Synthesis, antiviral, and anti-HIV-1 integrase activities of 3-aroyl-1,1-dioxo-1,4,2-benzodithiazines

Article abstract of DOI:10.1016/j.bmc.2004.04.024

HIV-1 integrase (IN) is an essential enzyme for effective viral replication and is an attractive target for selective blockade of viral infection. Previously, we identified a series of sulfones, sulfonamides, and mercaptosalicylhydrazides (MBSAs) as IN inhibitors with antiviral activities in cell-based assays. In an effort to optimize a series of our active site directed lead compounds, we designed and synthesized novel benzodithiazines starting from MBSAs. In contrast to all reported IN inhibitors benzodithiazines are essentially nontoxic. Significant antiviral potency was only observed at concentration exceedingly higher than that required to inhibit purified IN.

Full text of DOI:10.1016/j.bmc.2004.04.024

Bioorganic & Medicinal Chemistry 12 (2004) 3663–3672
Synthesis, antiviral, and anti-HIV-1 integrase activities of
3-aroyl-1,1-dioxo-1,4,2-benzodithiazinesq
Zdzislaw Brzozowski,a Franciszek Saczewski,a,* Tino Sanchez,b Chih-Ling Kuo,b
Maria Gdaniecc and Nouri Neamatib,*
aDepartment of Chemical Technology of Drugs, Medical University of Gdansk, Al. Gen. Hallera 107, 80-416 Gdansk, Poland
bDepartment of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, PSC 304BA,
Los Angeles, CA 90089, USA
cFaculty of Chemistry, A. Mickiewicz University, 60-780 Poznan, Poland
Received 26 November 2003; revised 12 April 2004; accepted 15 April 2004
Available online 18 May 2004
Abstract—HIV-1 integrase (IN) is an essential enzyme for effective viral replication and is an attractive target for selective blockade
of viral infection. Previously, we identified a series of sulfones, sulfonamides, and mercaptosalicylhydrazides (MBSAs) as IN
inhibitors with antiviral activities in cell-based assays. In an effort to optimize a series of our active site directed lead compounds, we
designed and synthesized novel benzodithiazines starting from MBSAs. In contrast to all reported IN inhibitors benzodithiazines are
essentially nontoxic. Significant antiviral potency was only observed at concentration exceedingly higher than that required to
inhibit purified IN.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
In our continuous efforts in developing IN inhibitors we
focused our efforts in developing compounds that (1)
lack hydroxylated aromatic moieties, which in most
cases has been associated with their notorious lack of
selectivity and toxicity, (2) represent novel chemotypes
that are easily amenable to derivatization, (3) exhibit
antiviral activity in cell-based assays. Among a series of
lead compounds we identified that satisfy all these
requirements are the mercaptobenzenesulfonamides
(MBSAs)8;9 and mercaptobenzenesalicylhydrazides
(MSHs).10 More recently a dipyrimidine class of com-
pound was identified as selective IN inhibitor and
among all the tested compounds the mercaptodipyrim-
Integration of viral DNA into the host cell chromo-
somal DNA to form a provirus is an essential step in the
viral life cycle. This process is mediated by IN, a 32 kDa
viral enzyme.1;2 During the past 10 years a plethora of
inhibitors have been identified and some were shown to
be selective against IN and block viral replication (for
recent reviews see Refs. 3–5). The two most predomi-
nant classes of inhibitors have been the catechol con-
taining hydroxylated aromatics6 and more recently the
diketoacid containing aromatics.7 Discovery of a bona
fide IN inhibitor has continuously been challenging and
is of paramount importance because currently there are
no FDA approved drugs targeting this enzyme. Inclu-
sion of IN inhibitors with the highly active antiretroviral
cocktails is expected to improve the efficacy of existing
drugs by lowering the viral load to undetectable level.
idine analog was found to be the most potent.11
A
common theme in these leads is the requirement for a
free mercaptoaryl group for antiviral activity as well as
anti-IN potency (Fig. 1).8;10;12 It was recently demon-
strated that compound MSH could possibly form a
ternary complex with magnesium and binds to the
Cysteine 65 on the active site of IN.10 A major problem
with these molecules in general is, their lack of in vitro
and in vivo stability due to dimerization and metabolism
that could potentially lead to their toxicity. Therefore,
optimization of these lead compounds requires signifi-
cant modification to enhance their stability under
physiological conditions.
Keyword: HIV-1 integrase inhibitors.
q Supplementary data associated with this article can be found, in the
online version, at doi:10.1016/j.bmc.2004.04.024
* Corresponding authors. Tel.: +1-323-442-2341; fax: +1-323-442-1390
(N.N.); tel.: +48-58-3493257; fax: +48-58-3493206 (F.S.); e-mail
addresses: saczew@amg.gda.pl; neamati@usc.edu
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.04.024

3664
Z. Brzozowski et al. / Bioorg. Med. Chem. 12 (2004) 3663–3672
reacted with thionyl chloride to give the target products
H
N
Cl
SH
O
O
H
O N
S
NO
SH
N
2
21–37 in 39–75% yield. The ring closure observed in the
reaction of benzenesulfonamides containing an aro-
ylmethylthio group at ortho-position with thionyl chlo-
ride represents a new type of reaction leading to
3-substituted 1,1-dioxo-1,4,2-benzodithiazine derivatives.
2
2
N
O
S
N
N
H
O
HS
NO
O
O
OH
N
OH
N
2.2. Single-crystal X-ray structure of 24
N
N
H
H
SH
In order, to design more potent analogs using high
throughput structure-based drug design we solved the X-
ray crystal structure of a representative compound (Fig.
2). Compound 24 was found to crystallize in monoclinic
HS
HS
O
SH
Figure 1. Structures of mercaptoaryl lead compounds where an SH
group is believed to be important for both antiviral and anti-integrase
activities.
space group P21/n, a ¼ 7:2102ð5Þ, b ¼ 13:6632ð7Þ,
3
ꢀ
ꢀ
c ¼ 15:6326ð8Þ A, b ¼ 91:406ð5Þꢁ, V ¼ 1539:57ð15Þ A ,
Z ¼ 4, dx ¼ 1:666 g cmꢀ3, T ¼ 155 K (Table 1). Final R
indices for 2808 reflections with I > 2rðIÞ and 244
refined parameters are: R1 ¼ 0:0388, wR2 ¼ 0:0921
(R1 ¼ 0:0455, wR2 ¼ 0:0964 for all 3112 data). Atom
labeling is shown in Figure 2. Compound 24 is virtually
flat due to a nearly planar conformation of the 1,4,2-
dithiazine ring and only small twists (ca. 7ꢁ) about the
Csp2 –Csp2 bonds to the carbonyl group. This molecular
conformation is stabilized by weak intramolecular C–
Hꢁ ꢁ ꢁN interaction between the phenyl ring and dithi-
Because arylsulfonamides represent a class of drugs with
a well-established safety profile (for a recent review see
Ref. 13) we developed a new method for the synthesis of
cyclic analogs of MBSAs (Scheme 1) incorporating aroyl
group in their structure. In this study we prepared a set
of closely related analogs in order to determine mini-
mum structural features that are required for anti-IN
and antiviral activity.
