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

Article abstract of DOI:10.1016/j.ejmech.2008.02.004

A series of novel 3-aroyl-2,3-dihydro-1,1-dioxo-1,4,2-benzodithiazines 15-28 as potential HIV-1 integrase (IN) inhibitors have been synthesized by the reduction of 3-aroyl-1,1-dioxo-1,4,2-benzodithiazines 1-14 with benzenesulfonyl hydrazide. All the compo

Full text of DOI:10.1016/j.ejmech.2008.02.004

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European Journal of Medicinal Chemistry 44 (2009) 190e196
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anti-HIV-1 integrase activities of
3-aroyl-2,3-dihydro-1,1-dioxo-1,4,2-benzodithiazines
b
Zdzis1aw Brzozowski a, Franciszek Sa˛czewski a, , Jaros1aw S1awinski ,
*
´
Tino Sanchez c, Nouri Neamati c
a
´
´
Department of Chemical Technology of Drugs, Medical University of Gdansk, Al. Gen. J. Hallera 107, 80-416 Gdansk, Poland
b Department of Organic Chemistry, Medical University of Gdan´sk, Al. Gen. J. Hallera 107, 80-416 Gdan´sk, Poland
c Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California,
1985 Zonal Avenue, PSC 304BA, Los Angeles, CA 90089, USA
Received 8 December 2007; received in revised form 7 February 2008; accepted 7 February 2008
Available online 4 March 2008
Abstract
A series of novel 3-aroyl-2,3-dihydro-1,1-dioxo-1,4,2-benzodithiazines 15e28 as potential HIV-1 integrase (IN) inhibitors have been synthe-
sized by the reduction of 3-aroyl-1,1-dioxo-1,4,2-benzodithiazines 1e14 with benzenesulfonyl hydrazide. All the compounds 15e28 inhibited
IN mediated strand transfer reaction with IC50 values ranging from 3 to 30 mM. The 3-(4-bromobenzoyl)-6-chloro-7-methyl-2,3-dihydro-1,1-
dioxo-1,4,2-benzodithiazine 17 with the IC50 values of 4 ꢀ 1 and 3 ꢀ 1 mM for 30-processing and strand transfer, respectively, was the most
potent. Compound 17 as well its analogues were 5e20-fold less potent in Y99S and H114A mutants, implicating these residues as potential
drug-binding site. This is a first report implicating Y99S and H114A of IN core domain in drug-binding interactions.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: 3-Aroyl-2,3-dihydro-1,1-dioxo-1,4,2-benzodithiazines; Synthesis; HIV-1 integrase inhibitors
1. Introduction
among the recently reported dipyridine inhibitors, only com-
pounds containing a free mercapto group showed antiviral
activity and inhibited IN (e.g., IV, Fig. 1) [17]. A common
theme in these leads is the requirement for a free mercaptoaryl
group for antiviral activity as well as anti-IN potency (IeIV,
Fig. 1) [10,12,13,15e17]. More recently, we found that cyclic
analogues of 2-mercaptobenzenesulfonamides (Fig. 1, struc-
ture V) also showed anti-HIV and anti-IN properties [18].
This led us to an assumption that expansion of a series of
candidate anti-IN agents of general formula VI (Fig. 1) may
shed light on the structural features contributing to the IN
inhibitors.
It is well known that compounds bearing arylsulfonamide
moiety have been applied in various strategies aimed at dis-
covery of novel antiviral agents [1e5]. HIV-1 integrase (IN)
is an attractive target for selective blockade of viral infection
since there are no known counterparts in the host cell [6].
Previously, we described the syntheses and anti-HIV activities
of various 4-chloro-2-mercaptobenzenesulfonamide deriva-
tives with the nitrogen atom of sulfonamide moiety attached
to a variety heterocyclic ring systems (Fig. 1, structure I)
[7e12]. Some of these compounds (I) [13,14], di(4-mer-
capto-3-nitrophenyl)sulfones (e.g., II) [15] and 1,2-di(2-mer-
captobenzoyl)hydrazines (e.g., III) [16] were described as
novel class of potent IN inhibitors (Fig. 1) [13e16]. In fact,
2. Results and discussion
2.1. Chemistry
The previously described methods were used to synthesize
starting compounds, 3-aroyl-1,1-dioxo-1,4,2-benzodithiazines
* Corresponding author. Tel.: þ48 58 3493250; fax: þ48 58 3493257.
