Synthesis and biological evaluation of nitric oxide-donating thalidomide analogues as anticancer agents

Article abstract of DOI:10.1002/cbdv.200800014

In search of more potent anticancer agents, 15 nitric oxide (NO)-donating thalidomide analogues, 6a, 6b, 8a-8e, and 13a-13h, were designed and synthesized. Cytotoxicity of these compounds was evaluated in vitro against three human tumor cell lines (HepG2, A549, and PC-3). The results indicated that 13a-13d exhibited notable anticancer activities comparable to or stronger than that of 5-fluorouracil (5-FU). Structure-activity relationships were also discussed, based on the experimental data obtained. Generally, the cytotoxic activity of target compounds is closely related to the type of NO donors, and the length of the spacers connecting to NO donors also appears important for the bioactivities.

Full text of DOI:10.1002/cbdv.200800014

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Synthesis and Biological Evaluation of Nitric Oxide-Donating Thalidomide
Analogues as Anticancer Agents
by Tao Wanga), Yi-Hua Zhang*a), Xiang-Wen Konga), Yi-Sheng Laia), Hui Ji*b), Yan-Ping Chenb),
and Si-Xun Penga)
a) Center of Drug Discovery, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009,
P. R. China (phone: þ86-25-83271186; fax: þ86-25-86635503; e-mail: zyhtgd@sohu.com)
b) Department of Pharmacology, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009,
P. R. China (e-mail: huijicpu@163.com)
In search of more potent anticancer agents, 15 nitric oxide (NO)-donating thalidomide analogues,
6a, 6b, 8a–8e, and 13a–13h, were designed and synthesized. Cytotoxicity of these compounds was
evaluated in vitro against three human tumor cell lines (HepG2, A549, and PC-3). The results indicated
that 13a–13d exhibited notable anticancer activities comparable to or stronger than that of 5-fluorouracil
(5-FU). Structure–activity relationships were also discussed, based on the experimental data obtained.
Generally, the cytotoxic activity of target compounds is closely related to the type of NO donors, and the
length of the spacers connecting to NO donors also appears important for the bioactivities.
Introduction. – Thalidomide (Fig.) was a sedative and/or hypnotic drug in the 1950s,
which was withdrawn from the market when its teratogenicity was discovered [1].
However, basic research on thalidomide did not come to a halt, and in earlier 1990s
thalidomide proved to be an immunomodulatory agent [2][3]. In response to this,
numerous researches have been carried out, especially for the treatment of tumors
[4–9].
Figure. The structures of thalidomide and NO-ASA
Nitric oxide (NO), naturally synthesized from l-arginine by the action of NO
synthase (NOS), is a key signaling molecule involved in the regulation of many
physiological processes. NO can also be generated from synthetic NO-releasing
compounds, such as nitrate, furoxan, diazeniumdiolate, and others [10][11]. During
recent years, NO-releasing derivatives have currently come into focus on the treatment
of cancer, inflammation, and vascular diseases [12–14]. Among these compounds, NO-
donating aspirin (NO-ASA; Fig.) is a representative drug for the treatment of cancer.
ꢀ 2009 Verlag Helvetica Chimica Acta AG, Zꢁrich

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In studies in vitro, NO-ASA inhibited the growth of colon, prostate, lung, and breast
cancer cell lines 10–6000-fold relative to their parent ASA [15–17].
Hybridization of two bioactive compounds is now accepted as an effective strategy
for designing ligands, inhibitors, and other types of drugs. Our group has implemented
the approach in drug discovery and achieved some promising results with varying
compound classes [18][19]. With these in mind, and also based on the diverse
bioactivities of the above-mentioned thalidomide as well as NO-ASA, we designed and
synthesized 15 NO-donating thalidomide analogues. In this article, we describe the
synthesis, in vitro cytotoxicity, and structure–activity relationships (SAR) of the target
compounds.
