Synthesis, anti-HIV-1 integrase, and cytotoxic activities of 4-chloro-N-(4-oxopyrimidin-2-yl)-2-mercaptobenzenesulfonamide derivatives

Article abstract of DOI:10.1016/j.ejmech.2007.08.013

Several 4-chloro-N-(4-oxopyrimidin-2-yl)-2-mercaptobenzenesulfonamide derivatives 13-28 and 35-44 have been synthesized and tested as potential HIV-1 integrase (IN) inhibitors. Compounds 15-17, 19, 21-28, 36 and 41 inhibited IN with IC₅₀ values in the range of 3.3-63.0 μM. The compounds 13, 15, 16, 21-24 and 26-28 were further tested at the US National Cancer Institute for their in vitro activity against a panel of 53-57 human tumor cell lines. The compounds 26-28 were inactive, whereas the other compounds exhibited high or reasonable activity (GI₅₀ < 0.01-20.0 μM) against one or more human tumor cell lines.

Full text of DOI:10.1016/j.ejmech.2007.08.013

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1188e1198
http://www.elsevier.com/locate/ejmech
Original article
Synthesis, anti-HIV-1 integrase, and cytotoxic activities of 4-chloro-
N-(4-oxopyrimidin-2-yl)-2-mercaptobenzenesulfonamide derivatives
a
a
a,
*
,
´
Zdzis1aw Brzozowski , Jaros1aw S1awinski , Franciszek Sa˛czewski
Tino Sanchez b, Nouri Neamati b
a
´
´
Department of Chemical Technology of Drugs, Medical University of Gdansk, Al. Gen. Hallera 107, 80-416 Gdansk, Poland
b Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal Avenue,
PSC 304 BA, Los Angeles, CA 90089, USA
Received 14 April 2007; received in revised form 26 July 2007; accepted 9 August 2007
Available online 11 September 2007
Abstract
Several 4-chloro-N-(4-oxopyrimidin-2-yl)-2-mercaptobenzenesulfonamide derivatives 13e28 and 35e44 have been synthesized and tested
as potential HIV-1 integrase (IN) inhibitors. Compounds 15e17, 19, 21e28, 36 and 41 inhibited IN with IC50 values in the range of 3.3e
63.0 mM. The compounds 13, 15, 16, 21e24 and 26e28 were further tested at the US National Cancer Institute for their in vitro activity against
a panel of 53e57 human tumor cell lines. The compounds 26e28 were inactive, whereas the other compounds exhibited high or reasonable
activity (GI50 < 0.01e20.0 mM) against one or more human tumor cell lines.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: 4-Chloro-N-(3,4-dihydro-4-oxopyrimidin-2-yl)-2-mercaptobenzenesulfonamide derivatives; Synthesis; Anti-HIV activity; Cytotoxic activity
1. Introduction
studies on the synthesis of a new series of 4-chloro-2-mercap-
tobenzenesulfonamide derivatives of type G (Fig. 1) with po-
tential anti-HIV-1 integrase and cytotoxic activities.
The arylsulfonamides constitute an important class of
compounds with several types of biological activities and well-
established safety profile [1]. Currently, there is significant
interest in the discovery and development of novel arylsulfona-
mides for the treatment of cancer and HIV infections [2e5]. As
part of a broad investigation of structures containing 4-chloro-
2-mercaptobenzenesulfonamide scaffold, several series of
novel sulfonamides with remarkable antitumor activity
(Fig. 1, structures: A [6e14], B [15e17], C [18e20] and D
[21]) or anti-HIV activity (Fig. 1, structures: A [6e8, 22e25],
B [26], C [20] E and F [27]) and HIV-1 integrase inhibitory ac-
tivity (Fig. 1, structures: A [22,24] and B [26]) were discovered
in our laboratories. In order to better understand the nature of
their cytotoxic versus antiviral properties, we extended our
2. Results and discussion
2.1. Chemistry
The previously described methods were employed for the
synthesis of compounds 2e9 and 13e20 [25]. Analogously,
we prepared novel methyl 3-(6-chloro-7-methyl-1,1-dioxo-
1,4,2-benzodithiazin-3-ylamino)thiophene-2-carboxylates 10e12
(Scheme 1).
The synthesis of the target 2-mercaptobenzenesulfonamides
21e28 were achieved by reacting corresponding methyl
o-(1,4,2-benzodithiazin-3-ylamino)aryl carboxylates 2, 4, 5,
6, 11 and 12 with benzyl- or furfurylamine in boiling toluene.
We propose a reaction sequence for the transformations as
shown in Scheme 2. Nucleophilic attack of amine at the
* Corresponding author. Tel.: þ48 58 349 3250; fax: þ48 58 349 3257.
E-mail address: saczew@amg.gda.pl (F. Sa˛czewski).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.08.013

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 43 (2008) 1188e1198
1189
R3
Cl
R1
Cl
R1
SH
Cl
R1
S
S
S
S
N
H
N
N
N
S
R2
R3
R2
R2
O O
O O
O
O
A
B
C
Cl
S
S
N
Cl
R1
S
S
N
Cl
S
S
N
N
R4
NH-Ar
N
N
N
R
R
R3
R2
O O
O
O
O O
D
E
F
R2
Cl
R1
S
H
N
N
O
S
O O
X = aromatic or heteroaromatic ring
X
HN
G
Fig. 1.
carbon C-3 atom of benzodithiazine ring results in guanidine
intermediate A, which undergoes a cyclocondensation reaction
leading to the formation of aminium salt of the final products
B, which after acidification with boiling glacial acetic acid
gave free 2-mercaptobenzenesulfonamides 21e28 in 67e
83% yields.
As shown in Scheme 3, the desired 2-benzylthiobenzene-
sulfonamides 35e44 were obtained by reacting the previously
described [13] N-(2-benzylthio-4-chlorobenzenesulfonyl)cya-
namide potassium salts 29e34 with the corresponding methyl
o-aminoaryl carboxylates in glacial acetic acid.
activity against purified IN, indicating that bulkier group at
this position is responsible for enhanced potency. Moreover,
the most active compounds 25e28 incorporate thieno[2,3-e]
pyrimidine ring with lipophylic tert-butyl, phenyl or thienyl
group at position 5 (IC50 values in the range of 6e22 mM
for 30-processing and 4e6 mM for strand transfer). These latter
compounds show potency comparable to those exhibited by
the well known 2-mercaptobenzenesulfonamide NSC 661073
[24].
The sulfonamides 13, 15, 16, 21e24 and 26e28 were fur-
ther tested at the National Cancer Institute (Bethesda, MD)
against a panel of approximately 60 human tumor cell lines
derived from nine different cancer types: leukemia, lung, co-
lon, CNS, melanoma, ovarian, renal, prostate and breast
(NCI 60) [28e30].