E-mail address: saczew@amg.gda.pl (F. Sa˛czewski).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.02.004

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 44 (2009) 190e196
191
O
O
O
O
SH
S
Cl
O2N
HS
S
NO2
SH
N
H
N
H
H
N
R
Me
S H
HS
O
O
I
II
III
NO2
O
O
H
OH
N
OH
N
Cl
R1
S
S
Cl
R1
S
S
Ar
Ar
N
N
O
O
R2
R2
O
O
O
SH
HS
IV
V
VI
Fig. 1. Structures of HIV-1 integrase inhibitors.
1e14 [18]. The synthesis of the target 3-aroyl-2,3-dihydro-
1,1-dioxo-1,4,2-benzodithiazines 15e28 was achieved by
reacting benzenesulfonyl hydrazide with the corresponding
aroylbenzodithiazines 1e14 in methanol.
coherence) spectrum showed the correlation between this
C3-H proton and the C-3 carbon atom, and were fully consis-
tent with the proposed structure.
To our knowledge the above reaction is the first example of
the reduction of C]N bond using arylsulfonylhydrazide. It
has been well known that arylsulfonylhydrazides upon heating
decompose to diimide which is convenient reducing agent for
symmetrical double bonds such as C]C, N]N or C^C.
Under this conditions, however, polar C]N bond has not
been reduced before [19]. Therefore, we propose two possible
mechanisms A and B which can be operative for the reduction
of compounds 1e14 incorporating formal a-imino-carbonyl
moiety (Scheme 1). According to mechanism A, the initial
step would consist in the formation of a spiro intermediate
of type A by [2 þ 2] cycloaddition of benzenesulfonyl hydra-
zide to the C3eC(]O)Ar bond of 3-aroyl-1,4,2-benzodithia-
zine 1,1-dioxides 1e14, followed by the 1,2-diazetidine ring
scission with subsequent loss of N2 and benzenesulfinic acid.
In the final stage of the reaction, the non-isolable enol B un-
dergoes tautomerization to afford the corresponding ketones
15e28. According to mechanism B, the addition
of hydrazone NH2 group to carbonyl C]O group of 1e14
may afford hemiaminal of type C, which undergoes a sponta-
neous retro-ene fragmentation with the formation of enol B
(Scheme 1).
2.2. Biology
The IN inhibitory activities of the newly prepared com-
pounds 15e28 compared to those of parent benzodithiazines
1e14 are presented in Table 1. All tested compounds are
significantly active against wild-type IN in the presences of
Mg2þ, albeit less effective as compared to Mn2þ, when added
as a co-factor. This is in agreement with our previous studies
[16].
For a series of compounds 1e7 with methyl group at posi-
tion 7 of benzodithiazine ring, partial hydrogenation leads to
compounds 15e21, inhibitory activity of which is equal (com-
pound 16, Ar ¼ 4-MeOPh), higher (15, Ar ¼ Ph; 17, Ar ¼ 4-
BrPh; 18, Ar ¼ 4-ClPh and 19, Ar ¼ 3-O2NPh) or lower (20,
Ar ¼ 3,4-diClPh and 21, Ar ¼ 2-naphthyl). Thus, electronic
and steric effects brought about by the Ar substituents seem
to be crucial for biological activity of these compounds. The
above pattern, however, is not observed for benzodithiazines
8e10 with 8-Me substituent, which upon hydrogenation
gave 22e24 with similar inhibitory activities.
Interestingly, upon hydrogenation of compounds 11e13
lacking the methyl group at both 7 and 8 position of benzodi-
thiazine ring, a very considerable improvement of inhibitory
activity is noted for 25 (Ar ¼ 4-ClPh) and 26 (Ar ¼ 3,4-
diClPh) thus obtained, while hydrogenated compound 27
(Ar ¼ 2-naphthyl) retains a relatively high potency of 13.