ꢀ
2. Results and discussion
2.1. Synthesis
azine moiety (Hꢁ ꢁ ꢁN 2.26 A, <C20–H20ꢁ ꢁ ꢁN13 124ꢁ).
It has been shown that 1,1-dioxo-1,2,4-dithiazine ring
can adopt planar15 and boat14 conformations whereas
1,4,2-dithiazine ring with the lone pairs of electrons on S
atom prefers the boat conformation; the planar con-
formation of this 8p-electron system is destabilized by
anti-aromaticity.15 The geometry of the 1,1-dioxo-1,2,4-
dithiazine system in 24 is very similar to that observed
in 5,6-dimethyl-3-(4-bromophenyl)-1,4,2-dithiazine 1,1-
dioxide15 with the only discrepancy in the C–C bond
The synthesis of a series of novel benzodithiazine com-
pounds is presented in Scheme 1. First, the 4-chloro-2-
mercaptobenzenesulfonamides 1–3 prepared according
to the previously described procedure14 were subjected
to the reaction with bromomethyl ketones in the pres-
ence of triethylamine to give the corresponding S-
substituted derivatives 4–20. The later compounds were
O
O
SH
NH
Cl
S
S
Cl
Cl
S
S
a, b
Ar
Ar
c
2
NH
N
R
S
2
1
R
R
1
1
O
O
R
2
O
O
O
R
2
O
R
2
1-3
4-20
21-37
Compd.
1, 4, 21
1, 5, 22
1, 6, 23
1, 7, 24
1, 8, 25
1, 9, 26
1, 10, 27
1, 11, 28
1, 12, 29
R1
R2
H
H
H
H
H
H
H
H
H
Ar
Ph
Compd.
1, 13, 30
2, 14, 31
2, 15, 32
2, 16, 33
3, 17, 34
3, 18, 35
3, 19, 36
3, 20, 37
R1
Me
H
R2
H
Ar
Me
Me
Me
Me
Me
Me
Me
Me
Me
2-naphthyl
4-ClPh
4-MeO Ph
4-BrPh
Me
Me
Me
H
H
3,4-diClPh
2-naphthyl
4-ClPh
4-Cl-Ph
4-FPh
H
H
4-O2NPh
3-O2NPh
3,4-diClPh
4-Ph-Ph
H
H
3-O2NPh
3,4-diClPh
2-naphthyl
H
H
H
H
Scheme 1. Synthesis of the benzenesulfonamides 4–20 and their transformation into benzodithiazines 21–37. Reagents, conditions, and yields: (a)
Et3N (1.1 M equiv), CH2Cl2, 18–24 ꢁC; (b) ArCOCH2Br (1.1 M equiv), 18–24 ꢁC, 1 h, reflux, 6 h, 59–85%; (c) SOCl2 (excess), reflux, 46 h, 39–75%.

Z. Brzozowski et al. / Bioorg. Med. Chem. 12 (2004) 3663–3672
3665
Figure 2. Ortep drawing of compound 24. Thermal ellipsoids were drawn at 50% probability level.
imity of the highly conserved D64, D116, and E152 triad
once mutated to a serine residue retains all it’s catalytic
activity similar to those of the wild-type IN. We have
used this mutant enzyme previously to demonstrate that
some compounds can potentially bind to this residue
and that this amino acid might be important for drug
resistance viral strains.10;16 In the current study we ob-
served significant differential potencies only with com-
pounds 21–23 and 35–37 against C65S mutant versus
those of the wild type. This may suggest that these latter
compounds could potentially interact with C65.
Table 1. Crystal data and structure refinement for compound 24
Empirical formula
Formula weight
Temperature
C15H9C12NO3S2
386.25
155(2) K
ꢀ
0.71073 A
Wavelength
Crystal system
Space group
Unit cell dimensions
Monoclinic
P21/n
ꢀ
a ¼ 7:2102ð5Þ A a ¼ 90ꢁ
ꢀ
b ¼ 13:6632ð7Þ A b ¼ 91:406ð5Þꢁ
ꢀ
c ¼ 15:6326ð8Þ A c ¼ 90ꢁ
3
ꢀ
1539.57(15) A
Volume
Z
Density (calculated)
Absorption coefficient
F (0 0 0)
Crystal size
h range for data collection
Index ranges
4
1.666 Mg/m3
0.705 mmꢀ1
784
We also observed that all compounds inhibited IN with
IC50 values below 100 lM (Fig. 3 and Table 2) while
their antiviral activities varied greatly. In general the
antiviral activities were observed only with concentra-
tions 1–10 folds higher than those against purified IN
(Table 2). Except for compound 33 all other compounds
showed cytotoxcity well above 100 lM ranges. It is also
important to note that although high concentration was
required for antiviral activity but in many instances
100% protection of HIV-1 infected CEM cells was
achieved (compounds designated as ‘A’, Table 2).
0.80 · 0.15 · 0.08 mm3
3.48–26.37ꢁ
)9 6 h 6 6, )17 6 k 6 16,
)17 6 l 6 19
8422
Reflections collected
Independent reflections
Completeness to h ¼ 26:37ꢁ
Absorption correction
Refinement method
3112 [RðintÞ ¼ 0:0283]
99.1%
None
Full-matrix least-squares on F 2
3112/0/244
1.118
Data/restraints/parameters
Goodness-of-fit on F 2
Final R indices [I > 2rðIÞ]
R indices (all data)
In conclusion, we have demonstrated that the cyclic
analogs of MBSAs or the benzodithiazines are novel
class of IN inhibitors, they exhibit antiviral activity al-
beit at high concentrations, and are essentially nontoxic.
Further structural modifications are underway to opti-
mize their potency.
R1 ¼ 0:0388, wR2 ¼ 0:0921
R1 ¼ 0:0455, wR2 ¼ 0:0964
ꢀ3
ꢀ
Largest diff. peak and hole
0.290 and )0.431 e A
length as a result of a difference in the C–C bond
character (aromatic vs double) in both compounds.
Interestingly, despite the planar molecular conformation
ꢀ
of 24 the Csp2 –Csp2 bond length of 1.533 A between the
benzoyl and heterocyclic moieties indicates no p-ele-
ctron conjugation for this bond.
3. Experimental
3.1. Chemistry. General
Melting points: Buchi 535 apparatus; IR spectra KBr
pellets, 400–4000 cmꢀ1 Perkin–Elmer 1600 FTIR spec-
trometer; H NMR spectra: Varian Gemini 200 appa-
2.3. In vitro biology
1
Previously, we discovered that MBSAs inhibit purified
IN and protected HIV-1 infected CEM cells.8;9 In that
study it was shown that aromatic mercapto group was
important for activity.8 More recently we provided evi-
dence that the MSHs class of compounds selectively
inhibit IN, chelate magnesium ion, and bind to the C65
residue on the active site of IN.10 In an effort to improve
upon the lack of stability of aforementioned compounds
we prepared a series of cyclic derivatives and tested their
inhibitory potency against wild-type IN as well as the
C65S mutant. The C65 residue located in close prox-
ratus (chemical shifts are expressed as d values relative
to Me4Si as standard). 13C NMR spectra were taken on
a Varian Unity 500 spectrometer. Analyses of C, H, N,
were within ꢂ0.4% of the theoretical values.