The structures of the newly obtained compounds 10e12,
21e28 and 35e44 were confirmed by elemental analyses
(C, H, N) and spectroscopic data presented in Section 4.
First of all, it should be pointed out that the compounds
26e28, which exhibited highest HIV-1 integrase inhibitory
2.2. Biology
activity, proved to be inactive in cytotoxicity tests (GI50
>
All the final sulfonamides 13e28 and 35e44 were tested as
potential HIV-1 integrase inhibitors. On the basis of data pre-
sented in Table 1, several pertinent features emerge to be im-
portant for IN inhibition. Compounds 13e20 with a free
mercapto group and the NH2 substituent at N-3 nitrogen
atom of oxopyrimidine moiety possess rather weak activity
(IC50 values above 55 mM for 30-processing and above
35 mM for strand transfer inhibition). Similarly, compounds
35e43 with benzyl-protected mercapto group and no substitu-
ent at N-3 nitrogen atom are either inactive (compound 37,
IC50 values above 100 mM) or possess weak activity (IC50
values above 47 and 33 mM for 30-processing and strand trans-
fer inhibition, respectively).
100 mM), and therefore, they can be considered for further
development as anti-HIV agents.
On the other hand, the other tested compounds exhibited
structure dependent high, reasonable or weak activity
(GI50 < 0.01e99.8 mM) against 25e96% of the 53e57 human
tumor cell lines (Table 2). The data of the most susceptible cell
lines (GI50 < 20.0 mM) recorded in Table 2 indicate the fol-
lowing rank order of potency: 22 (against nine cell lines,
GI50 ¼ 3.9e19.9 mM), 23 (against nine cell lines,
GI50 ¼ 14.2e19.9 mM), 21 (six cell lines, GI50 < 0.01e
19.5 mM), 13 (six cell lines, GI50 ¼ 7.2e19.7 mM) 24, (two
cell lines, GI50 ¼ 9.4 ¼ 19.4 mM), 15 (against two cell lines,
GI50 ¼ 15.0e17.7 mM) and 16 (GI50 > 20.0 mM for all cell
lines).
On the other hand, the series of 2-mercaptobenzenesulfona-
mides 21e28 with benzyl or furfuryl substituent at N-3 nitro-
gen atom of oxopyrimidine moiety showed very promising
Analysis of the structureeactivity relationship suggests that
electronic effects of substituents Y affect considerably their

1190
Z. Brzozowski et al. / European Journal of Medicinal Chemistry 43 (2008) 1188e1198
X
N
Cl
S
S
SMe
Cl
S
S
a [25], b
c, d [25]
H
N
N
Me
Me
O O
O O
1
2-9 [25], 10-12
Cl
SH
Cl
SH
H
H
N
N
N
Me
S
X
Me
S
Y
O O
O O
HN
NH2
13-20 [25]
Compd
X
Y
N
O
2, 13
3, 14
4, 15
N
H2N
O
N
OMe
OMe
OMe
OMe
OMe
O
O
N
H2N
O
N
OMe
OMe
S
S
N
H2N
O
N
O
O
5, 16
6, 17
7, 18
8, 19
N
S
OMe
S
H2N
O
N
N
S
OMe
S
S
H2N
O
N
O
Me
EtO
EtO
N
H2N
M
O
N
S
S
O
S
Me
Me
Me
N
H2N
Me
O
N
N
N
NH
NH
9,20
N
O
H2N
O
OEt
O
O
O
10
S
Cl
OMe
11
S
OMe
12
S
S
OMe
Scheme 1. Synthesis of alkyl o-(6-chloro-7-methy-1,1-dioxo-1,4,2-benzodithiazin-3-ylamino)arylcarboxylates 2e12 and 4-chloro-2-mercapto-5-methylbenzene-
sulfonamides 13e20. Reagents, conditions and yields: (a, for 9, 10) alkyl o-aminoaryl carboxylate (1 molar equiv), pyridine (1.33 molar equiv), toluene, reflux,
55e91% (1e9); (b) 3-amino-5-(phenyl-, 4-chlorophenyl- or 3-thienyl)thiophene-2-carboxylate (1 molar equiv), pyridine (1.33 molar equiv), toluene, reflux 45e
60 h, 44e72%; (c, for 13e17) hydrazine hydrate (3.4 molar equiv), methanol, room temperature, 60e64 h, 90e96%; (d, for 18e20) hydrazine hydrate (2.2 molar
equiv), toluene, room temperature 12 h, reflux 8 h, 41e81%.

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 43 (2008) 1188e1198
1191
X
N
Ar
H3N
Cl
S
S
Cl
S
S
a
H
H
N
N
N
Me
Me
X
Ar = Ph or 2-furyl
O O
O O
HN
Ar
2, 4-6, 10-12
Cl
SH
H2N
Ar
b
Cl
SH
H
S
O O
H
N
N
Me
S
Y
Me
Y
O O
21-28
Compd
X
Y
N
O
O
2, 21
N
N
O
O
N
OMe
OMe
S
S
4, 22
O
N
O
O
5, 23
5, 24
6, 25
N
N
N
S
S
OMe
OMe
O
N
S
S
S
S
S
O
O
N
O
O
OMe
O
N
10, 26
11, 27
12, 28
N
N
N
S
O
N
OMe
OMe
O
O
Cl
S
S
S
S
Cl
O
N
S
S
O
O
OMe
Scheme 2. Synthesis of 4-chloro-2-mercapto-5-methylbenzenesulfonamides 21e28. Reagents, conditions and yields: (a) benzyl or furfurylamine (2.3 molar equiv),
toluene, reflux 6 h; (b) glacial acetic acid, reflux 3 min, 67e83%.
cytotoxic potency, specificity and selectivity. Thus, compound
13 (Y ¼ 3-amino-3,4-dihydro-4-oxoquinazolin-2-yl) exhibited
the highest inhibitory activity (GI50 ¼ 7.2e19.7 mM) against
six cell lines derived from three different human cancer types
(Table 2). Replacement of substituent Y in compound 13 by
even stronger electron-withdrawing 3-amino-3,4-dihydro-4-
oxothieno[3,4-e]pyrimidin-2-yl moiety yielded analogue 15
with antitumor activity limited to SF-268 and SNB-75 cell
lines of CNS cancer (GI50 ¼ 15.0e17.7 mM). Similarly,
replacement of Y ¼ 3-benzyl-3,4-dihydro-4-oxothieno[2,3-e]
pyrimidin-2-yl in one of the most active 23 (nine cell lines
from four different human cancer types, GI50 ¼ 14.2e
19.9 mM) by stronger electron-withdrawing substituents
(Y ¼ 3-furfuryl-3,4-dihydro-4-oxothieno[2,3-e]pyrimidin-2-yl
or 3-benzyl-3,4-dihydro-4-oxo-6-phenylthieno[2,3-e]pyrimi-
din-2-yl) resulted in distinguished decrease of cytotoxic
potency (24: two cell lines from leukemia) till the complete
loss of activity in case of 26 (Table 2).