Summing up, all newly prepared compounds showed signif-
icant inhibition of purified IN with IC50 values ranging from 3
to 30 mM for strand transfer. The most active 3-(4-bromoben-
zoyl)-6-chloro-7-methyl-2,3-dihydro-1,1-dioxo-1,4,2-benzodi-
thiazine (17) with IC50 values of 4 ꢀ 1 and 3 ꢀ 1 mM for
1
The H NMR spectrum of the product 18 containing an
aroyl group at position 3 of benzodithiazine ring showed
two doublet signals attributed to the C3-H proton at
d 7.29 ppm and NH proton at d 9.13 ppm with the coupling
constant J ¼ 11.5 Hz. Furthermore, the correlation between
NH proton and both C-3 carbon atom at d 64.04 and C]O
1
carbon atom at d 190.16 was found in the He13C heterocor-
related spectrum (HMBC-heteronuclear multiple bond correla-
tion), while the HSQC (heteronuclear single quantum

192
Z. Brzozowski et al. / European Journal of Medicinal Chemistry 44 (2009) 190e196
O
Ar
HO
NH
Ar
Cl
R1
S
S
Cl
R1
S
Mechanism A
S
Ar
N
O
N
H
O
N
S
R2
O
O
O
R2
O
1-14
A
Mechanism B
-[ PhSO2N=NH
N2 + PhSO2H ]
OH
HO
N
Ar
Cl
Cl
S
S
S
retro-ene
Ar
NH
NH
R1
SO2Ph
R1
-[ PhSO2N=NH
N2 + PhSO2H ]
S
H
R2
R2
O
O
O
O
C
B
O
H
Cl
S
S
Ar
N
R1
R2
O
O
15-28
Compds
1, 15
2, 16
3, 17
4, 18
5, 19
6, 20
7, 21
X
Cl
Cl
Cl
Cl
Cl
Cl
Cl
R1
R2
Ar
Compds
8, 22
X
R1
H
H
H
H
H
H
H
R2
Me
Me
Me
H
Ar
Me
Me
Me
Me
Me
Me
Me
H
Ph
Cl
Cl
Cl
Cl
Cl
Cl
H
4-ClPh
H
H
H
H
H
H
4-MeOPh
9, 23
3,4-diClPh
2-naphthyl
4-ClPh
4-BrPh
10, 24
11, 25
12, 26
13, 27
14, 28
4-ClPh
3-O2NPh
3,4-diClPh
2-naphthyl
H
3,4-diClPh
2-naphthyl
3,4-diClPh
H
H
Scheme 1. Synthesis of 3-aroyl-2,3-dihydro-1,1-dioxo-1,4,2-benzodithiazines 15e28 and proposed mechanism of their formation. Reagents, conditions and yields:
(a) benzenesulfonyl hydrazide (1.33 molar equiv), methanol, room temperature, 20 h, reflux, 12 h, 62e91%.
30-processing and strand transfer, respectively, may serve as
useful lead compound for further development of powerful
anti-HIV agents.
sites. Further studies are underway to provide a direct proof
for these observations.
In our biological investigations we further used Y99S and
H114A mutants of the enzyme. Importantly, the Y99S mutant
in general was about fivefold more resistant than the H114A
(Table 1). This implies that the tested compounds most likely
bind to these novel sites. This is the first report implicating
Y99 and H114 residues of IN catalytic site as drug-binding
3. Experimental protocols
3.1. Synthesis
The following instruments and parameters were used:
(melting points) Buchi 535 apparatus; (IR spectra) KBr
¨

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 44 (2009) 190e196
193
Table 1
Structure and anti-HIV integrase activities of previously described 3-aroyl-1,1-dioxo-1,4,2-benzodithiazines 1e14 [12], and their novel 2,3-dihydro analogues 15e
28
O
O
X
S
S
X
S
S
Ar
Ar
N
N
R1
R1
H
R2
O
O
R2
O
O
substrates 1-14
products 15-28
Compd
X
R1
R2
Ar
Ph
Anti-HIV integrase activities
Compd 1e14
Compd 15e28
IC50 (mM)a
Y99Sc
Clv. ST
H114Ad
Clv. ST
IC50 (mM)a
Cleavage
(30-processing)
Integration
(strand transfer)
Cleavage
(30-processing)
Integration
(strand transfer)
1, 15
Cl
Me
H
12
12
7 ꢀ 1 (60)b
5 ꢀ 1 (27)b
90
18
15
9
2, 16
3, 17
Cl
Cl
Me
Me
H
H
4-MeOPh
4-BrPh
30
30
33
30
38 ꢀ 11
4 ꢀ 1
17 ꢀ 2
3 ꢀ 1
75
20
90
20
20
18
18
16
4, 18
Cl
Me
H
4-ClPh
30
65
6 ꢀ 2 (63)b
4 ꢀ 1 (40)b
5, 19
6, 20
7, 21
8, 22
9, 23
10, 24
Cl
Cl
Cl
Cl
Cl
Cl
Me
Me
Me
H
H
3-O2NPh
3,4-diClPh
2-Naphthyl
4-ClPh
57
15
28
22
17
22
50
7
32 ꢀ 1
28 ꢀ 6
H
20 ꢀ 13
57 ꢀ 14
23 ꢀ 5
10 ꢀ 6
H
3
30 ꢀ 13
14 ꢀ 4
Me
Me
Me
25
25
7
H
3,4-diClPh
2-Nahpthyl
20 ꢀ 1
14 ꢀ 4
H
9 ꢀ 1 (83)b
6 ꢀ 1 (51)b
70
23
33
23
20
18
18
18
9
11, 25
12, 26
13, 27
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
4-ClPh
100
61.2
8
90
124.0
8
8 ꢀ 1 (51)b
9 ꢀ 3 (58)b
8 ꢀ 2 (55)b
22 ꢀ 3
4 ꢀ 1 (25)b
5 ꢀ 3 (25)b
3 ꢀ 1 (28)b
15 ꢀ 4
100
20
3,4-diClPh
2-Naphthyl
3,4-diClPh
90
20
100
15
15
14, 28
NT
NT
a
Inhibitory concentration (mM) 50% required to reduce HIV-1 IN mediated 30-processing (cleavage) or strand transfer (integration) reactions (carried out in the
presences of Mnþ2 as a co-factor).
b
Values in the parenthesis were generated from experiments carried out in the presence of Mgþ2 as a co-factor.