3.2. General procedure for the preparation of 2-[(2-aryl-
oxoethyl) sulfanyl]benzenesulfonamides 4–20
To a suspension of the corresponding 2-mercaptobenz-
enesulfonamide 1, 2, or 3 (0.02 mol) in dichloromethane

3666
Z. Brzozowski et al. / Bioorg. Med. Chem. 12 (2004) 3663–3672
Figure 3. (A) A representative gel showing inhibition of purified IN by selected compounds. A 21-mer blunt-end oligonucleotide corresponding to
the U5 end of the HIV-1 LTR, 50 end-labeled with 32P, is reacted with purified IN. The initial step involves nucleolytic cleavage of two bases from the
30-end, resulting in a 19-mer oligonucleotide. Subsequently, 30 ends are covalently joined at several sites to another identical oligonucleotide that
serves as the target DNA. This reaction is referred to as strand transfer and the products formed migrate slower than the original substrate (shown in
the figure as STP for strand transfer products). Drug concentrations in lM are indicated above each lane. (B) Quantitation of panel A.
(30 mL), triethylamine (2.23 g, 0.022 mol) was added
with stirring. The solution thus obtained was treated
portionwise with the appropriate aryl bromomethyl
ketone (0.022 mol) at 18–24 ꢁC (water–ice bath). Then,
the reaction mixture was stirred at room temperature for
1 h followed by reflux for 6 h. After cooling to room
temperature and standing overnight the precipitate of
the adequate S-substituted 2-mercaptobenzenesulfona-
mide was filtered off and washed successively with di-
chloromethane (3 · 3 mL), 2-propanol (2 · 1.5 mL),
water (4 · 5 mL), and 2-propanol (2 · 2 mL).
2-mercaptobenzenesulfonamide 1 (4.76 g) and 4-meth-
oxyphenacyl bromide (5.0 g); yield: 6.0 g (77%); mp 154–
155 ꢁC; IR (KBr) 3295, 3200 (NH2), 1670 (C@O), 1350,
1
1325, 1165 (SO2) cmꢀ1; H NMR (DMSO-d6) d 2.36 (s,
3H, CH3), 3.89 (s, 3H, CH3O), 4.85 (s, 2H, CH2), 7.11
(d, J ¼ 8:9 Hz, 2H, 4CH3OPhCO), 7.43 (s, 2H, NH2H),
7.71 (s, 1H, H-3, PhSO2), 7.85 (s, 1H, H-6, PhSO2), 8.07
(d, J ¼ 8:9 Hz, 2H, 4-CH3OPhCO) ppm. Anal.
(C16H16ClNO3S2) C, H, N.
3.2.3. 4-Chloro-2-{[2-(4-bromophenyl)-2-oxoethyl]sulfan-
yl}-5-methylbenzenesulfonamide (6). Starting from
2-mercaptobenzenesulfonamide 1 (4.76 g) and 4-bromo-
phenacyl bromide (6.1 g); yield: 6.2 g (71%); mp 185–
186 ꢁC; IR (KBr) 3365, 3255 (NH2), 1675 (C@O), 1350,
1330, 1160, (SO2) cmꢀ1; 1H NMR (DMSO-d6) d 2.34 (s,
3H, CH3), 4.88 (s, 2H, CH2), 7.40 (s, 2H, NH2), 7.66 (s,
1H, H-3, PhSO2), 7.78 (d, J ¼ 8:3 Hz, 2H, 4-BrPhCO),
7.80 (s, 1H, H-6, PhSO2), 7.99 (d, J ¼ 8:3 Hz, 2H, 4-
BrPHCO) ppm. Anal. (C15H13BrClNO3S2) C, H, N.
In this manner the following sulfonamide were obtained:
3.2.1. 4-Chloro-2-[(2-phenyl-2-oxoethyl)sulfanyl]-5-meth-
ylbenzenesulfonamide (4). Starting from 2-mercapto-
benzenesulfonamide 1 (4.76 g) and phenacyl bromide
(4.4 g); yield: 4.2 g (59%); mp 159–161 ꢁC; IR (KBr)
3300, 3200 (NH2), 1685 (C@O), 1355, 1330, 1160 (SO2)
cmꢀ1; 1H NMR (DMSO-d6) d 2.34 (s, 3H, CH3), 4.91 (s,
2H, CH2), 7.40 (s, 2H, NH2), 7.53–7.68 (m, 4H, arom.),
7.83 (s, 1H, H-6, PhSO2), 8.06 (d, J ¼ 7:5 Hz, 2H,
PhCO) ppm. Anal. (C15H14ClNO3S2) C, H, N.
3.2.4. 4-Chloro-2-{[2-(4-chlorophenyl)-2-oxoethyl]sulfan-
yl}-5-methylbenzenesulfonamide (7). Starting from
2-mercaptobenzenesulfonamide 1 (4.76 g) and 4-chloro-
phenacyl bromide (5.1 g); yield: 5.9 g (75%); mp 169–
171 ꢁC; IR (KBr) 3295, 3205 (NH2), 1685 (C@O), 1350,
3.2.2. 4-Chloro-2{[2-(4-methoxyphenyl)-2-oxoethyl]sulfan-
yl}-5-methylbenzenesulfonamide (5). Starting from

Z. Brzozowski et al. / Bioorg. Med. Chem. 12 (2004) 3663–3672
Table 2. Antiviral and anti-HIV-1 integrase activities of novel benzodithiazines 21–37
3667
Inhibition of integrase
catalitic activities
Antiviral activitiesa
Compd
30-process
Integration
IC50 (lM)b
WTg/C65Sh
EC50 (lM)c
CC50 (lM)d
TIe
Maximum of Protection
Commentsf
IC50 (lM)b
Dose (lM)
%
WTg/C65Sh
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
12
90
30
>100
30
65
30
33
57
75
NT
11
57
95
15
20
11
18
28
30
27
60
17
8
12
90
33
30
30
65
65
33
42
17
NT
11
50
80
7
80.9
205.8
NTj
287.5
>1000.0
NTj
3.5
100.0
103
99
A
A
>4.8
1000.0
i
>159.0
161.0
52.5
159.0
NR
>1.25
2.4
200.0
200.0
63.3
25
60
I
>200.0
125.0
M
M
A
I
60
77.5
183.0
2.4
200.0
200.0
200.0
200.0
1000.0
200.0
200.0
100.0
100.0
63.3
102
31
>200.0
>200.0
>200.0
256.7
>200.0
>200.0
72.7
143.0
NRi
NR
NR
>3.9
NR
NR
>2.7
4.7
10
10
20
3
110.0
15
I
>200.0
>1000.0
>200.0
49.2
47
I
15
25
65
25
3
106
12
A
I
22
20
100
100
25
62
19
47
8
7
18
I
20
90
100
20
80
19
20
8
>200.0
230.7
113
104
52
A
A
M
M
49.0
61.2
124.0
2.0
54.5
170.0
3.1
100.0
83
65
35
a Data obtained from the NCI’s in vitro anti-HIV primary screen.
b IC50: Inhibitory concentration 50% (inhibition of purified integrase).
c EC50: Effective concentration 50% (protection of HIV-1 infected CEM cells).
d CC50: Cytotoxic concentration 50% (toxicity to uninfected CEM cells).
e TI: Therapeutic index ¼ CC50/EC50
.
f Comments: NCI designated activity: A (confirmed active); M (confirmed moderate); I (confirmed inactive).
g The top values in each row are samples tested against wild-type HIV-1 integrase.
h The bottom values in each row are samples tested against active-site mutant C65S. The values are presented as an average of two to four
experiments with the standard error of less than 5%.
i NR: No therapeutic benefit reached due to cytotoxicity.
j NT: not tested.