1192
Z. Brzozowski et al. / European Journal of Medicinal Chemistry 43 (2008) 1188e1198
Ph
Ph
Ph
+ MeCOOK
Cl
S
S
Cl
S
S
Cl
S
S
a
H
N
H
K
N
H
N
N
R
Ar
R
R
Y
N
O O
O O
O O
NH
29-34
35-43
b (R = Me)
Ph
S
Ph
+ MeCOOK
Cl
S
S
Cl
Me
N
Me
N
H
N
H
N
Me
Me
S
O O
O O
NH
N
O
OMe
O
44
Compd
29, 35
R
Ar
Y
N
O
Me
HN
HN
OMe
O
N
Cl
Cl
29, 36
Me
O
O
O
OMe
OMe
OMe
O
N
H2NCO
PhNHCO
30, 37
HN
HN
O
N
31, 38
O
N
O
O
32, 39
4-MePhNHCO
HN
HN
OMe
OMe
O
N
33, 40
4-MeOPhNHCO
O
N
O
34, 41
29, 42
29, 43
4-ClPhNHCO
HN
HN
HN
OMe
OMe
O
N
S
S
Me
Me
O
O
O
N
S
S
OMe
O
Scheme 3. Synthesis of 2-benzylthio-4-chlorobenzenesulfonamides 35e44. Reagents, conditions and yields: (a) methyl o-aminoaryl carboxylate (1.0 molar equiv),
glacial acetic acid, reflux, 15 h, 18e63%; (b) methyl 2-methylaminobenzoate (1.0 molar equiv), glacial acetic acid, reflux, 15 h, 43%.

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 43 (2008) 1188e1198
1193
Table 1
Anti-HIV integrase activity of novel 4-chloro-2-mercaptobenzenesulfonamide
4.1.1. General procedure for the preparation of methyl
3-(6-chloro-7-methyl-1,1-dioxo-1,4,2-benzodithiazin-
3-ylamino)thiophene-2-carboxylates (10e12)
derivatives 13e28 and 35e44 and the reference 4-chloro-N-(3-amino-1H-
1,2,4-triazol-5-yl)-2-mercapto-5-methylbenzenesulfoamide (NSC 661073)
[24]
To a solution of 6-chloro-7-methyl-3-methylthio-1,4,2-ben-
zodithiazine 1,1-dioxide 1 (4.4 g, 0.015 mol) and the appro-
priate methyl 3-aminothiophene-2-carboxylate (0.015 mol) in
dry toluene (15 ml) was added anhydrous pyridine (1.6 g,
0.02 mol). The reaction mixture was refluxed with stirring un-
til the evolution of MeSH had ceased (45e60 h) (CAUTION:
because of high toxicity, MeSH should be trapped in an
aqueous NaOH solution). After cooling to room temperature
the suspension was left overnight. The precipitate was col-
lected by filtration, washed successively with toluene
(4 ꢁ 2.5 ml) and methanol (4 ꢁ 2.5 ml), dried and recrystal-
lized from DMF.
a
IC50 (mM)
Compd
30-procesing
Strand transfer
13
14
>100
>100
69 ꢃ 6
65 ꢃ 2
60 ꢃ 12
>100
55 ꢃ 5
>100
78 ꢃ 15
80 ꢃ 35
53 ꢃ 7
62 ꢃ 4
22 ꢃ 11
9 ꢃ 2
>100
>100
15
16
43 ꢃ 2
53 ꢃ 3
35 ꢃ 2
81 ꢃ 9
39 ꢃ 2
>100
17
18
19
20
21
22
42 ꢃ 8
31 ꢃ 10
29 ꢃ 13
53 ꢃ 3
4 ꢃ 1
In this manner, the following carboxylates were obtained.
23
24
25
26
4.1.1.1. Methyl 3-(6-chloro-7-methyl-1,1-dioxo-1,4,2-benzodi-
thiazin-3-ylamino)-5-phenylthiophene-2-carboxylate (10). Start-
ing from methyl 3-amino-5-phenylthiophene-2-carboxylate
(3.5 g), the title compound 10 was obtained (5.2 g, 72%);
m.p. 261e262 ꢂC. IR (KBr) 3210 (NH), 1675 (C]O), 1325,
6 ꢃ 1
27
28
6 ꢃ 3
4 ꢃ 2
8 ꢃ 2
6 ꢃ 2
35
36
>100
47 ꢃ 10
>100
73 ꢃ 28
>100
>100
100
83 ꢃ 30
33 ꢃ 14
>100
1165 (SO2) cmꢀ1 1H NMR (DMSO-d6) d 2.44 (s, 3H,
;
37
38
36 ꢃ 3
77 ꢃ 23
93 ꢃ 7
52 ꢃ 27
78 ꢃ 6
70 ꢃ 9
90 ꢃ 10
4.90
CH3), 3.82 (s, 3H, CH3O), 7.47e7.51 (m, 3H, Ph), 7.73e
7.78 (m, 2H, Ph), 7.84 (s, 1H, H-5, benzodithiazine), 8.03
(s, 1H, H-8, benzodithiazine), 8.05 (s, 1H, H-4, thiophene),
11.42 (s, 1H, NH) ppm. Anal. Calcd. for C20H15ClN2O4S3
(478.98): C, 50.14; H, 3.15; N, 5.85. Found: C, 50.10; H,
3.19; N, 6.01.
39
40
41
42
>100
>100
>100
4.79
43
44
NSC 661073
a
IC50: Inhibitory concentration 50% (inhibition of purified integrase).
4.1.1.2. Methyl 3-(6-chloro-7-methyl-1,1-dioxo-1,4,2-benzodi-
thiazin-3-ylamino)-5-(4-chlorophenyl)thiophene-2-carboxylate
(11). Starting from methyl 3-amino-5-(4-chlorophenyl)thio-
phene-2-carboxylate (4.02 g), the title compound 11 was
obtained (5.4 g, 70%); m.p. 280e282 ꢂC dec. IR (KBr)
3. Conclusions
3205 (NH), 1675 (C]O), 1325, 1160 (SO2) cmꢀ1
;
1H
We have developed methods for the synthesis of various 4-
chloro-N-(4-oxopyrimidin-2-yl)-2-mercaptobenzenesulfonamide
derivatives containing the pyrimidine moiety fused with an
aromatic or heteroaromatic ring. Some of these sulfonamides
depending on their structure exhibited either HIV-1 integrase
inhibitory activity (25e28) or cytotoxic activity (13, 15 and
21e24). Moreover, the sulfonamide 21 acting as potent inhib-
itor toward CNS cancer SNB-19 cell line (GI50 < 0.01 mM)
may serve as useful lead compound for the search of more
powerful selective antineoplastic agents.