Results and Discussion. – 1. Synthesis. Compounds 6a and 6b were prepared
according to the procedures illustrated in Scheme 1. The appropriate (hydroxyme-
thyl)phenols, 1a and 1b, were transformed into the corresponding chloro derivatives 2a
and 2b, respectively, by chlorination with SOCl2 in anhydrous CHCl3. The subsequent
reaction with AgNO3 in MeCN gave the nitrates 3a and 3b, which reacted with acyl
halide of 5 in anhydrous CH2Cl2 to afford 6a and 6b, respectively. Compound 5 was
prepared from phthalic anhydride 4, which was condensed with 4-aminobenzoic acid in
refluxing AcOH to give 5.
Scheme 1
a) SOCl2, CHCl3, 08, 0.5 h. b) AgNO3, MeCN, 708, 12 h. c) 4-Aminobenzoic acid, AcOH, reflux, 3 h. d) 1.
SOCl2, DMF (cat.), reflux, 1 h; 2. 3a,3b, CHCl3, r.t., 2 h.
To investigate the influence of replacing the aryl residue in the NO-donor moiety of
6a and 6b by an alkyl group on anticancer activity, compounds 8a–8e were prepared.
The synthesis of 8a–8e is depicted in Scheme 2. Compound 5 was first treated with
dibromoalkanes bearing two to six C-atoms in the presence of Et3N in acetone to

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
generate 7a–7e in 70–88% yields. The subsequent reaction with AgNO3 in MeCN
produced the nitrates 8a–8e.
Scheme 2
a) Brꢀ(CH2)nꢀBr (n¼2–6), Et3N, reflux, 6 h. b) AgNO3, MeCN, 708, 4 h.
Subsequently, to compare the effects of a furoxan (¼ forazan 2-oxide) with those of
a nitrate on anticancer activity, compounds 13a–13h were synthesized directly from 5
as depicted in Scheme 3.
Scheme 3
a) 1. NaOH (aq.), ClCH2CO2H, 1408, 2 h; 2. 6n HCl. b) 1. 30% H2O2, AcOH, r.t., 3 h; 2. fuming HNO3,
908, 4 h. c) HOꢀXꢀOH, 50% NaOH, THF, r.t., 2 h. d) 5, 1-Ethyl-3-[3-(dimethylamino)propyl]carbo-
diimide hydrochloride (EDCI), 4-(dimethylamino)pyridine (DMAP), CH2Cl2, r.t., 24 h.
The substituted furoxans were prepared in a three-step sequence. The starting
thiophenol (9) was converted to 2-(phenylsulfanyl)acetic acid (10) by treatment with

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
469
ClCH2CO2H. Compound 10 was oxidized with 30% H2O2 solution, followed by
treatment with fuming HNO3 to give bis(phenylsulfonyl)furoxans 11 [20]. The latter
were then converted to various mono(phenylsulfonyl)furoxans 12a–12h by treatment
with corresponding diol. Finally, the resulting furoxans were reacted with 5 to give 13a–
13h.
2. Cytotoxicity Evaluation. The pharmacological results, i.e., the cytotoxic activities
against HepG2 (human hepatoma cells), A549 (human lung carcinoma cells), and
PC-3 (human prostate cancer cells), were summarized in the Table.