IC50 values in mM refer to cleavage (top) and strand transfer (bottom) using Y99S mutant.
IC50 values in mM refer to cleavage (top) and strand transfer (bottom) using H114A mutant.
c
d
pellets, 400e4000 cmꢁ1 PerkineElmer 1600 FTIR spectro-
photometer; (1H and 13C NMR spectra) Varian Gemini 200
apparatus at 200 and 50 MHz, respectively (chemical shifts
are expressed at d values relative to Me4Si as standard).
HSQC and HMBC spectra were taken on Varian Unity 500
spectrometer. The results of elemental analyses for C, H and
N were within ꢀ0.4% of theoretical values.
and standing overnight the precipitate of the adequate 2,3-
dihydro-1,4,2-benzodithiazine was collected by filtration,
washed with methanol (4 ꢂ 2 ml) and dried.
In this manner, the following 2,3-dihydro-1,4,2-benzodi-
thiazines were obtained.
3.1.1.1. 3-Benzoyl-6-chloro-7-methyl-2,3-dihydro-1,1-dioxo-1,
4,2-benzodithiazine (15). Starting from benzodithiazine 1
(1.06 g); yield: 0.79 g (74%); m.p. 165e166 ꢃC; IR (KBr)
3.1.1. General procedure for the selective reduction of
3-aroyl-1,1-dioxo-1,4,2-benzodithiazines into 3-aroyl-2,
3-dihydro-1,1-dioxo-1,4,2-benzodithiazines (15e28)
3228 (NH), 1690 (C]O), 1345, 1165 (SO2) cmꢁ1
;
2.36 (s, 3H, CH3), 7.20 (d,
1H
NMR (DMSO-d6)
d
J ¼ 11.5 Hz, 1H, H-3), 7.57e7.78 (m, 4H, arom.), 7.92 (s,
1H, H-8), 7.98e8.07 (m, 2H, arom.), 9.11 (d, J ¼ 11.5 Hz,
1H, NH) ppm; 13C NMR (DMSO-d6) d 19.18, 63.93,
127.64, 127.89, 129.17, 129.77, 132.22, 133.04, 133.38,
A mixture of benzenesulfonyl hydrazide (0.69 g, 4 mmol)
and the corresponding benzodithiazines 1e14 (3 mmol) in
methanol (15 ml) was stirred at room temperature for 20 h,
followed at reflux for 12 h. After cooling to room temperature

194
Z. Brzozowski et al. / European Journal of Medicinal Chemistry 44 (2009) 190e196
134.09, 134.75, 137.90, 190.90 ppm. Anal. (C15H12ClNO3S2)
C, H, N.
(m, 3H, 3,4-diClPh), 8.22 (s, 1H, H-8), 9.16 (d, J ¼ 11.3 Hz,
1H, NH) ppm; 13C NMR (DMSO-d6) d 19.18, 63.96,
127.67, 127.94, 129.67, 131.43, 131.62, 132.15, 132.20,
133.02, 133.35, 134.27, 137.52, 138.00, 189.45 ppm. Anal.
(C15H10Cl3NO3S2) C, H, N.
3.1.1.2. 6-Chloro-3-(4-methoxybenzoyl)-7-methyl-2,3-dihydro-
1,1-dioxo-1,4,2-benzodithiazine (16). Starting from benzodi-
thiazine 2 (1.15 g); yield: 0.98 g (85%); m.p. 172e174 ꢃC;
IR (KBr) 3230 (NH), 1665 (C]O), 1355, 1345, 1170 (SO2)
3.1.1.7. 6-Chloro-7-methyl-3-(2-naphthoyl)-2,3-dihydro-1,1-
dioxo-1,4,2-benzodithiazine (21). Starting from benzodithia-
zine 7 (1.21 g); yield: 1.1 g (90%); m.p. 191e192 ꢃC; IR
1
cmꢁ1; H NMR (DMSO-d6) d 2.35 (s, 3H, CH3), 3.88 (s,
3H, CH3O), 7.11 (d, J ¼ 11.7 Hz, 1H, H-3), 7.14 (d,
J ¼ 8.8 Hz, 2H, 4-MeOPh), 7.63 (s, 1H, H-5), 7.93 (s, 1H,
H-9), 8.04 (d, J ¼ 8.8 Hz, 2H, 4-MeOPh), 9.06 (d,
J ¼ 11.7 Hz, 1H, NH) ppm; 13C NMR (DMSO-d6) d 19.17,
56.05, 63.64, 114.55, 125.60, 127.64, 127.76, 127.85,
132.27, 133.54, 134.03, 137.85, 164.47, 188.87 ppm. Anal.