NMR (CDCl3) d 2.49 (s, 3H, CH3), 3.96 (s, 3H, CH3O),
7.44 (d, J ¼ 8.7 Hz, 2H, 4-ClPh), 7.49 (s, 1H, H-5, benzodi-
thiazine), 7.62 (d, J ¼ 8.7 Hz, 2H, 4-ClPh), 8.07 (s, 1H, H-8,
benzdithiazine), 8.42 (s, 1H, H-4, thiophene), 10.83 (s, 1H,
NH) ppm; 13C NMR (CDCl3) d 22.09, 54.58, 113.30,
120.72, 128.78, 129.60, 129.72, 129.80, 131.44, 131.92,
132.93, 127.79, 140.61, 140.91, 145.36, 151.03, 161.37,
166.87 ppm. Anal. Calcd. for C20H14Cl2N2O4S3 (513.43):
C, 46.78; H, 2.75; N, 5.45. Found: C, 46.71; H, 2.86;
N, 5.40.
4. Experimental protocols
4.1.1.3. Methyl 3-(6-chloro-7-methyl-1,1-dioxo-1,4,2-benzodithia-
zin-3-ylamino)-5-(3-thienyl)thiophene-2-carboxylate (12). Starting
from methyl 3-amino-5-(3-thienyl)thiophene-2-carboxylate (3.6 g),
the title compound 12 was obtained (3.2 g, 44%); m.p. 245e
247 ꢂC. IR (KBr) 3215 (NH), 1670 (C]O), 1325, 1160
4.1. Synthesis
The following instruments and parameters were used:
1
(melting points) Buchi 535 apparatus; (IR spectra) KBr pel-
¨
(SO2) cmꢀ1; H NMR (CDCl3) d 2.49 (s, 3H, CH3), 3.94 (s,
lets, 400e4000 cmꢀ1 PerkineElmer 1600 FTIR spectropho-
tometer; (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).
3H, CH3O), 7.36e7.44 (m, 2H, 3-thienyl), 7.49 (s, 1H, H-5,
benzodithiazine), 7.64 (dd, J2e4 ¼ 1.3 Hz, J2e5 ¼ 2.7 Hz, 1H, H-
2, 3-thienyl), 8.07 (s, 1H, H-8, benzodithiazine), 8.32 (s, 1H, H-
thiophene), 10.86 (s, 1H, NH) ppm. Anal. Calcd. for

1194
Z. Brzozowski et al. / European Journal of Medicinal Chemistry 43 (2008) 1188e1198
Table 2
Inhibition of in vitro human cancer cell lines growth by 4-chloro-2-mercaptobenzenesulfonamide derivatives 13, 15, 16, 21e24 and 26e28a
Compd
Number of the cell lines
Most sensitive cell lines (GI50 < 20.0 mM)
TGIc (mM)
b
GI50 (mM)
Investigated Giving positive GI50 and TGI
Panel
Cell line
TGIc (mM)
b
GI50 (mM)
No
51
Range
No
10
Range
13
53
7.2e71.1
41.1e92.7
Non-small cell lung cancer
CNS cancer
EKVX
NCI-H522
SF-268
14.8
19.0
7.2
29.2
50.8
40.1
47.2
32.7
49.2
SNB-19
SNB-75
UACC-62
15.7
13.3
19.7
Melanoma
15
54
48
8.1e96.6
6
48.5e90.4
CNS cancer
SF-268
SNB-75
17.7
15.0
68.1
58.5
16
21
53
57
29
53
31.3e94.9
1
8
88.0
e
e
e
e
<0.01e99.8
40.7e88.8
Leukemia
MOLT-4
SR
18.6
19.5
<0.01
16.1
15.7
15.0
70.6
40.7
88.8
59.3
51.9
81.4
CNS cancer
Melanoma
Breast cancer
SNB-19
LOXIMVI
BT-549
T-47D
22
56
55
57
51
53
14
3.9e81.6
14.2e87.6
9.9e99.5
8
25
2
32.5e90.8
34.2e98.7
29.7e84.2
Leukemia
CCRF-CEM
MOLT-4
HOP-62
14.0
3.9
47.6
32.5
39.3
45.9
68.5
74.7
51.6
54.5
40.8
Non-small cell lung cancer
Colon cancer
16.5
18.2
18.9
19.9
18.2
16.6
4.5
NCI-H322 M
HCC-2998
KM 12
Melanoma
Renal cancer
Breast cancer
LOXIMVI
SN12C
T-47D
23
Non-small cell lung cancer
Colon cancer
HOP-62
19.7
19.5
19.2
19.9
19.9
19.8
12.5
19.5
14.2
46.6
48.8
37.7
48.2
56.1
49.6
34.2
44.4
49.3
NCI-H322 M
HCT-116
KM 12
Melanoma
MALME-3M
M14
SK-MEL-5
UACC-62
A498
Renal cancer
Leukemia
24
MOLT-4
SR
19.4
9.9
84.2
29.7
a
Data obtained from the in vitro cancer cell lines screen performed at the NCI [28e30]. Compounds 26e28 were inactive (GI50 > 100.0 mM).
GI50: molar concentration that inhibits 50% net cell growth.
TGI: molar concentration giving total growth inhibition.
b
c
C18H13ClN2O4S4 (485.03): C, 44.57; H, 2.70; N, 5.77. Found: C,
44.62; H, 2.79; N, 5.81.
for 3 min. After cooling to room temperature, the precipitate
of the adequate sulfonamide obtained was collected by filtra-
tion, washed successively with acetic acid (2 ꢁ 2 ml) and
toluene (4 ꢁ 2 ml), and dried at temperature gradually
increasing to 90 ꢂC.
4.1.2. General procedure for the preparation of 4-chloro-
N-(3,4-dihydro-4-oxopyrimidin-2-yl)-2-mercapto-5-
methylbenzenesulfonamide derivatives (21e28)
4.1.2.1. 4-Chloro-N-(3-furfuryl-3,4-dihydro-4-oxoquinazolin-
2-yl)-2-mercapto-5-methylbenzenesulfonamide (21). Starting
from methyl 2-(6-chloro-7-methyl-1,1-dioxo-1,4,2-benzodi-
thiazin-3-ylamino)benzoate 2 (1.98 g) and furfurylamine,
the title compound 21 was obtained (1.7 g, 74%); m.p.
169e170 ꢂC. IR (KBr) 3275 (NH), 2540 (SH), 1710
To a solution of benzylamine (1.24 g, 0.0115 mol) or fur-
furylamine (1.12 g, 0.0115 mol) in dry toluene (30 ml), the
corresponding methyl o-(1,4,2-benzodithiazin-3-ylamino)aryl-
carboxylate 2, 4, 5, 6, 10, 11 or 12 (0.005 mol) was added and
stirred at reflux for 6 h. After cooling to room temperature, the
aminium salt of the appropriate product that precipitated was
separated by suction, washed with toluene (2 ꢁ 2 ml) and
immediately acidified in boiling glacial acetic acid (30 ml)
1
(C]O), 1350, 1160 (SO2) cmꢀ1; H NMR (CDCl3) d 2.39
(s, 3H, CH3), 4.39 (s, 1H, SH), 5.43 (s, 2H, CH2), 6.19

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 43 (2008) 1188e1198
1195
(s, 2H, furan), 7.28 (s, 1H, furan), 7.36 (t, J ¼ 7.8 Hz, 2H,
4.1.2.5.