Table. Cytotoxic Activity (IC50 [mm]) of Target Compounds against Human Tumor Cell Lines
Compound
HepG2
25.9ꢁ2.5
28.2ꢁ1.6
>100
>100
PC-3
38.5ꢁ1.9
27.8ꢁ0.7
>100
>100
>100
>100
>100
A549
32.6ꢁ1.4
37.5ꢁ1.7
>100
>100
>100
>100
>100
6a
6b
8a
8b
8c
8d
8e
23.9ꢁ2.0
>100
>100
13a
13b
13c
13d
13e
13f
30.3ꢁ1.9
24.8ꢁ2.6
35.8ꢁ1.1
19.6ꢁ1.7
12.8ꢁ2.1
24.9ꢁ1.8
41.9ꢁ2.2
39.5ꢁ2.1
46.3ꢁ1.4
44.6ꢁ1.9
52.9ꢁ1.3
19.8ꢁ1.15
13.5ꢁ1.3
7.2ꢁ2.2
16.8ꢁ1.0
10.4ꢁ1.1
28.6ꢁ2.9
34.2ꢁ0.5
41.9ꢁ2.6
38.1ꢁ2.5
49.2ꢁ0.7
47.5ꢁ1.8
25.3ꢁ0.63
27.4ꢁ1.2
30.4ꢁ1.1
33.5ꢁ3.1
47.7ꢁ2.1
41.5 ꢁ0.8
33.4ꢁ1.3
20.4ꢁ1.25
13g
13h
Thalidomide
5-FUa)
a) 5-Fluorouracil, used as a positive control. The data were the meanꢁSD obtained from three
independent experiments.
As can be seen from the Table, most target compounds showed certain anticancer
activities against the tumor cells (HepG2, A549, and PC-3) as compared with the
control 5-fluorouracil (5-FU). Among these compounds, 13a and 13d were as potent as
5-FU, while 13b and 13c were even more potent than 5-FU.
According to the activities mentioned above, some structure–activity relationships
of target compounds can be inferred. First, the glutarimide ring of thalidomide is not
essential for cytotoxicity against tumor cells lines, since substitution of the glutarimide
moiety by phenyl ring does not produce biologically inactive analogues. Furthermore,
the results in the Table indicate the bioactivity of target compounds is closely related to
the type of NO donors. The compounds 13a–13d with furoxan moiety displayed more
potent anticancer activities than those with nitrate groups. Additionally, the length of
the spacers connecting to NO donors also appears important for the bioactivities.
Compounds 13b and 13c with a C3 spacer was more potent than 13a or 13d with shorter
or longer spacers, respectively. As a matter of fact, 13c showed the highest anticancer
activity than others.

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Conclusions. – In search of more potent anticancer agents, we have designed and
synthesized 15 NO-donating thalidomide analogues, 6a and 6b, 8a–8e, and 13a–13h.
Preliminary screening experiments revealed that 13a–13d showed anticancer activities
similar to or better than that of 5-fluorouracil (5-FU). Further in vivo biological
evaluation and mechanistic studies on this novel class of anticancer agents are currently
in progress and will be reported in due course.
Experimental Part
1. General. Reagents were from Shanghai Chemical Reagent Company and used without further
purification. Column chromatography (CC): silica gel 60 (SiO2; 200–300 mesh). TLC: SiO2 60 F254
plates (250 mm; Qingdao Ocean Chemical Company, P. R. China). M.p.: RDCSY-I melting point
1
apparatus; uncorrected. IR Spectra: Shimadzu FTIR-8400S spectrometer; in cmꢀ1. H-NMR Spectra:
Bruker ACF-300Q apparatus at 300 MHz, in CDCl3 unless otherwise indicated; d in ppm rel. to Me4Si, J
in Hz. MS: Hewlett-Packard 1100 LC/MSD spectrometer; in m/z. Elemental analysis: Elementar Vario
EL III instrument.
3-Hydroxybenzyl Chloride (2a). SOCl2 (0.22 ml, 12.0 mmol) was added dropwise to a cold soln. of 3-
(hydroxymethyl)phenol (1a; 496 mg, 4.0 mmol) in CHCl3 (14 ml). The mixture was stirred for 1 h at r.t.
The CHCl3 layer was washed with H2O and dried (Na2SO4), and the solvent was removed to afford 2a
(306 mg, 54%). Yellow oil. ESI-MS: 143 ([MþH]þ ).