(C16H14ClNO4S2) C, H, N.
(KBr) 3300 (NH), 1685 (C]O), 1330, 1165 (SO2) cmꢁ1
;
1H NMR (DMSO-d6) d 2.37 (s, 3H, CH3), 7.32 (d,
J ¼ 11.4 Hz, 1H, H-3, benzodithiazine), 7.64e7.79 (m, 3H,
arom.), 7.96e8.30 (m, 5H, arom.), 8.80 (d, J ¼ 5.5 Hz, 1H,
arom.), 9.13 (d, J ¼ 11.4 Hz, 1H, NH) ppm. Anal.
(C19H14ClNO3S2) C, H, N.
3.1.1.3. 3-(4-Bromobenzoyl)-6-chloro-7-methyl-2,3-dihydro-
1,1-dioxo-1,4,2-benzodithiazine (17). Starting from benzodi-
thiazine 3 (1.29 g); yield: 1.18 g (91%); m.p. 202e204 ꢃC;
IR (KBr) 3230 (NH), 1675 (C]O), 1345, 1335, 1165 (SO2)
3.1.1.8. 6-Chloro-3-(4-chlorobenzoyl)-8-methyl-2,3-dihydro-
1,1-dioxo-1,4,2-benzodithiazine (22). Starting from benzodi-
thiazine 8 (1.16 g); yield: 0.82 g (70%); m.p. 201e203 ꢃC;
IR (KBr) 3235 (NH), 1680 (C]O), 1330, 1165 (SO2) cmꢁ1
;
cmꢁ1
;
1H NMR (DMSO-d6) d 2.36 (s, 3H, CH3), 7.19
1H NMR (DMSO-d6) d 2.63 (s, 3H, CH3), 7.19 (d,
J ¼ 12.0 Hz, 1H, H-3), 7.33 (d, J ¼ 1.8 Hz, 1H, H-7), 7.54
(d, J ¼ 1.8 Hz, 1H, H-5), 7.70 (d, J ¼ 8.5 Hz, 2H, 4-ClPh),
8.05 (d, J ¼ 8.5 Hz, 2H, 4-ClPh), 9.30 (d, J ¼ 12.0 Hz, 1H,
NH) ppm. Anal. (C15H11Cl2NO3S2) C, H, N.
(d, J ¼ 11.6 Hz, 1H, H-3), 7.66 (s, 1H, H-5), 7.84 (d,
J ¼ 8.4 Hz, 2H, 4-BrPh), 7.86 (s, 1H, H-8), 7.96 (d,
J ¼ 8.4 Hz, 2H, 4-BrPh), 9.13 (d, J ¼ 11.6 Hz, 1H, NH)
ppm; 13C NMR (DMSO-d6) d 19.18, 64.00, 127.65, 127.76,
127.91, 128.96, 131.63, 132.17, 132.31, 133.20, 134.16,
137.95, 190.29 ppm. Anal. (C15H11BrClNO3S2) C, H, N.
3.1.1.9. 6-Chloro-3-(3,4-dichlorobenzoyl)-8-methyl-2,3-dihy-
dro-1,1-dioxo-1,4,2-benzodithiazine
(23). Starting
from
3.1.1.4. 6-Chloro-3-(4-chlorobenzoyl)-7-methyl-2,3-dihydro-
1,1-dioxo-1,4,2-benzodithiazine (18). Starting from benzodi-
thiazine 4 (1.16 g); yield: 0.96 g (82%); m.p. 185e186 ꢃC;
IR (KBr) 3235 (NH), 1680 (C]O), 1345, 1335, 1170 (SO2)
benzodithiazine 9 (1.26 g); yield: 1.07 g (84%); m.p. 183e
185 ꢃC; IR (KBr) 3250 (NH), 1690 (C]O), 1335, 1165
1
(SO2) cmꢁ1; H NMR (DMSO-d6) d 2.63 (s, 3H, CH3), 7.23
(d, J ¼ 11.7 Hz, 1H, H-3), 7.34e7.36 (m, 1H, arom.), 7.56
(d, J ¼ 1.9 Hz, 1H, arom.), 7.90e7.97 (m, 1H, arom.), 8.23
(d, J ¼ 1.6 Hz, 1H, arom.), 9.33 (d, J ¼ 11.7 Hz, 1H, NH)
ppm; 13C NMR (DMSO-d6) d 20.71, 63.37, 125.51, 129.16,
129.69, 131.20, 131.61, 131.96, 132.12, 133.16, 136.23,
136.71, 137.62, 139.96, 189.52 ppm. Anal. (C15H10Cl3NO3S2)
C, H, N.