1
1330, 1160 (SO2) cmꢀ1; H NMR (DMSO-d6) d 2.34 (s,
3H, CH3), 4.89 (s, 2H, CH2), 7.40 (s, 2H, NH2). 7.62 (d,
J ¼ 8:6 Hz, 2H, 4ClPhCO), 7.66 (s, 1H, H-3, PhSO2),
7.83 (s, 1H, H-6, PhSO2), 8.7 (d, J ¼ 8:6 Hz, 2H, 4-
ClPhCO) ppm. Anal. (C15H13Cl2NO3S2) C, H, N.
3H, CH3), 4.88 (s, 2H, CH2), 7.35–7.44 (m, 4H, NH2
and 4-FPhCO), 7.66 (s, 1H, H-3, PhSO2), 7.83 (s, 1H, H-
6, PhSO2), 8.11–8.19 (m, 2H, 4-FPhSO2) ppm; 13C
NMR (DMSO-d6) d 19.28, 115.91, 116.35, 129.67,
130.30, 131.84, 132.03, 132.33, 133.42, 134.03, 137.27,
140.48,
(C15H13ClFNO3S2) C, H, N.
163.09,
168.12,
193.32 ppm.
Anal.
3.2.5. 4-Chloro-2-{[2-(4-fluorophenyl)-2-oxoethyl]sulfan-
yl}-5-methylbenzenesulfonamide (8). Starting from
2-mercaptobenzenesulfonamide 1 (4.76 g) and 4-fluoro-
phenacyl bromide (4.8 g); yield: 5.5 g (73%); mp 138–
139 ꢁC; IR (KBr) 3305, 3225 (NH2), 1680 (C@O), 1345,
3.2.6. 4-Chloro-2-{[2-(4-nitrophenyl)-2-oxoethyl]sulfan-
yl}-5-methylbenzenesulfonamide (9). Starting from
2-mercaptobenzenesulfonamide 1 (4.76 g) and 4-nitro-
phenacyl bromide (5.4 g); yield: 4.9 g (61%); mp
1
1325, 1160 (SO2) cmꢀ1; H NMR (DMSO-d6) d 2.34 (s,

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Z. Brzozowski et al. / Bioorg. Med. Chem. 12 (2004) 3663–3672
195–196 ꢁC; IR (KBr) 3375, 3280 (NH2), 1690 (C@O),
phenacyl bromide (5.1 g); yield: 5.4 g (69%); mp 154–
156 ꢁC; IR (KBr) 3280, 3185 (NH2), 1680 (C@O), 1335,
1
1350, 1150 (SO2) cmꢀ1; H NMR (DMSO-d6) d 2.35 (s,
1
3H, CH3), 4.98 (s, 2H, CH2), 7.41 (s, 2H, NH2), 7.61 (s,
1H, H-3, PhSO2), 7.84 (s, 1H, H-6, PhSO2), 8.28 (d,
J ¼ 8:7 Hz, 2H, 4-O2NPhCO), 8.38 (d, J ¼ 8:7 Hz, 2H,
4-O2NPhCO) ppm. Anal. (C15H13ClN2O5S2) C, H, N.
1160 (SO2) cmꢀ1; H NMR (DMSO-d6) d 2.59 (s, 3H,
CH3), 4.84 (s, 2H, CH2), 7.27 (s, 2H, NH2), 7.48 (s, 1H,
H-5, PhSO2), 7.51 (s, 1H, H-3, PhSO2), 7.64 (d,
J ¼ 8:5 Hz, 2H, 4-ClPhCO), 8.08 (d, J ¼ 8:5 Hz, 2H, 4-
ClPhCO) ppm. Anal. (C15H13Cl2NO3S2) C, H, N.
3.2.7. 4-Chloro-2-[{2-(3-nitriphenyl)-2-oxoethyl]sulfanyl}-
5-methylbenzenesulfonamide (10). Starting from 2-
mercaptobenzenesulfonamide 1 (4.76 g) and 3-nitro-
phenacyl bromide (5.4 g); yield: 6.5 g (81%); mp 176–
178 ꢁC; IR (KBr) 3365, 3270 (NH2), 1685 (C@O), 1350,
3.2.12. 4-Chloro-2-{[2-(3,4-dichlorophenyl)-2-oxoethyl]-
sulfanyl}-6-methylbenzenesulfonamide (15). Starting
from 2-mercaptobenzenesulfonamide 2 (4.76 g) and 3,4-
dichlorophenacyl bromide (5.8 g); yield: 6.6 g (78%); mp
189–190 ꢁC; IR (KBr) 3290, 3190 (NH2), 1680 (C@O),
1
1150 (SO2) cmꢀ1; H NMR (DMSO-d6) d 2.34 (s, 3H,
1
CH3), 5.01 (s, 2H, CH2), 7.39 (s, 2H, NH2), 7.70 (s, 1H, H-
3, PhSO2), 7.83–7.90 (m, 2H, arom.), 8.45–8.52 (m, 2H,
arom.), 8.73 (t, J ¼ 1:8 Hz, 1H, H-2, 3-O2NPhCO) ppm:
13C NMR (DMSO-d6) d 19.32, 123.21, 128.13, 129.93,
130.35, 130.87, 133.58, 135.04, 136.82, 137.26, 140.70,
148.31, 193.25 ppm. Anal, (C15H13ClN2O5S2) C, H, N.
1330, 1165 (SO2) cmꢀ1; H NMR (DMSO-d6) d 2.59 (s,
3H, CH3), 4.86 (s, 2H, CH2), 7.28 (d, J ¼ 1:8 Hz, 1H,
arom.), 7.49 (s, 3H, NH2 and 1H arom.), 7.83–8.02 (m,
2H, arom.), 8.30 (d, J ¼ 1:8 Hz, 1H, arom.) ppm. Anal.
(C15H12Cl3NO3S2) C, H, N.