4-Hydroxybenzyl Chloride (2b). As described for 2a, with SOCl2 (0.22 ml, 12.0 mmol) and 4-
(hydroxymethyl)phenol (1b; 496 mg, 4.0 mmol): 2b (273 mg, 48%). Yellow oil. ESI-MS: 143 ([MþH]þ ).
3-Hydroxybenzyl Nitrate (3a). A soln. of 2a (284 mg, 2.00 mmol) in a small amount of MeCN was
added to a stirred soln. of AgNO3 (680 mg, 4.00 mmol) in MeCN (8 ml). Stirring was continued for 1 h at
r.t. in the dark, and then the precipitate was filtered off, and the solvent was evaporated. The crude
product was purified by CC (SiO2; AcOEt/petroleum ether (PE) 1:1) to give 3a (118 mg, 35%). Yellow
oil. ESI-MS: 170 ([MþH]þ ).
4-Hydroxybenzyl Nitrate (3b). As described for 3a, with 2b (284 mg, 2.00 mmol) and AgNO3
(680 mg, 4.00 mmol): 3b (91 mg, 27%). Yellow oil. ESI-MS: 170 ([MþH]þ ).
4-(2,3-Dihydro-1,3-dioxo-1H-isoindol-2-yl)benzoic Acid (5). A soln. of 4-aminobenzoic acid (10 g,
72.9 mmol) and phthalic anhydride (10 g, 67.5 mmol) in glacial AcOH (50 ml) and was heated to reflux
for 1 h. Compound 5 solidified upon cooling. An almost white powder appeared that was filtered and
washed twice with H2O (50 ml), to give 5 (15.48 g, 80%). White powder. M.p. 293–2948. 1H-NMR
((D6)DMSO): 13.71–12.92 (br. s, 1 H); 8.09 (d, J¼8.7, 2 H); 8.01–7.91 (m, 4 H); 7.62 (d, J¼8.4, 2 H).
ESI-MS: 266 ([MꢀH]ꢀ ).
3-(Nitrooxymethyl)phenyl 4-(2,3-Dihydro-1,3-dioxo-1H-isoindol-2-yl)benzoate (6a). A soln. of 5
(267 mg, 1.00 mmol) in freshly dist. SOCl2 (4 ml) and cat. amount of DMF was vigorously stirred under
reflux for 1 h. Then, the solvent was carefully evaporated at reduced pressure, and a soln. of 3a (200 mg,
1.20 mmol) in CH2Cl2 (10 ml) was added. The mixture was stirred for 30 min at r.t., then poured in H2O
(20 ml) and extracted with CH2Cl2 (3ꢂ15 ml). The org. layer was dried (Na2SO4) and evaporated at
reduced pressure to give 6a (196 mg, 47%). M.p. 122–1268. IR (KBr): 1778, 1762, 1733, 1718, 1635.
1H-NMR: 8.05 (d, J¼8.7, 2 H); 7.96–7.91 (m, 4 H); 7.86 (d, J¼9.3, 2 H); 7.54–7.23 (m, 4 H); 5.41 (s, 2 H).
ESI-MS: 419 ([MþH]þ ). Anal. calc. for C22H14N2O7: C 63.16, H 3.37, N 6.70; found: C 63.37, H 3.56, N
6.51.
4-(Nitrooxymethyl)phenyl 4-(2,3-Dihydro-1,3-dioxo-1H-isoindol-2-yl)benzoate (6b). As described
for 6a, with 3b (1.20 mmol) and 5 (267 mg, 1.00 mmol): 6b (176 mg, 42%). M.p. 101–1048. IR (KBr):
1
1781, 1759, 1738, 1712, 1633. H-NMR: 8.01 (d, J¼9.0, 2 H), 7.98–7.87 (m, 4 H); 7.87 (d, J¼9.0, 2 H);
7.49–7.28 (m, 4 H); 5.47 (s, 2 H). ESI-MS: 419 ([MþH]þ ). Anal. calc. for C22H14N2O7: C 63.16, H 3.37, N
6.70; found: C 63.30, H 3.19, N 6.44.
