690 (CˆO); 1340, 1165 (SO2). 1H NMR
(DMSO-d6) d 11.55 (bs, 1H, NH); 8.55 (s, 1H, thio-
phene); 7.31 (s, 5H, benzene); 4.92 (s, 2H, CH2). Anal.
(C12H9ClN2O3S2) C, H, N, S.
1
1307, 1167 (SO2). H NMR (DMSO-d6) d 8.66 (d, 1H,
J=3.3 Hz, thiophene); 7.64 (bs, 1H, NH); 7.32±7.25 (m,
6H, benzene and NH); 7.21 (d, 1H, J=3.3 Hz, thio-
phene CH); 4.94 (s, 2H, CH2); 4.47 (s, 2H, CH2).
4-Benzyl-2-(carbamoylmethyl)-1,1,3-trioxo-2H,4H-thi-
eno[3,4-e][1,2,4]thiadiazine (7c) and 4-(N-benzylamino)-
3-(N-cyanomethyl)sulfamoylthiophene (8). To a solution
of 6n (0.10 g, 0.3 mmol) in dichloromethane (0.2 mL)
containing tetrabutylammonium hydrogen sulfate
(0.02 g, 0.06 mmol) were added dropwise a 30% hydro-
gen peroxide solution (0.14 mL) and a 20% aqueous
solution of sodium hydroxide (0.11 mL), maintaining the
temperature below 10ꢀC. The reaction mixture was stir-
red at room temperature for 2 h and was then extracted
with dichloromethane. The organic layer was washed
with a saturated NaCl solution, dried (Na2SO4) and
evaporated to dryness. The residue was puri®ed by col-
umn chromatography using chloroform:ethanol 30:1 as
eluent. The ®rst fractions yielded 8 (20 mg, 22%) as an
oil. 1H NMR (DMSO-d6) d 8.57 (t, 1H, J=6.0 Hz, NH);
8.08 (d, 1H, J=3.5 Hz, thiophene); 7.41±7.23 (m, 5H,
benzene); 6.11 (d, 1H, J=3.5 Hz, thiophene); 5.54 (t, 1H,
J=5.1 Hz, NH); 4.27 (d, 2H, J=5.1 Hz, CH2); 4.11 (d,
2H, J=6.0 Hz, CH2). The slowest moving fractions gave
2-Benzyl-4-methyl-5-nitro-1,1,3-trioxo-2H,4H-thieno-
[3,4-e][1,2,4]thiadiazine (11). This compound was
obtained in an 80% yield from the reaction of 10a with
methyl iodide following the general procedure used for
the preparation of thiadiazines 6a±n; mp 167±169ꢀC
(EtOH±H2O). IR (KBr, cm 1) 1695 (CˆO); 1340, 1187
1
(SO2). H NMR (DMSO-d6) d 8.94 (s, 1H, thiophene);
7.33 (s, 5H, benzene); 4.93 (s, 2H, CH2); 3.44 (s, 3H,
CH3). Anal. (C13H11N3O5S2) C, H, N, S.
2-Benzyl-4-methyl-1,1,3-trioxo-2H,4H-benzo[1,2,4]thi-
adiazine (13a). This compound was obtained in a 75%
yield from the reaction of 2-benzyl-1,1,3-trioxo-2H,4H-
1,2,4-benzothiadiazine (12)52 with methyl iodide; mp
110±111ꢀC (EtOH). H NMR (DMSO-d6) d 7.95 (dd,
1
1H, J=7.7 Hz, J=1.5 Hz, benzene H-8); 7.83 (ddd, 1H,
J=8.4 Hz, J=7.7 Hz, J=1.5 Hz, benzene H-6); 7.57 (d,
1H, J=8.4 Hz, benzene H-5); 7.43 (t, 1H, J=7.7 Hz,
benzene H-7); 7.31 (s, 5H, benzene); 4.98 (s, 2H, CH2);
3,47 (s, 3H, CH3). Anal. (C15H14N2O3S) C, H, N, S.