cmꢁ1 1H NMR (DMSO-d6) d 2.38 (s, 3H, CH3), 7.29
;
(d, J ¼ 11.5 Hz, 1H, H-3), 7.66 (s, 1H, H-5), 7.77 (d,
J ¼ 8.6 Hz, 2H, 4-ClPh), 7.95 (s, 1H, H-8), 8.04 (d,
J ¼ 8.6 Hz, 2H, 4-ClPh), 9.13 (d, J ¼ 11.5 Hz, 1H, NH)
ppm; 13C NMR (DMSO-d6) d 19.18, 64.04, 127.65, 127.91,
129.35, 131.61, 131.82, 132.18, 133.22, 134.17, 137.95,
139.67, 190.16 ppm. Anal. (C15H11Cl2NO3S2) C, H, N.
3.1.1.10. 6-Chloro-8-methyl-3-(2-naphthoyl)-2,3-dihydro-1,1-
dioxo-1,4,2-benzodithiazine (24). Starting from benzodithia-
zine 10 (1.21 g); yield: 1.08 g (89%); m.p. 190e191 ꢃC; IR
3.1.1.5. 6-Chloro-3-(3-nitrobenzoyl)-7-methyl-2,3-dihydro-1,1-
dioxo-1,4,2-benzodithiazine (19). Starting from benzodithia-
zine 5 (1.19 g); yield: 0.85 g (71%); m.p. 203e205 ꢃC; IR
(KBr) 3250 (NH), 1705 (C]O), 1345, 1330, 1170 (SO2)
(KBr) 3205 (NH), 1685 (C]O), 1325, 1165 (SO2) cmꢁ1
;
1H NMR (DMSO-d6) d 2.65 (s, 3H, CH3), 7.33 (d,
J ¼ 11.6 Hz, 1H, H-3, benzodithiazine), 7.46e7.84 (m, 4H,
arom.), 8.03e8.14 (m, 4H, arom.), 8.82 (d, J ¼ 8.0 Hz, 1H,
arom.), 9.34 (d, J ¼ 11.6 Hz, 1H, NH) ppm; 13C NMR
(DMSO-d6) d 20.77, 63.03, 124.66, 125.39, 127.65, 128.17,
128.73, 129.03, 129.79, 130.04, 130.20, 132.11, 132.32,
134.40, 135.78, 136.15, 137.17, 139.92, 190.69 ppm. Anal.
(C19H14ClNO3S2) C, H, N.
1
cmꢁ1; H NMR (DMSO-d6) d 2.39 (s, 3H, CH3), 7.36 (d,
J ¼ 11.3 Hz, 1H, H-3), 7.72 (s, 1H, H-5), 7.79e7.91 (m, 2H,
H-8, benzodithiazine and H-5, 3-O2NPh), 8.41e8.46 (m, 1H,
H-6, 3-O2NPh), 8.55e8.60 (m, 1H, H-4, 3-O2NPh), 8.77 (t,
J ¼ 1.8 Hz, 1H, H-2, 3-O2NPh), 9.22 (d, J ¼ 11.3 Hz, 1H,
NH) ppm. Anal. (C15H11ClN2O5S2) C, H, N.
3.1.1.6. 6-Chloro-3-(3,4-dichlorobenzoyl)-7-methyl-2,3-dihy-
dro-1,1-dioxo-1,4,2-benzodithiazine
(20). Starting
from
3.1.1.11. 6-Chloro-3-(4-chlorobenzoyl)-2,3-dihydro-1,1-dioxo-
1,4,2-benzodithiazine (25). Starting from benzodithiazine 11
(1.12 g); yield: 0.70 g (62%); m.p. 212e214 ꢃC; IR (KBr)
benzodithiazine 6 (1.26 g); yield: 1.0 g (78%); m.p. 211e
212 ꢃC; IR (KBr) 3230 (NH), 1680 (C]O), 1340, 1170
1
1
(SO2) cmꢁ1; H NMR (DMSO-d6) d 2.36 (s, 3H, CH3), 7.24
3245 (NH), 1690 (C]O), 1365, 1340, 1170 (SO2) cmꢁ1; H
(d, J ¼ 11.3 Hz, 1H, H-3), 7.68 (s, 1H, H-5), 7.90e8.00
NMR (DMSO-d6) d 7.24 (d, J ¼ 11.5 Hz, 1H, H-3), 7.42

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 44 (2009) 190e196
195
(dd, Jortho ¼ 8.5 Hz, Jmeta ¼ 2.1 Hz, 1H, H-7), 7.70 (d, Jmeta
¼
the point mutations according to the manufacturer’s
instructions.