3.2.13. 4-Chloro-2-{[2-(2-naphthyl)-2-oxoethyl]sulfanyl}-
6-methylbenzenesulfonamide (16). Starting from 2-
mercaptobenzenesulfonamide 2 (4.76 g) and bromo-
methyl 2-naphthyl ketone (5.5 g); yield: 6.9 g (85%); mp
186–187 ꢁC; IR (KBr) 3290, 3190 (NH2), 1680 (C@O),
3.2.8. 4-Chloro-2-{[2-(3,4-dichlorophenyl)-2-oxoethyl]sul-
fanyl}-5-methylbenzenesulfonamide (11). Starting from
2-mercaptobenzenesulfonamide 1 (4.76 g) and 3,4-di-
chlorophenacyl bromide (5.8 g); yield: 5.6 g (66%); mp
179–180 ꢁC; IR (KBr) 3290, 3200 (NH2), 1695 (C@O),
1
1330, 1160 (SO2) cmꢀ1; H NMR (DMSO-d6) d 2.59 (s,
1
1350, 1325, 1165 (SO2) cmꢀ1; H NMR (DMSO-d6) d
3H, CH3), 4.98 (s, 2H, CH2), 7.27 (d, J ¼ 2:0 Hz, 1H,
arom.), 7.51 (s, 2H, NH2), 7.59 (d, J ¼ 2:0 Hz, 1H,
arom.), 7.62–7.74 (m, 2H, arom.), 8.00–8.17 (m,
4H, arom.), 8.83 (s, 1H, H-1, 2-naphthoyl) ppm. Anal.
(C19H16ClNO3S2) C, H, N.
2.50 (s, 3H, CH3), 5.05 (s, 2H, CH2), 7.57 (s, 2H, NH2),
7.82 (s, 1H, H-3, PhSO2), 7.98–8.16 (m, 3H, arom.), 8.42
(s, 1H, H-6, PhSO2) ppm; 13C NMR (DMSO-d6) d
19.30, 128.76, 129.82, 130.32, 130.77, 131.39, 132.11,
133.54, 133.65, 135.77, 136.84, 137.26, 140.59,
192.91 ppm. Anal. (C15H12Cl3NO3S2) C, H, N.
3.2.14. 4-Chloro-2-{[2-(4-chlorophenyl)-2-oxoethyl]sulfan-
yl}benzenesulfonamide (17). Starting from 2-mercapto-
benzenesulfonamide 3 (4.47 g) and 4-chlorophenacyl
bromide (5.1 g); yield: 4.9 g (65%); mp 161–162 ꢁC; IR
(KBr) 3280, 3200 (NH2), 1690 (C@O), 1330, 1160 (SO2)
3.2.9. 4-Chloro-2-{[2-(4-biphenylyl)-2-oxoethyl]sulfanyl}-
5-methylbenzenesulfonamide (12). Starting from 2-
mercaptobenzenesulfonamide 1 (4.76 g) and 4-phenyl-
phenacyl bromide (6.0 g); yield: 5.3 g (61%); mp 199–
200 ꢁC; IR (KBr) 3355, 3260 (NH2), 1660 (C@O), 1355,
1
cmꢀ1; H NMR (DMSO-d6) d 4.97 (s, 2H, CH2), 7.43
(dd, Jortho ¼ 8:5 Hz, Jmeta ¼ 1:9 Hz, 1H, H-5, PhSO2),
7.50 (s, 2H, NH2), 7.62–7.68 (m, 3H, arom.), 7.87 (d,
Jortho ¼ 8:5 Hz, 1H, H-6, PhSO2), 8.11 (d, J ¼ 8:5 Hz,
2H, arom.) ppm. Anal. (C14H11Cl2NO3S2) C, H, N.
1
1330, 1160 (SO2) cmꢀ1; H NMR (DMSO-d6) d 2.35 (s,
3H, CH3), 4.93 (s, 2H, CH2), 7.41 (s, 2H, NH2), 7.44–
7.57 (m, 3H, arom.), 7.70 (s, 1H, H-3, PhSO2), 7.76–7.89
(m, 5H, arom.), 8.14 (d, J ¼ 8:4 Hz, 2H, arom.) ppm.
Anal. (C21H18ClNO3S2) C, H, N.
3.2.15. 4-Chloro-2-{[2-(3-nitrophenyl)-2-oxoethyl]sulfan-
yl}benzenesulfonamide (18). Starting from 2-mercapto-
benzenesulfonamide 3 (4.47 g) and 3-nitrophenacyl
bromide (5.4 g); yield: 4.6 g (59%); mp 196–197 ꢁC; IR
(KBr) 3300, 3210 (NH2), 1705 (C@O), 1350, 1330, 1165
3.2.10. 4-Chloro-2-{[2-(2-naphthyl)-2-oxoethyl]sulfanyl}-
5-methylbenzenesulfonamide (13). Starting from 2-
mercaptobenzenesulfonamide 1 (4.76 g) and bromo-
methyl 2-naphthyl ketone (5.5 g); yield: 6.1 g (75%); mp
170–171 ꢁC; IR (KBr) 3265, 3170 (NH2), 1675 (C@O),
1
(SO2) cmꢀ1; H NMR (DMSO-d6) d 5.06 (s, 2H, CH2),
7.41 (dd, Jortho ¼ 8:5 Hz, Jmeta ¼ 1:9 Hz, 1H, H-5,
PhSO2), 7.46 (s, 2H, NH2), 7.69 (d, Jmeta ¼ 1:9 Hz, 1H,
H-3, PhSO2), 7.82–7.90 (m, 2H, arom.), 8.46–8.52 (m,
2H, arom.), 8.75 (d, J ¼ 1:9 Hz, 1H arom.) ppm. Anal.
(C14H11ClN2O5S2) C, H, N.
1
1350, 1160 (SO2) cmꢀ1; H NMR (DMSO-d6) d 2.34 (s,
3H, CH3), 5.02 (s, 2H, CH2), 7.41 (s, 2H, NH2), 7.64–7.71
(m, 2H, arom.), 7.73 (s, 1H, H-3, PhSO2), 7.84 (s, 1H, H-
6, PhSO2), 8.00–8.16 (m, 4H, arom.), 8.81 (s, 1H, H-1, 2-
naphthoyl) ppm. Anal. (C19H16ClNO3S2) C, H, N.
3.2.16. 4-Chloro-2-{[2-(3,4-dichlorophenyl)-2-oxoethyl]-
sulfanyl}benzenesulfonamide (19). Starting from 2-
mercaptobenzenesulfonamide 3 (4.47 g, 0.02 mol) and
3,4-dichlorophenacyl bromide (5.8 g, 0.022 mol), the title
3.2.11. 4-Chloro-2-{[2-(4-chlorophenyl)-2-oxoethyl]sulfan-
yl}-6-methylbenzenesulfonamide (14). Starting from
2-mercaptobenzenesulfonamide 2 (4.76 g) and 4-chloro-

Z. Brzozowski et al. / Bioorg. Med. Chem. 12 (2004) 3663–3672
3669
compound 19 was obtained (5.3 g, 64%): mp 171–173 ꢁC;
IR (KBr) 3390, 3240 (NH2), 1685 (C@O), 1340, 1165
3.3.3. 3-(4-Bromobenzoyl)-6-chloro-7-methyl-1,1-dioxo-
1,4,2-benzodithiazine (23). Starting from benzenesulf-
onamide 6 (4.35 g); yield: 2.5 g (58%); mp 257–258 ꢁC;
IR (KBr) 1660 (C@O), 1580 (C@N), 1330, 1165 (SO2)
1
(SO2) cmꢀ1; H NMR (DMSO-d6) d 5.00 (s, 2H, CH2),
7.45 (dd, Jortho ¼ 8:5 Hz,, Jmeta ¼ 1:8 Hz, 1H, H-5,
PhSO2), 7.53 (s, 2H, NH2), 7.70 (d, Jmeta ¼ 1:8 Hz, 1H,
H-3, PhSO2), 7.89 (d, J ¼ 8:5 Hz, 2H, arom.), 8.04 (dd,
Jortho ¼ 8:5 Hz, Jmeta ¼ 1:8 Hz, 1H, H-5, PhCO), 8.34, d,
Jmeta ¼ 1:8 Hz, 1H, H-2, PhCO) ppm. Anal.