N-(3-Benzyl-6-(tert-butyl)-3,4-dihydro-4-oxothieno
quinazoline), 7.39 (s, 1H, H-3, PhSO2), 7.71 (t, J ¼ 7.8 Hz,
1H, quinazoline), 7.97 (s, 1H, H-6, PhSO2), 8.21 (d,
J ¼ 8.3 Hz, 1H, quinazoline), 10.89 (s, 1H, NH) ppm; 13C
NMR (CDCl3) d 19.50, 38.31, 109.87, 110.31, 15.28,
115.97, 125.23, 128.63, 130.66, 130.74, 131.82, 133.79,
135.79, 136.55, 137.37, 138.47, 142.15, 148.75, 148.93,
159.95 ppm. Anal. Calcd. for C20H16ClN3O4S2 (461.92):
C, 52.00; H, 3.49; N, 9.09. Found: C, 51.98; H, 3.53; N,
9.12.
[2,3-e]pyrimidin-2-yl)-4-chloro-2-mercapto-5-methylbenzene-
sulfonamide (25). Starting from 5-(tert-butyl)-3-(6-chloro-7-
methyl-1,1-dioxo-1,4,2-benzodithiazin-3-ylamino)thiophene-
2-carboxylate 6 (2.29 g) and benzylamine, the title compound
25 was obtained (2.0 g, 75%); m.p. 216e217 ꢂC dec. IR (KBr)
3270 (NH), 2560 (SH), 1690 (C]O), 1360, 1340, 1160
(SO2) cmꢀ1 1H NMR (CDCl3) d 1.41 (s, 9H, tert-butyl),
;
2.36 (s, 3H, CH3-5, PhSO2), 4.13 (s, 1H, SH), 5.27 (s, 2H,
CH2), 6.79 (s, 1H, H-3, PhSO2), 7.03e7.26 (m, 5H, Ph),
7.30 (s, 1H, H-6, PhSO2), 7.88 (s, 1H, H-7, thienopyrimidine),
11.18 (s, 1H, NH) ppm; 13C NMR (CDCl3) d 21.54, 33.86,
37.86, 47.25, 114.04, 129.58, 130.12, 131.02, 132.41,
132.66, 134.04, 135.83, 137.98, 139.58, 140.32, 143.87,
144.02, 151.93, 158.47, 172.66 ppm. Anal. Calcd. for
C24H24ClN3O3S3 (534.11): C, 53.96; H, 4.53; N, 7.86. Found:
C, 53.87; H, 4.68; N, 7.88.
4.1.2.2. N-(3-Benzyl-3,4-dihydro-4-oxothieno[3,4-e]pyrimidin-
2-yl)-4-chloro-2-mercapto-5-methylbenzenesulfonamide (22).
Starting from 3-(6-chloro-7-methyl-1,1-dioxo-1,4,2-benzodi-
thiazin-3-ylamino)thiophene-4-carboxylate
4 (2.01 g) and
benzylamine, the title compound 22 was obtained (1.6 g,
67%); m.p. 168e170 ꢂC dec. IR (KBr) 3270 (NH), 2545
1
(SH), 1690 (C]O), 1370, 1340, 1140 (SO2) cmꢀ1; H NMR
(CDCl3) d 2.37 (s, 3H, CH3), 4.19 (s, 1H, SH), 5.26 (s, 2H,
CH2), 6.95 (d, J ¼ 3.3 Hz, 1H, H-7, thienopyrimidine),
7.05e7.29 (m, 5H, Ph), 7.31 (s, 1H, H-3, PhSO2), 7.89 (s,
1H, H-6, PhSO2), 8.29 (d, J ¼ 3.3 Hz, 1H, H-5, thienopyrimi-
dine), 10.88 (s, 1H, NH) ppm. Anal. Calcd. for
C20H16ClN3O3S3 (477.99): C, 50.25; H, 3.37; N, 8.79. Found:
C, 50.19; H, 3.44; N, 8.73.
4.1.2.6. N-(3-Benzyl-3,4-dihydro-4-oxo-6-phenylthieno[2,3-e]
pyrimidin-2-yl)-4-chloro-2-mercapto-5-methylbenzenesulfona-
mide (26). Starting from 3-(6-chloro-7-methyl-1,1-dioxo-1,4,
2-benzodithiazin-3-ylamino)-5-phenylthiophene-2-carboxylate
10 (2.39 g) and benzylamine, the title compound 26 was ob-
tained (2.3 g, 83%); m.p. 267e268 ꢂC dec. IR (KBr) 3290
(NH), 2555 (SH), 1675 (C]O), 1355, 1340, 1155
1
(SO2) cmꢀ1; H NMR (CDCl3) d 2.38 (s, 3H, CH3), 4.12 (s,
4.1.2.3. N-(3-Benzyl-3,4-dihydro-4-oxothieno[2,3-e]pyrimidin-
2-yl)-4-chloro-2-mercapto-5-methylbenzenesulfonamide (23).
Starting from 3-(6-chloro-7-methyl-1,1-dioxo-1,4,2-benzodi-
1H, SH), 5.30 (s, 2H, CH2), 7.11e748 (m, 10H, Ph-2 and
PhCH2), 7.64 (s, 1H, H-3, PhSO2), 7.66 (s, 1H, H-6,
PhSO2), 7.91 (s, 1H, H-7, thienopyrimidine), 11.29 (s, 1H,
NH) ppm. Anal. Calcd. for C26H20ClN3O3S3 (554.08): C,
56.35; H, 3.64; N, 7.58. Found: C, 56.42; H, 3.71; N, 7.51.
thiazin-3-ylamino)thiophene-2-carboxylate
5 (2.01 g) and
benzylamine, the title compound 23 was obtained (1.8 g,
75%); m.p. 193e195 ꢂC dec. IR (KBr) 3245 (NH), 2548
1
(SH), 1680 (C]O), 1355, 1335, 1145 (SO2) cmꢀ1; H NMR
4.1.2.7.