2.1 Hz, 1H, H-5), 7.72 (d, J ¼ 8.7 Hz, 2H, 4-ClPh), 7.92 (d,
Jortho ¼ 8.5 Hz, 1H, H-5), 8.03 (d, J ¼ 8.7 Hz, 2H, 4-ClPh),
9.18 (d, J ¼ 11.5 Hz, 1H, NH) ppm; 13C NMR (DMSO-d6)
d 64.08, 126.34, 127.40, 127.63, 129.38, 131.60, 131.80,
132.27, 136.85, 137.44, 139.71, 190.07 ppm. Anal.
(C14H9Cl2NO3S2) C, H, N.
Nucleotide replacements were confirmed by sequencing at
the USC/Norris Comprehensive Cancer Center Microchemical
Core Facility (University of Southern California). The IN plas-
mid was expressed in Escherichia coli BL21(De3) PLYS S
expression strain (Invitrogen) after induction by IPTG
(1 mM) at an absorbance of 0.6e0.8 optical density at
595 nm. Protein purification was performed as described in
Ref. [20].
3.1.1.12. 6-Chloro-3-(3,4-dichlorobenzoyl)-2,3-dihydro-1,1-di-
oxo-1,4,2-benzodithiazine (26). Starting from benzodithiazine
12 (1.22 g); yield: 0.80 g (65%); m.p. 185e186 ꢃC; IR (KBr)
1
3225 (NH), 1675 (C]O), 1335, 1170 (SO2) cmꢁ1; H NMR
3.2.3. Preparation of oligonucleotide substrates
The oligonucleotides 21top, 50-GTGTGGAAAATCTC-
TAGCAGT-30 and 21bot, 50-ACTGCTAGAGATTTTCCA-
CAC-30 were purchased from the Norris Cancer Center
Microsequencing Core Facility (University of Southern Cali-
fornia) 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 g
[32P]-ATP (Amersham Biosciences or ICN). The kinase was
heat-inactivated and 21bot was added in 1.5-molar excess.
The mixture was heated at 95 ꢃC, allowed to cool slowly to
room temperature, and run through a spin 25 mini-column
(USA Scientific) to separate annealed double-stranded oligo-
nucleotide from unincorporated material.
(DMSO-d6) d 7.27 (d, J ¼ 11.2 Hz, 1H, H-3), 7.42 (dd, Jortho
¼
8.4 Hz, Jmeta ¼ 1.9 Hz, 1H, H-7), 7.72 (d, Jmeta ¼ 1.9 Hz, 1H,
H-5), 7.84e7.95 (m, 3H, H-5,6 of 3,4-diClPh and H-8 of ben-
zodithiazine), 8.23 (s, 1H, H-2, 3,4-diClPh), 9.22 (d,
J ¼ 11.2 Hz, 1H, NH) ppm; 13C NMR (DMSO-d6) d 63.99,
126.42, 127.42, 127.66, 129.67, 131.43, 131.65, 132.18,
132.29, 133.33, 136.66, 137.49, 137.57, 189.35 ppm. Anal.
(C14H8Cl3NO3S2) C, H, N.
3.1.1.13.
6-Chloro-3-(2-naphthoyl)-2,3-dihydro-1,1-dioxo-
1,4,2-benzodithiazine (27). Starting from benzodithiazine 13
(1.16 g); yield: 0.86 g (73%); m.p. 178e180 ꢃC; IR (KBr)
3190 (NH), 1675 (C]O), 1350, 1325, 1170, 1160 (SO2)
1
cmꢁ1; H NMR (DMSO-d6) d 7.36 (d, J ¼ 11.6 Hz, 1H, H-
3), 7.40 (dd, Jortho ¼ 8.4 Hz, Jmeta ¼ 1.7 Hz, 1H, H-7), 7.63e
7.77 (m, 3H, arom.), 7.95 (d, Jortho ¼ 8.4 Hz, 1H, H-8),
8.01e8.29 (m, 4H, arom.), 8.82 (d, J ¼ 6.6 Hz, 1H, arom.),
9.19 (d, J ¼ 11.6 Hz, 1H, NH) ppm. Anal. (C18H12ClNO3S2)
C, H, N.