2-Bromoethyl 4-(2,3-Dihydro-1,3-dioxo-1H-isoindol-2-yl)benzoate (7a). To a stirred soln. of 5
(534 mg, 2.00 mmol) in acetone (6 ml), 1,2-dibromoethane (744 mg, 4.00 mmol) and Et3N (0.4 ml) in
acetone were added dropwise. The mixture was heated at reflux for 4 h and cooled to r.t. Solvent and

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
471
excess Et3N were evaporated in vacuo, and the residue was purified by CC (SiO2; AcOEt/PE 1:5) to give
1
7a (610 mg, 83%). M.p. 195–1988. H-NMR: 8.18 (d, J¼7.8, 2 H); 8.05–8.00 (m, 4 H); 7.66 (d, J¼7.8,
2 H); 4.69 (t, J ¼ 6.0, 2 H); 3.65 (t, J ¼ 6.0, 2 H).
3-Bromopropyl 4-(2,3-Dihydro-1,3-dioxo-1H-isoindol-2-yl)benzoate (7b). As described for 7a, with
1,3-dibromopropane (800 mg, 4.00 mmol) and 5 (534 mg, 2.00 mmol): 7b (302 mg, 78%). M.p. 172–1748.
1H-NMR: 8.19 (d, J¼7.8, 2 H); 7.99–7.83 (m, 4 H); 7.61 (d, J¼7.8, 2 H); 4.57 (t, J ¼ 6.0, 2 H); 3.58 (t, J¼
6.3, 2 H); 2.43–2.34 (m, 2 H).
4-Bromobutyl 4-(2,3-Dihydro-1,3-dioxo-1H-isoindol-2-yl)benzoate (7c). As described for 7a, with
1,4-dibromobutane (856 mg, 4.00 mmol) and 5 (534 mg, 2.00 mmol): 7c (706 mg, 88%). M.p. 138–1408.
1H-NMR: 8.18 (d, J¼8.4, 2 H); 7.99–7.81 (m, 4 H); 7.61 (d, J¼8.7, 2 H); 4.39 (t, J ¼ 6.0, 2 H); 3.49 (t, J¼
6.0, 2 H); 2.10–1.94 (m, 2 H).
5-Bromopentyl 4-(2,3-Dihydro-1,3-dioxo-1H-isoindol-2-yl)benzoate (7d). As described for 7a, with
1,5-dibromopentane (912 mg, 4.00 mmol) and 5 (534 mg, 2.00 mmol): 7d (680 mg, 82%). M.p. 103–1058.
1H-NMR: 8.18 (d, J¼8.7, 2 H); 8.00–7.81 (m, 4 H); 7.61 (d, J¼8.7, 2 H); 4.37 (t, J ¼ 6.3, 2 H); 3.45 (t, J¼
6.6, 2 H); 2.00–1.78 (m, 4 H); 1.68–1.60 (m, 4 H).
6-Bromohexyl 4-(2,3-Dihydro-1,3-dioxo-1H-isoindol-2-yl)benzoate (7e). As described for 7a, with
1,6-dibromohexane (968 mg, 4.00 mmol) and 5 (534 mg, 2.00 mmol): 7e (600 mg, 70%). M.p. 126–1288.
1H-NMR: 8.17 (d, J¼8.4, 2 H); 7.99–7.81 (m, 4 H); 7.26 (d, J¼9.0, 2 H); 4.36 (t, J ¼ 6.6, 2 H); 3.43 (t, J¼
6.6, 2 H); 1.93–1.79 (m, 4 H); 1.60–1.52 (m, 4 H).