N-[3-Benzyl-6-(4-chlorophenyl)-3,4-dihydro-4-oxo-
(CDCl3) d 2.36 (s, 3H, CH3), 4.14 (s, 1H, SH), 5.29 (s, 2H,
CH2), 7.03 (d, J ¼ 5.2 Hz, 1H, H-7, thienopyridine), 7.06e
7.32 (m, 6H, Ph and H-3, PhSO2), 7.79 (d, J ¼ 5.2 Hz, 1H,
H-6, thienopyridine), 7.89 (s, 1H, H-6, PhSO2), 11.30 (s,
1H, NH) ppm; 13C NMR (CDCl3) d 19.46, 45.32, 117.03,
127.62, 128.10, 128.88, 129.01, 130.37, 130.62, 131.95,
133.81, 135.69, 136.57, 137.30, 138.38, 141.91, 149.80,
156.55 ppm. Anal. Calcd. for C20H16ClN3O3S3 (477.99): C,
50.25; H, 3.37; N, 8.79. Found: C, 50.22; H, 3.36; N, 8.84.
thieno[2,3-e]pyrimidin-2-yl]-4-chloro-2-mercapto-5-methyl-
benzenesulfonamide (27). Starting from 3-(6-chloro-7-methyl-1,
1-dioxo-1,4,2-benzodithiazin-3-ylamino)-5-(4-chlorophenyl)-
thiophene-2-carboxylate 11 (2.57 g) and benzylamine, the title
compound 27 was obtained (2.4 g, 81%); m.p. 267e268 ꢂC
dec. IR (KBr) 3280 (NH), 2560 (SH), 1700 (C]O), 1350,
1165 (SO2) cmꢀ1 1H NMR (CDCl3) d 2.37 (s, 3H, CH3),
;
4.09 (s, 1H, SH), 5.29 (s, 2H, CH2), 7.08e7.20 (m, 5H, Ph),
7.27 (s, 1H, H-3, PhSO2), 7.32 (s, 1H, H-6, PhSO2), 7.45 (d,
J ¼ 8.1 Hz, 2H, 4-ClPh), 7.58 (d, J ¼ 8.1 Hz, 2H, 4-ClPh),
7.91 (s, 1H, H-7, thienopyrimidine), 11.29 (s, 1H, NH) ppm.
Anal. Calcd. for C26H19Cl2N3O3S3 (588.52): C, 53.05; H,
3.25; N, 7.14. Found: C, 53.14; H, 3.28; N, 7.07.
4.1.2.4. 4-Chloro-N-(3-furfuryl-3,4-dihydro-4-oxothieno[2,3-e]-
pyrimidin-2-yl)-2-mercapto-5-methylbenzenesulfonamide (24).
Starting from 3-(6-chloro-7-methyl-1,1-dioxo-1,4,2-benzodi-
thiazin-3-ylamino)thiophene-2-carboxylate 5 (2.01 g) and fur-
furylamine, the title compound 24 was obtained (1.9 g, 82%);
m.p. 212e213 ꢂC dec. IR (KBr) 3270 (NH), 2545 (SH), 1690
4.1.2.8. 4-Chloro-N-[3-furfuryl-3,4-dihydro-4-oxo-6-(3-thie-
nyl)thieno[2,3-e]pyrimidin-2-yl]-2-mercapto-5-methylbenzene-
sulfonamide (28). Starting from 3-(6-chloro-7-methyl-1,
1-dioxo-1,4,2-benzodithiazin-3-ylamino)-5-(3-thienyl)thiophene-
2-carboxylate 12 (2.42 g) and furfurylamine, the title com-
pound 28 was obtained (2.1 g, 76%); m.p. 259e261 ꢂC. IR
(KBr) 3250 (NH), 2552 (SH), 1670 (C]O), 1350, 1320,
(C]O), 1355, 1340, 1150 (SO2) cmꢀ1 1H NMR (CDCl3)
;
d 2.39 (s, 3H, CH3), 4.35 (s, 1H, SH), 5.32 (s, 2H, CH2),
6.18 (s, 2H, H-3 and H-4, furan), 7.03 (d, J ¼ 5.0 Hz, 1H,
H-7, thienopyrimidine), 7.28 (s, 1H, H-5, furan), 7.39 (s, 1H,
H-3, PhSO2), 7.81 (d, J ¼ 5.0 Hz, 1H, H-6, thienopyrimidine),
7.96 (s, 1H, H-6, PhSO2), 11.29 (s, 1H, NH) ppm. Anal. Calcd.
for C18H14ClN3O4S3 (467.98): C, 46.19; H, 3.02; N, 8.98.
Found: C, 46.27; H, 3.20; N, 9.07.
1
1155 (SO2) cmꢀ1; H NMR (DMSO-d6) d 2.32 (s, 3H, CH3),
3.50 (s, 1H, SH), 5.16 (s, 2H, CH2), 6.08 (d, J ¼ 3.2 Hz, 1H,
H-3, furan), 6.24 (d, J ¼ 3.2 Hz, 1H, H-4, furan), 7.44

1196
Z. Brzozowski et al. / European Journal of Medicinal Chemistry 43 (2008) 1188e1198
(s, 1H, H-3, PhSO2), 7.50e7.77 (m, 5H, arom.), 7.89 (s, 1H,
H-7, thienopyrimidine), 8.13 (s, 1H, NH) ppm. Anal. Calcd.
for C22H16ClN3O4S4 (550.08): C, 48.03; H, 2.93; N, 7.64.
Found: C, 48.00; H, 3.11; N, 7.72.
m.p. 310e311 ꢂC dec. IR (KBr) 3371, 3195 (NH), 1710
(C]O), 1685 (C]O), 1307, 1147 (SO2) cmꢀ1 1H NMR
;
(DMSO-d6) d 4.38 (s, 2H, SCH2), 7.06e7.09 (m, 2H, He
Ar), 7.12e7.14 (m, 1H, HeAr), 7.18e7.21 (m, 2H, HeAr),
7.35e7.39 (m, 1H, HeAr), 7.52e7.53 (m, 1H, HeAr), 7.69
(s, 1H, H-3, PhSO2), 7.73 (s, 1H, CONHa), 7.76e7.79 (m,
1H, HeAr), 7.98 (br s, 2H, CONHb, HeAr), 8.03 (s, 1H,
H-6, PhSO2), 11.39 (s, 1H, NH), 11.86 (s, 1H, SO2NH) ppm;
13C NMR (DMSO-d6) d 36.40, 107.38, 115.83, 117.69,
124.98, 127.15, 127.58, 128.51, 128.61, 129.00, 133.28,
134.06, 135.94, 136.02, 138.55, 139.55, 139.56, 149.57,
160.12, 166.88 ppm. Anal. Calcd. for C22H17ClN4O4S2
(500.99): C, 52.74; H, 3.42; N, 11.18. Found: C, 52.88; H,
3.32; N, 10.91.