(C14H10Cl3NO3S2) C, H, N.
1
cmꢀ1; H NMR (CDCl3) d 2.55 (s, 3H, CH3), 7.57 (s,
1H, H-5), 7.69 (d, J ¼ 8:8 Hz, 2H, 4-BrPh), 8.08 (s, 1H,
H-8), 8.12 (d, J ¼ 8:8 Hz, 2H, 4BrPh) ppm; 13C NMR
(CDCl3) d 20.83, 112.90, 125.96, 128.25, 128.59, 130.86,
131.84, 132.86, 133.12, 140.68, 141.20, 169.95,
185.70 ppm. Anal. (C15H9BrClNO3S2) C, H, N.
3.2.17. 4-Chloro-2-{[2-(2-naphthyl)-2-oxoethyl]sulfanyl}-
benzenesulfonamide (20). Starting from 2-mercaptobenz-
enesulfonamide 3 (4.47 g) and bromomethyl 2-naphthyl
ketone (5.5 g); yield: 5.2 g (66%); mp 171–172 ꢁC; IR
(KBr) 3355, 3205 (NH2), 1680 (C@O), 1345, 1165 (SO2)
3.3.4. 6-Chloro-3-(4-chlorobenzoyl)-7-methyl-1,1-dioxo-
1,4,2-benzodithiazine (24). Starting from benzenesulf-
onamide 7 (3.9 g); yield: 2.2 g (57%); mp 241–242 ꢁC; IR
(KBr) 1655 (C@O), 1580 (C@N), 1330, 1170 (SO2)
1
1
cmꢀ1; H NMR (CDCl3) d 2.54 (s, 3H, CH3), 7.52 (d,
cmꢀ1; H NMR (DMSO-d6) d 5.08 (s, 2H, CH2), 7.41
J ¼ 8:8 Hz, 2H, 4-ClPh), 7.57 (s, 1H, H-5), 8.09 (s, 1H,
H-8), 8.20 (d, J ¼ 8:8 Hz, 2H, 4-ClPh) ppm; 13C NMR
(CDCl3) d 20.59, 125.34, 128.00, 128.33, 129.00, 129.62,
130.28 132.93, 140.31, 140.96, 142.66, 169.61,
184.98 ppm. Anal. (C15H9Cl2NO3S2) C, H, N.
(dd, Jortho ¼ 8:5 Hz, Jmeta ¼ 2:0 Hz, 1H, H-5, PhSO2),
7.49 (s, 2H, NH2), 7.60–7.73 (m, 3H, arom.), 7.85 (d,
Jortho ¼ 8:5 Hz, 1H, H-6, PhSO2), 7.99–8.16 (m, 4H,
arom.), 8.84 (s, 1H, H-1, 2-naphthoyl) ppm. Anal.
(C18H14ClNO3S2), C, H, N.
3.3.5. 6-Chloro-3-(4-fluorobenzoyl)-7-methyl-1,1-dioxo-
1,4,2-benzodithiazine (25). Starting from benzenesulf-
onamide 8 (3.76 g); yield: 1.8 g (48%); mp 194–195 ꢁC;
IR (KBr) 1655 (C@O), 1590 (C@N), 1340,1170 (SO2)
3.3. General procedure for the preparation of 3-aroyl-1,1-
dioxo-1,4,2-benzodithiazines 21–37
A solution of the corresponding benzenesulfonamide 4–
20 (0.01 mol) in thionyl chloride (45 mL) was refluxed
for 46 h. Excess thionyl chloride was evaporated under
reduced pressure (toluene (10 mL) was added to facili-
tate the removal of thionyl chloride). Then, the resulting
solid residue was refluxed in toluene (20–25 mL) for
0.5 h. The insoluble material was separated by filtration,
and the pure product (21–37) that precipitated from the
filtrate upon cooling to room temperature was collected
by filtration, washed with toluene (3 · 3 mL), and dried.
1
cmꢀ1; H NMR (CDCl3) d 2.54 (s, 3H, CH3), 7.18–7.27
(m, 2H, 4-FPh), 7.57 (s, 1H., H-5), 8.10 (s, 1H, H-8),
8.28–8.35 (m, 2H, 4-FPh) ppm; MS m=z 369 (Mþ). Anal.
(C15H9ClFNO3S2) C, H, N.
3.3.6.
6-Chloro-7-methyl-3-(4-nitrobenzoyl)-1,1-dioxo-
1,4,2-benzodithiazine (26). Starting from benzenesulf-
onamide 9 (4.0 g); yield: 1.9 g (48%); mp 236–237 ꢁC; IR
(KBr) 1675 (C@O), 1613 (NO2), 1580 (C@N), 1350,
1175 (SO2) cmꢀ1; 1H NMR (CDCl3) d 2.56 (s, 3H, CH3),
7.60 (s, 1H, H-5), 8.11 (s, 1H, H-8), 8.39 (s, 4H, 4-
O2NPhCO) ppm. Anal. (C15H9ClN2O5S2) C, H, N.
In this manner the following benzodithiazines were
obtained:
3.3.1. 3-Benzoyl-6-chloro-7-methyl-1,1-dioxo-1,4,2-ben-
zodithiazine (21). Starting from benzenesulfonamide 4
(3.56 g); yield: 1.6 g (46%); mp 159–160 ꢁC; IR (KBr)
3.3.7.
6-Chloro-7-methyl-3-(3-nitrobenzoyl)-1,1-dioxo-
1,4,2-benzodithiazine (27). Starting from benzenesulf-
onamide 10 (4.0 g); yield: 2.0 g (50%); mp 226–227 ꢁC;
IR (KBr) 1670 (C@O), 1610 (NO2), 1575 (C@N), 1345,
1170 (SO2) cmꢀ1; 1H NMR (CDCl3) d 2.55 (s, 3H, CH3),
7.60 (s, 1H, H-5), 7.77 (t, J ¼ 8:0 Hz, 1H, 3-O2NPh),
8.11 (s, 1H, H-8), 8.52–8.63 (m, 2H, 3-O2NPh), 9.00 (t,
J ¼ 1:7 Hz, 1H, 3-O2NPh) ppm; 13C NMR (CDCl3) d
20.86, 125.57, 126.40, 128.34, 128.68, 128.85, 129.62,
130.68, 133.64, 137.22, 140.75, 141.53, 148.95, 168.76,
184.95 ppm. Anal. (C15H9ClN2O5S2) C, H, N.