3.2.4. IN assays
To determine the extent of 30-processing and strand trans-
fer, wild-type 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 mM EDTA, 50 mM dithio-
threitol, 10% glycerol (w/v), 7.5 mM MnCl2, 0.1 mg/ml
bovine serum albumin, 10 mM 2-mercaptoethanol, 10%
dimethyl sulfoxide, and 25 mM MOPS, pH 7.2) at 30 ꢃC for
30 min. Then, 20 nM of the 50-end 32P-lebeled linear oligonu-
cleotide substrate was added, and incubation at 30 ꢃC was
continued for an additional 1 h. Reactions were quenched by
the addition of an equal volume (16 ml) of loading dye (98%
deionized formamide, 10 mM EDTA, 0.025% xylene cyanol
and 0.025% bromophenol blue). An aliquot (5 ml) was electro-
phoresed on denaturing 20% polyacrylamide gel (0.09 M
Triseborate pH 8.3, 2 mM EDTA, 20% acrylamide, 8 M
urea). Gels were dried, exposed in a PhosphorImager cassette,
analyzed using a Typhoon 8610 Variable Mode Imager (Amer-
sham Biosciences), and quantitated using ImageQuant 5.2.
Percent inhibition (%I) was calculated using the following
equation:
3.1.1.14. 3-(3,4-Dichlorobenzoyl)-2,3-dihydro-1,1-dioxo-1,4,2-
benzodithiazine (28). Starting from benzodithiazine 14
(1.12 g); yield: 0.8 g (71%); m.p. 206e207 ꢃC; IR (KBr)
1
3205 (NH), 1685 (C]O), 1345, 1185 (SO2) cmꢁ1; H NMR
(DMSO-d6) d 7.24 (d, J ¼ 11.3 Hz, 1H, H-3), 7.37 (t,
J ¼ 7.4 Hz, 1H, H-6), 7.47e7.56 (m, 2H, arom.), 7.91 (t,
J ¼ 7.4 Hz, 1H, H-7), 7.93e8.00 (m, 2H, arom.), 8.23 (s,
1H, H-2, 3,4-diClPh), 9.20 (d, J ¼ 11.3 Hz, 1H, NH) ppm.
Anal. (C14H9Cl2NO3S2) C, H, N.
3.2. HIV-1 IN inhibition assay
3.2.1. Materials
For biological testing all compounds were dissolved in
DMSO and the stock solutions were stored at ꢁ20 ꢃC. The
g [32P]-ATP was purchased from either Amersham Biosci-
ences or ICN.
%I ¼ 100½1 ꢁ ðD ꢁ CÞ=ðN ꢁ CÞꢄ
3.2.2. Site directed mutagenesis
Site directed mutagenesis was conducted on the pET-15b-
IN plasmid, a generous gift from Dr. Robert Craigie, Labora-
tory of Molecular Biology, NIDDK, NIH, Bethesda, MD. The
plasmid contains full length IN fused to a N-terminal histidine
tag downstream from a T7 prompter. A Quickchange site
directed mutagenesis kit (Stratagene) was employed to make
where C, N, and D are the fractions of 21-mer substrate
converted to 19-mer (30-processing product) or strand transfer
products for DNA alone, DNA plus IN, and IN plus drug,
respectively. The IC50 values were determined by plotting
the logarithm of drug concentration versus percent inhibition
to obtain the concentration that produced 50% inhibition.

196
Z. Brzozowski et al. / European Journal of Medicinal Chemistry 44 (2009) 190e196
[10] Z. Brzozowski, Acta Polon. Pharm. Drug Res. 53 (1997) 49e53.
[11] Z. Brzozowski, Acta Polon. Pharm. Drug Res. 55 (1998) 473e480.
Acknowledgement
´
[12] Z. Brzozowski, J. S1awinski, F. Sa˛czewski, T. Sanchez, N. Neamati, Eur.
Work in Dr. Neamati’s laboratory was supported by funds
from the Campbell Foundation.
J. Med. Chem. (2007). doi:10.1016/j.ejmech.2007.08.013.
[13] N. Neamati, A. Mazumder, S. Sunder, J.H. Owen, R.J. Schutz,
Y. Pommier, Antiviral Chem. Chemother. 8 (1997) 485e495.
´
[14] L. Ch.Kuo, H. Assefa, Z. Brzozowski, J. S1awinski, F. Sa˛czewski,
J.K. Buolamwini, N. Neamati, J. Med. Chem. 47 (2004) 385e399.
[15] N. Neamati, A. Mazumder, H. Zaho, S. Sunder, T.R. Burke Jr.,
R.J. Schultz, Y. Pommier, Antimicrob. Agents Chemother. 41 (1997)
385e393.
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