4.1.3. General procedure for the preparation of
2-benzylthio-4-chloro-N-(3,4-dihydro-4-oxopyrimidin-
2-yl)benzenesulfonamide derivatives (35e43)
A suspension of the corresponding N-(2-benylthio-4-chlor-
obenzenesulfonyl)cyanamide potassium salt 29e34 (3 mmol)
and appropriate methyl o-aminoaryl carboxylate (3 mmol) in
glacial acetic acid (15 ml) was refluxed with stirring for 15e
20 h. After cooling the reaction mixture was left to stand over-
night at room temperature. The precipitate was collected by
filtration, washed with glacial acetic acid (2 ꢁ 0.5 ml) and
dried. The crude reaction product was purified by recrystalli-
zation from ethanol.
4.1.3.4. 4-Benzylothio-2-chloro-5-(3,4-dihydro-4-oxoquinazolin-
2-ylaminosulfonyl)-N-(phenyl)benzamide (38). Starting from
cyanamide potassium salt 31 (1.46 g) and methyl 2-aminoben-
zoate (0.45 g) the title compound 38 was obtained (0.8 g,
47%); m.p. 282e283 ꢂC. IR (KBr) 3289, 3236 (NH), 1726
In this manner, the following compounds were obtained.
4.1.3.1. 2-Benzylthio-4-chloro-N-(3,4-dihydro-4-oxoquinazo-
lin-2-yl)-5-methylbenzenesulfonamide (35). Starting from
cyanamide potassium salt 29 (1.17 g) and methyl 2-aminoben-
zoate (0.45 g), the title compound 35 was obtained (0.6 g,
46%); m.p. 191e192 ꢂC. IR (KBr) 3283, 3227 (NH), 1701
(C]O), 1684 (C]O), 1303, 1143 (SO2) cmꢀ1 1H NMR
;
(DMSO-d6) d 4.47 (s, 2H, SeCH2), 7.17e7.27 (m, 5H, He
Ar), 7.37e7.44 (m, 3H, HeAr), 7.59e7.62 (m, 2H, HeAr),
7.72e7.75 (m, 3H, HeAr), 7.83 (s, 1H, H-3, PhSO2), 8.02e
8.05 (m, 1H, HeAr), 8.15 (s, 1H, H-6, PhSO2), 10.66 (s,
1H, CONH), 11.50 (s, 1H, NH), 11.88 (s, 1H, SO2NH) ppm.
Anal. Calcd. for C28H21ClN4O4S2 (577.09): C, 58.27; H,
3.67; N, 9.70. Found: C, 58.32; H, 3.75; N, 9.57.
(C]O), 1302, 1130 (SO2) cmꢀ1
;
1H NMR (DMSO-d6)
d 2.35 (s, 3H, CH3), 4.30 (s, 2H, SCH2), 6.99e7.17 (m, 5H,
Ph), 7.38 (t, 1H, H-6, quinazoline), 7.51 (d, J ¼ 8.3 Hz, 1H,
H-8, quinazoline), 7.65 (s, 1H, H-3, PhSO2), 7.78 (t,
J ¼ 8.3 Hz, 1H, H-7, quinazoline), 7.95 (s, 1H, H-6, PhSO2),
7.96e8.00 (m, 1H, H-5, quinazoline), 11.33 (s, 1H, NH),
11.86 (s, 1H, SO2NH) ppm; 13C NMR (DMSO-d6) d 19.25,
36.98, 115.68, 117.46, 124.94, 127.18, 127.45, 128.51,
128.96, 129.17, 130.61, 133.26, 135.64, 136.07, 136.22,
137.61, 139.16, 139.43, 149.38, 159.95 ppm. Anal. Calcd.
for C22H18ClN3O3S2 (471.99): C, 55.98; H, 3.84; N, 8.90.
Found: C, 56.08; H, 3.98; N, 8.92.
4.1.3.5. 4-Benzylothio-2-chloro-5-(3,4-dihydro-4-oxoquinazolin-
2-ylaminosulfonyl)-N-(4-methylphenyl)benzamide (39). Starting
from cyanamide potassium salt 32 (1.53 g) and methyl 2-amino-
benzoate (0.45 g), the title compound 39 was obtained (0.8 g,
46%); m.p. 271e274 ꢂC. IR (KBr) 3278, 3242 (NH), 1726
(C]O), 1685 (C]O), 1303, 1143 (SO2) cmꢀ1 1H NMR
;
(DMSO-d6) d 2.29 (s, 3H, CH3), 4.44 (s, 2H, SCH2), 7.15e
7.24 (m, 7H, HeAr), 7.36e7.44 (m, 2H, HeAr), 7.57e7.60
(m, 3H, HeAr), 7.79 (m, 1H, H-3, PhSO2), 7.99e8.02 (m, 1H,
HeAr), 8.11 (s, 1H, H-6, PhSO2), 10.55 (s, 1H, CONH), 11.46
(s, 1H, NH), 11.86 (s, 1H, SO2NH) ppm; 13C NMR (DMSO-
d6) d 20.81, 36.36, 115.86, 117.72, 120.00, 125.05, 127.15,
127.61, 128.64, 128.80, 129.02, 129.48, 133.16, 133.35,
134.34, 135.97, 136.10, 136.44, 138.66, 139.47, 140.49,
149.56, 160.12, 163.41 ppm. Anal. Calcd. for C29H23ClN4O4S2
(591.11): C, 58.93; H, 3.92; N, 9.48. Found: C, 59.03; H, 3.99;
N, 9.62.
4.1.3.2. 2-Benzylthio-4-chloro-N-(7-chloro-3,4-dihydro-4-oxo-
quinazolin-2-yl)-5-methylbenzenesulfonamide (36). Starting
from cyanamide potassium salt 29 (1.17 g) and methyl 2-
amino-4-chlorobenzoate (0.56 g), the title compound 36 was
obtained (0.3 g, 18%); m.p. 230e232 ꢂC. IR (KBr) 3304,
1
3265, 3135 (NH), 1692 (C]O), 1306, 1130 (SO2) cmꢀ1; H
NMR (DMSO-d6) d 2.39 (s, 3H, CH3), 4.34 (s, 2H, SCH2),
7.07e7.23 (m, 5H, Ph), 7.42e7.47 (m, 1H, quinazoline),
7.68 (s, 2H, H-3 and H-6, PhSO2), 7.97e8.02 (m, 2H, quina-
zoline), 11.52 (s, 1H, NH), 11.83 (s, 1H, SO2NH) ppm; 13C
NMR (DMSO-d6) d 19.26, 37.04, 114.68, 117.09, 125.08,
127.49, 128.52, 129.01, 129.18, 130.63, 133.28, 135.80,
136.21, 137.72, 138.96, 140.39, 140.48, 149.49, 159.46 ppm.
Anal. Calcd. for C22H17Cl2N3O3S2 (506.43): C, 52.18; H,
3.38; N, 8.29. Found: C, 52.25; H, 3.44; N, 8.29.