Ma E. Arranz et al. / Bioorg. Med. Chem. 7 (1999) 2811±2822
2821
2-Benzyl-4-cyanomethyl-1,1,3-trioxo-2H,4H-benzo[1,2,4]-
thiadiazine (13b). This compound was obtained in a
59% yield from the reaction of 12 with 2-chloro-
acetonitrile. The crude product was puri®ed by column
chromatography using CH2Cl2 as eluent; mp 141±
143ꢀC. 1H NMR (DMSO-d6) d 8.02 (dd, 1H, J=7.8 Hz,
J=1.4 Hz, benzene H-8); 7.93 (ddd, 1H, J=8.4 Hz,
J=7.8 Hz, J=1.4 Hz, benzene H-6); 7.66 (d, 1H,
J=8.4 Hz, benzene H-5); 7.52 (t, 1H, J=7.8 Hz, ben-
zene H-7); 7.34 (s, 5H, benzene); 5.25 (s, 2H, CH2); 5.01
(s, 2H, CH2). Anal. (C16H13N3O3S) C, H, N, S.
Madrid, Spain (research grant SAF 96-0111) is grate-
fully acknowledged. We thank the Consejerõa de Edu-
cacion y Cultura de la Comunidad de Madrid for a
graduate fellowship and the Sociedad Espanola de Quõ-
mica Terapeutica for a Scienti®c Award to M.E.A. We
also thank the Biomedical Research Programme of the
European Commission. We want to acknowledge Kris-
tien Erven, Ann Absillis and Lizette van Berckelaer for
excellent technical assistance with the anti-HIV and
anti-RT assays, Juan J. Bustos for preparation of the
starting materials and Francisco Caballero for editorial
assistance.
Anti-HIV activity assays
References
The human immunode®ciency virus strain used was
HIV-1 (IIIB).57 Anti-HIV activity and cytotoxicity mea-
surements were carried out in parallel. They were based
on the viability of MT-4 cells that had been infected
with HIV and then exposed to various concentrations of
the test compounds. After the MT-4 cells were allowed
to proliferate for 5 days, the number of viable cells was
quanti®ed by a tetrazolium-based colorimetric 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) procedure in 96-well microtrays.58 In all of these
assays, viral input (viral multiplicity of infection, MOI)
was 0.01, or 100 times the 50% cell culture infective
dose (CCID50). The 50% antivirally e€ective con-
centration (EC50) was de®ned as the compound con-
centration required to protect 50% of the virus-infected
cells against viral cytopathicity. The 50% cytotoxic
concentration (CC50) was de®ned as the compound
concentration required to reduce the viability of mock-
infected cells by 50%. The > symbol is used to indicate
the highest concentration at which the compounds were
tested and still found to be non-cytotoxic. Average EC50
and CC50 values for several separate experiments are
presented as de®ned above. As a rule, the individual
values did not deviate by more than two fold up or
down from the EC50 and CC50 values indicated in the
tables. On the other hand, the inhibitory activity of
TTDs against wild-type HIV-1 and mutant strains in
CEM cell cultures was examined as follows. CEM cells
were suspended at 250,000±350,000 cells per mL of cul-
ture medium and infected with approximately 100
CCID50 (1 CCID50 being the 50% cell culture infective
dose) of HIV-1 (IIIB). Then, 100 mL of the infected cell
suspensions was added to 200-mL-microtiter plate wells
containing 100 mL of an appropriate dilution of the test
compounds. The inhibitory e€ect of the test compounds
on HIV-1-induced syncytium formation in CEM cells
was examined on day 4 post infection. The 50% e€ec-
tive concentration (EC50) was determined as the com-
pound concentration required to inhibit syncytium
formation by 50%. The kinetics of inhibition of HIV-1
RT by several TTDs were performed as described pre-
viously.59 The template/primer was poly rC.dG and the
radiolabelled substrate [2.8-3H]dGTP.
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