1
1660 (C@O), 1590 (C@N), 1345, 1175 (SO2) cmꢀ1; H
NMR (CDCl3) d 2.54 (s, 3H, CH3), 7.50–7.58 (m, 4H,
arom.), 8.10 (s, 1H, H-8), 8.19–8.24 (m, 2H, arom.)
ppm. Anal. (C15H10ClNO3S2) C, H, N.
3.3.2.
6-Chloro-7-methyl-3-(4-methoxybenzoyl)-1,1-di-
oxo-1,4,2-benzodithiazine (22). Starting from benzene-
sulfonamide 5 (3.86 g); yield: 1.5 g (39%); mp 202–
203 ꢁC; IR (KBr) 1665 (C@O), 1595 (C@N), 1345, 1340,
1180, 1165 (SO2) cmꢀ1; 1H NMR (CDCl3) d 2.53 (s, 3H,
CH3), 3.91 (s, 3H, CH3O), 6.99 (d, J ¼ 9:0 Hz, 2H, 4-
CH3OPh), 7.54 (s, 1H, H-5), 8.09 (s, 1H, H-8), 8.26 (d,
J ¼ 9:0 Hz, 2H, 4CH3OPh) ppm; 13C NMR (CDCl3):
20.79, 56.24, 114.90, 124.89, 125.74, 128.18, 128.52,
129.72, 134.57, 140.35, 140.84, 166.12, 170.06,
183.90 ppm. Anal. (C16H12ClNO4S2) C, H, N.
3.3.8. 6-Chloro-3-(3,4-dichlorobenzoyl)-7-methyl-1,1-di-
oxo-1,4,2-benzodithiazine (28). Starting from benzene-
sulfonamide 11 (4.25 g); yield: 1.9 g (45%); mp 207–
209 ꢁC; IR (KBr) 1680 (C@O), 1580 (C@N), 1325, 1170
(SO2) cmꢀ1 1H NMR (CDCl3) d 2.55 (s, 3H, CH3),
;
7.57–7.56 (m, 2H, arom.), 8.10 (s, 1H, H-8), 8.11–8.16
(m, 1H, arom.), 8.30 (d, J ¼ 2:0 Hz, 1H, arom.) ppm.

3670
Z. Brzozowski et al. / Bioorg. Med. Chem. 12 (2004) 3663–3672
EIMS (70 eV): m=z ¼ 420 (Mþ). Anal. (C15H8Cl3NO3S2)
8.16–8.23 (m, 3H, arom.). Anal. (C14H7Cl2NO3S2) C, H,
N.
C, H, N.
3.3.15. 6-Chloro-3-(3-nitrobenzoyl)-1,1-dioxo-1,4,2-ben-
zodithiazine (35). Starting from benzenesulfonamide 18
(3.87 g); yield: 1.9 g (50%); mp 176–177 ꢁC; IR (KBr)
3.3.9.
6-Chloro-7-methyl-3-biphenylyl-1,1-dioxo-1,4,2-
benzodithiazine (29). Starting from benzenesulfonamide
12 (4.32 g); yield: 1.7 g (40%); mp 208–209 ꢁC; IR (KBr)
1
1695 (C@O), 1610 (C@N), 1350, 1175 (SO2) cmꢀ1; H
1655 (C@O), 1600 (C@N), 1330, 1170 (SO2) cmꢀ1; H
1
NMR (DMSO-d6) d 7.86–7.94 (m, 2H, arom.), 8.21–
8.30 (m, 2H, arom.), 8.42–8,59 (m, 2H, arom.), 8.85 (t,
J ¼ 1:8 Hz, 1H, H-2, 3-O2NPh) ppm. Anal.
(C14H7ClN2O5S2) C, H, N.
NMR (CDCl3) d 2.55 (s, 3H, CH3), 7.44–7.55 (m, 3H,
biPh), 7.58 (s, 1H, H-5), 7.62–7.69 (m, 2H, biPh), 7.76
(d, J ¼ 8:5 Hz, 2H, biPH), 8.12 (s, 1H, H-8), 8.32 (d,
J ¼ 8:5 Hz, 2H, biPh) ppm. Anal. (C21H14ClNO3S2) C,
H, N.
3.3.16. 6-Chloro-3-(3,4-dichlorobenzoyl)-1,1-dioxo-1,4,2-
benzodithiazine (36). Starting from benzenesulfonamide
19 (4.1 g); yield: 2.1 g (51%); mp 199–201 ꢁC; IR (KBr)
3.3.10.
6-Chloro-7-methyl-3-(2-naphthoyl)-1,1dioxo-
1,4,2-benzodithiazine (30). Starting from benzenesulf-
onamide 13 (4.06 g); yield: 1.6 g (40%); mp 216–217 ꢁC;
IR (KBr) 1660 (C@O), 1620 (C@N), 1330, 1165 (SO2)
1
1665 (C@O), 1630 (C@N), 1330, 1170 (SO2) cmꢀ1; H
NMR (CDCl3) d 7.59–7.66 (m, 2H, arom.), 7.72 (dd,
Jortho ¼ 6:6Hz, Jmeta ¼ 1:9 Hz, 1H, H-6, benzodithiaz.),
8.12–8.21 (m, 2H, arom.), 8.32 (d, J ¼ 2:0 Hz, 1H, H-2,
PhCO) ppm. Anal. (C14H6Cl3NO3S2) C, H, N.
1
cmꢀ1; H NMR (CDCl3) d 2.56 (s, 3H, CH3), 756–7.59
(m, 3H, arom.), 7.64–8.05 (m, 3H, arom.), 8.13–8.18 (m,
2H, arom.), 8.89 (s, 1H, H-1, naphthyl) ppm. Anal.
(C19H12ClNO3S2) C, H, N.
3.3.17. 6-Chloro-3-(2-naphthoyl)-1,1-dioxo-1,4,2-benzo-
dithiazine (37). Starting from benzenosulfonamide 20
(3.92 g); yield: 2.0 g (51%); IR (KBr) 1660 (C@O), 1610
3.3.11. 6-Chloro-3-(4-chlorobenzoyl)-8-methyl-1,1-dioxo-
1,4,2-benzodithiazine (31). Starting from benzenesulf-
onamide 14 (3.9 g); yield: 2.9 g (75%); mp 229–230 ꢁC;
IR (KBr) 1660 (C@O), 1595 (C@N), 1330, 1170 (SO2)
cmꢀ1; 1H NMR (DMSO-d6) d 2.71 (s, 3H, CH3), 7.71 (d,
J ¼ 8:6 Hz, 2H, 4-ClPh), 7.81 (d, J ¼ 1:8 Hz, 1H, H-7),
8.04 (d, J ¼ 1:8 Hz, 1H, H-5), 8.12 (d, J ¼ 8:6 Hz, 2H, 4-
ClPh) ppm. Anal. (C15H9ClNO3S2) C, H, N.