4.1.3.6. 4-Benzylothio-2-chloro-5-(3,4-dihydro-4-oxoquinazolin-
2-ylaminosulfonyl)-N-(4-methoxyphenyl)benzamide (40). Starting
from cyanamide potassium salt 33 (1.58 g) and methyl 2-amino-
benzoate (0.45 g) the title compound 40 was obtained (1.0 g,
55%); m.p. 295e297 ꢂC dec. IR (KBr) 3283, 3242 (NH), 1725
(C]O), 1681 (C]O), 1303, 1143 (SO2) cmꢀ1 1H NMR
;
4.1.3.3. 4-Benzylthio-2-chloro-5-(3,4-dihydro-4-oxoquinazolin-
2-ylaminosulfonyl)benzamide (37). Starting from cyanamide
potassium salt 30 (1.26 g) and methyl 2-aminobenzoate
(0.45 g), the title compound 37 was obtained (0.7 g, 48%);
(DMSO-d6) d 3.74 (s, 3H, OCH3), 4.42 (s, 2H, SCH2), 6.91e
6.95 (m, 2H, HeAr), 7.12e7.22 (m, 5H, HeAr), 7.32e7.48
(m, 1H, HeAr), 7.58e7.61 (m, 3H, HeAr), 7.73 (br s, 2H,
H-3, PhSO2 and HeAr), 7.97e8.01 (m, 1H, HeAr), 8.09

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 43 (2008) 1188e1198
1197
(s, 1H, H-6, PhSO2), 10.47 (s, 1H, CONH), 11.46 (s, 1H, NH),
11.84 (s, 1H, SO2NH) ppm. Anal. Calcd. for C29H23ClN4O5S2
(607.11): C, 57.37; H, 3.82; N, 9.23. Found: C, 57.44; H, 3.90;
N, 9.37.
1H, HeAr), 7.68e7.75 (m, 2H, H-3 PhSO2 and HeAr),
7.88e8.11 (m, 3H, H-6, PhSO2 and 2HeAr), 11.03 (s, 1H,
NH) ppm. Anal. Calcd. for C23H20ClN3O3S2 (486.01): C,
56.84; H, 4.15; N, 8.65. Found: C, 56.95; H, 4.21; N, 8.84.
4.1.3.7. 4-Benzylothio-2-chloro-N-(4-chlorophenyl)-5-(3,4-dihy-
dro-4-oxoquinazolin-2-ylaminosulfonyl)benzamide (41). Starting
from cyanamide potassium salt 41 (1.59 g) and methyl 2-amino-
benzoate (0.45 g), the title compound 41 was obtained (0.7 g,
41%); m.p. 263e264 ꢂC. IR (KBr) 3236 (NH), 1727 (C]O),
4.2. Integrase assay
To determine the extent of 30-processing and strand transfer,
HIV-1 IN was preincubated at a final concentration of 200 nM
with inhibitor in reaction buffer (50 mM NaCl, 1 mM HEPES,
pH 7.5, 50 mM EDTA, 50 mM dithiothretiol, 10% glycerol (w/
v), 7.5 mM MnCl2, 0.1 mg/ml bovine serum albumin, 10 mM
2-mercaptoethanol, 10% DMSO and 25 mM MOPS, pH 7.2)
at 30 ꢂC for 30 min. Then, 20 nM of the 50-end 32P-labeled linear
oligonucleotide substrate was added and incubation was contin-
ued for an additional 1 h. Reactions were quenched by an addi-
tion of (8 ml) loading dye (98% deionized formamide, 10 mM
EDTA, 0.025% xylene cyanol and 0.025% bromophenol
blue). An aliquot (5 ml) was electrophoresed on a denaturing
20% polyacrylamide gel (0.09 M triseborate pH 8.3, 2 mM
EDTA, 20% acrylamide, 8 M urea). Gels were dried, exposed
on a PhosphorImager cassette, read on a Typhoon 8610 Variable
mode Imager (Amersham Biosciences), and quantitated using
ImageQuant 5.2 (Amersham Biosciences).
1
1681 (C]O), 1304, 1143 (SO2) cmꢀ1; H NMR (DMSO-d6)
d 4.46 (s, 2H, SeCH2), 7.14e7.39 (m, 5H, HeAr), 7.43e7.48
(m, 3H, HeAr), 7.57e7.62 (m, 1H, HeAr), 7.73e7.85 (m,
4H, H-3, HeAr), 8.00e8.05 (m, 1H, HeAr), 8.16 (s, 1H, H-6,
PhSO2), 10.79 (s, 1H, CONH), 11.48 (s, 1H, NH), 11.87 (s,
1H, SO2NH) ppm. Anal. Calcd. for C28H20Cl2N4O4S2 (611.53):
C, 54.99; H, 3.30; N, 9.16. Found: C, 55.10; H, 3.36; N, 9.18.
4.1.3.8. 2-Benzylthio-4-chloro-N-(3,4-dihydro-4-oxothieno[3,4-e]
pyrimidin-2-yl)-5-methylbenzenesulfonamide (42). Starting from
cyanamide potassium salt 29 (1.17 g) and methyl 3-aminothio-
phene-4-carboxylate (0.47 g) the title compound 42 was obtained
(0.9 g, 63%); m.p. 237e239 ꢂC dec. IR (KBr) 3294, 3228, 3191
1
(NH), 1692 (C]O), 1363, 1341, 1132 (SO2) cmꢀ1; H NMR
(DMSO-d6) d 2.34 (s, 3H, CH3), 4.30 (s, 2H, SCH2), 7.12e
7.17 (m, 3H, Ph), 7.21e7.23 (m, 2H, Ph), 7.39e7.40 (d,
J ¼ 3.4 Hz, 1H, H-7, thienopyrimidine), 7.61 (s, 1H, H-3,
PhSO2), 7.96 (s, 1H, H-6, PhSO2), 8.50 (d, J ¼ 2.9 Hz, 1H, H-
5, thienopyrimidine), 11.35 (s, 1H, NH), 11.58 (s, 1H,
SO2NH) ppm. Anal. Calcd. for C20H16ClN3O3S3 (487.01): C,
50.25; H, 3.37; N, 8.79. Found: C, 52.40; H, 3.43; N, 8.64.
Acknowledgements
This work was supported by the Polish State Committee for
Scientific Research (Grant No. 2 P05F 035 27). The authors
are very grateful to Dr. V.L. Narayanan, Chief of Drug Synthe-
sis, Chemistry Branch, National Cancer Institute (Bethesda,
MD) for the in vitro screening.
4.1.3.9. 2-Benzylthio-4-chloro-N-(3,4-dihydro-4-oxothieno[2,3-e]
pyrimidin-2-yl)-5-methylbenzenesulfonamide (43). Starting from
cyanamide potassium salt 29 (1.17 g) and methyl 3-aminothio-
phene-2-carboxylate (0.47 g), the title compound 43 was
obtained (0.5 g, 35%); m.p. 212e214 ꢂC. IR (KBr) 3303,
3208, 3173, 3113, 3102 (NH), 1684 (C]O), 1340, 1127, 1105
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