1
(C@N), 1340, 1175 (SO2) cmꢀ1; H NMR (CDCl3) d
7.57–7.74 (m, 4H, arom.), 7.90–8.05 (m, 3H, arom.),
8.14–8.23 (m, 2H, arom.), 8.89 (s, 1H, H-1, naphthyl)
ppm. Anal. (C18H10ClNO3S2) C, H, N.
3.4. Materials, chemicals, and enzymes
All compounds were dissolved in DMSO and the stock
solutions were stored at )20 ꢁC. The c [32P]-ATP was
purchased from either Amersham Biosciences or ICN.
The expression systems for the wild-type IN, soluble
mutant INF185KC280S, and the truncated mutant IN50-212
(F185K) were generous gifts of Dr. Robert Craigie,
Laboratory of Molecular Biology, NIDDK, NIH,
Bethesda, MD. The expression systems for the cysteine
mutant was a generous gift of Dr. Anna Marie Skalka,
Fox Chase Cancer Center, Philadelphia, PA and the
enzymes were prepared as described.17;18
3.3.12. 6-Chloro-3-(3,4-dichlorobenzoyl)-8-methyl-1,1-di-
oxo-1,4,2-benzodithiazine (32). Starting from benzene-
sulfonamide 15 (4.25 g); yield: 2.2 g (52%); mp 248–
250 ꢁC; IR (KBr) 1655 (C@O), 1590 (C@N), 1335, 1170
1
(SO2) cmꢀ1; H NMR (DMSO-d6) d 2.71 (s, 3H, CH3),
7.82 (d, J ¼ 1:4 Hz, 1H, arom.), 7.93 (d, J ¼ 8:4 Hz, 1H,
arom.), 8.02–8.08 (m, 2H, arom.), 8.29 (d, J ¼ 1:9 Hz,
1H, arom.) ppm. Anal. (C15H8Cl3NO3S2) C, H, N.
3.3.13.
6-Chloro-8-methyl-3-(2-naphthoyl)-1,1-dioxo-
1,4,2-benzodithiazine (33). Starting from benzenesulf-
onamide 16 (4.06 g); yield: 2.6 g (65%); mp 198–200 ꢁC;
IR (KBr) 1655 (C@O), 1620 (C@N), 1335, 1170 (SO2)
3.5. Preparation of oligonucleotide substrates
1
cmꢀ1; H NMR (CDCl3) d 2.86 (s, 3H, CH3), 7.31 (d,
The oligonucleotides 21top, 50-GTGTGGAAAATCT-
CTAGCAGT-30 and 21bot, 50-ACTGCTAGAGAT-
TTTCCACAC-30 were purchased from Norris Cancer
Center Core Facility (University of Southern California)
and purified by UV shadowing on polyacrylamide gel.
To analyze the extent of 30-processing and strand
transfer using 50-end labeled substrates, 21top was 50-
end labeled using T4 polynucleotide kinase (Epicentre,
Madison, WI) and [32P]-ATP (Amersham Biosciences or
ICN). The kinase was heat-inactivated and 21bot was
added in 1.5-M excess. The mixture was heated at 95 ꢁC,
allowed to cool slowly to room temperature, and run
through a spin 25 mini-column (USA Scientific) to
J ¼ 1:4 Hz, 1H, H-7, benzodithiaz.),7.42 (d, J ¼ 1 Hz,
1H, H-5, benzodithiaz.), 7.54–7.73 (m, 2H, naphthyl),
7.89–8.20 (m, 4H, naphthyl), 8.95 (s, 1H, H-1, naphthyl)
ppm. Anal. (C19H12ClNO3S2) C, H, N.
3.3.14. 6-Chloro-3-(4-chlorobenzoyl)-1,1-dioxo-1,4,2-ben-
zodithiazine (34) . Starting from benzenesulfonamide 17
(3.76 g); yield: 1.8 g (48%); mp 214–215 ꢁC; IR (KBr)
1
1665 (C@O), 1580 (C@N), 1340, 1175 (SO2) cmꢀ1; H
NMR (CDCl3) d 7.50–7.60 (m, 3H, arom.), 7.79 (dd,
Jortho ¼ 6:5 Hz, Jmeta ¼ 2:0 Hz, 1H, H-6, benzodithiaz.),

Z. Brzozowski et al. / Bioorg. Med. Chem. 12 (2004) 3663–3672
3671
separate annealed double-stranded oligonucleotide from
unincorporated material.
3.8. X-ray structure analysis of 24
Crystal data for C15H9C12NO3S2: monoclinic, space
group P21/n, a ¼ 7:2102ð5Þ, b ¼ 13:6632ð7Þ, c ¼
3.6. Integrase assays
3
ꢀ
ꢀ
15:6326ð8Þ A, b ¼ 91:406ð5Þꢁ, V ¼ 1539:57ð15Þ A , Z ¼
4, dx ¼ 1:666 g cmꢀ3, T ¼ 155 K. Data were collected for
a crystal with dimensions 0.8 · 0.15 · 0.08 mm with a
KumaCCD diffractometer using graphite monochro-
mated Mo Ka radiation. Final R indices for 2808 reflec-
tions with I > 2rðIÞ and 244 refined parameters are:
R1 ¼ 0:0388, wR2 ¼ 0:0921 (R1 ¼ 0:0455, wR2 ¼ 0:0964
for all 3112 data). Atom labeling is shown in Figure 2.
To determine the extent of 30-processing and strand
transfer, IN was preincubated at a final concentration of
200 nM with the inhibitor in reaction buffer (50 mM
NaCl, 1 mM HEPES, pH 7.5, 50 lM EDTA, 50 lM
dithiothreitol, 10% glycerol (w/v), 7.5 mM MnCl2,
0.1 mg/mL bovine serum albumin, 10 mM 2-mercapto-
ethanol, 10% dimethyl sulfoxide, and 25 mM MOPS,
pH 7.2) at 30 ꢁC for 30 min. Then, 20 nM of the 50-end 32P-
labeled linear oligonucleotide substrate was added, and
incubation was continued for an additional 1 h. Reactions
were quenched by the addition of an equal volume (16 lL)
of loading dye (98% deionized formamide, 10 mM
EDTA, 0.025% xylene cyanol, and 0.025% bromophenol
blue). An aliquot (5 lL) was electrophoresed on a dena-
turing 20% polyacrylamide gel (0.09 M tris-borate pH 8.3,
2 mM EDTA, 20% acrylamide, 8 M urea).
Supporting information available: Detailed atomic coor-
dinates, isotropic displacement parameters, bond length,
bond angles, H-bond coordinates, and torsion angles
(Tables 3–7) are available upon request.
Acknowledgements
We would like to thank the Drug Synthesis and
Chemistry Branch, Developmental Therapeutics of the
National Cancer Institute for the antiviral testing. The
work in N.N.’s lab was supported by funds from
the GlaxoSmithKline Drug Discovery Award.
Gels were dried, exposed in a PhosphorImager cassette,
and analyzed using a Typhoon 8610 Variable Mode
Imager (Amersham Biosciences) and quantitated using
ImageQuant 5.2. Percent inhibition (% I) was calculated
using the following equation:
References and notes
%I ¼ 100 ꢃ ½1 ꢀ ðD ꢀ CÞ=ðN ꢀ CÞꢄ
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