2-(Nitrooxy)ethyl 4-(2,3-Dihydro-1,3-dioxo-1H-isoindol-2-yl)benzoate (8a). To a suspension of 7a
(373 mg, 1.00 mmol) in MeCN (10 ml), AgNO3 (340 mg, 2.00 mmol) was added, and the mixture was
refluxed for 4 h. The precipitate was filtered off, and the solvent was carefully evaporated. The residue
was taken up in AcOEt, washed with H2O and brine, dried (Na2SO4), and evaporated. The crude residue
was purified by CC (SiO2; AcOEt/PE 1:5) to afford 8a (146 mg, 41%) as a pale-yellow solid that was
further purified by twice crystallizations with 95% EtOH. M.p. 173–1758. IR (KBr): 1758, 1740, 1698,
1633. 1H-NMR: 8.19 (d, J¼8.7, 2 H); 8.00–7.83 (m, 4 H); 7.63 (d, J¼9.0, 2 H); 4.74 (t, J¼6.0, 2 H); 4.64
(t, J ¼ 6.0, 2 H). ESI-MS: 357 ([MþH]þ ). Anal. calc. for C17H12N2O7: C 57.31, H 3.39, N 7.86; found: C
57.65, H 3.55, N 7.56.
3-(Nitrooxy)propyl 4-(2,3-Dihydro-1,3-dioxo-1H-isoindol-2-yl)benzoate (8b). As described for 8a,
with AgNO3 (340 mg, 2.00 mmol) and 7b (387 mg, 1.00 mmol): 8b (193 mg, 52%). M.p. 142–1448. IR
(KBr): 1789, 1732, 1701, 1629. 1H-NMR: 8.18 (d, J¼8.4, 2 H); 7.99–7.81 (m, 4 H); 7.62 (d, J¼9.3, 2 H);
4.65 (t, J ¼ 6.3, 2 H); 4.47 (t, J¼6.0, 2 H); 2.28–2.20 (m, 2 H). ESI-MS: 371 ([MþH]þ ). Anal. calc. for
C18H14N2O7: C 58.38, H 3.81, N 7.56; found: C 58.10, H 3.57, N 7.78.
4-(Nitrooxy)butyl 4-(1,3-Dioxo-2,3-dihydro-1H-2-isoindolyl)benzoate (8c). As described for 7a,
with AgNO3 (340 mg, 2.00 mmol) and 7c (401 mg, 1.00 mmol): 8c (177 mg, 46%). M.p. 118–1198. IR
(KBr): 1770, 1729, 1710, 1643. 1H-NMR: 8.24 (d, J¼8.7, 2 H); 7.98–7.82 (m, 4 H); 7.62 (d, J¼9.0, 2 H);
4.56 (t, J ¼ 6.6, 2 H); 4.28 (t, J¼6.3, 2 H); 2.09–2.02 (m, 4 H). ESI-MS: 385 ([MþH]þ ). Anal. calc. for
C19H16N2O7: C 59.38, H 4.20, N 7.29; found: C 59.14, H 3.88, N 7.57.
5-(Nitrooxy)pentyl 4-(2,3-Dihydro-1,3-dioxo-1H-isoindol-2-yl) benzoate (8d). As described for 7a,
with AgNO3 (340 mg, 2.00 mmol) and 7d (415 mg, 1.00 mmol): 8d (195 mg, 49%). M.p. 91–948. IR
(KBr): 1792, 1736, 1708, 1656. 1H-NMR: 8.17 (d, J¼8.4, 2 H); 7.98–7.82 (m, 4 H); 7.63 (d, J¼8.4, 2 H);
4.55 (t, J ¼ 6.0, 2 H); 4.40 (t, J¼6.0, 2 H); 2.28–2.20 (m, 4 H); 1.62–1.56 (m, 2 H). ESI-MS: 399 ([Mþ
H]þ ). Anal. calc. for C20H18N2O7: C 60.30, H 4.55, N 7.03; found: C 60.46, H 4.69, N 6.87.
6-(Nitrooxy)hexyl 4-(2,3-Dihydro-1,3-dioxo-1H-isoindol-2-yl) benzoate (8e). As described for 7a,
with AgNO3 (340 mg, 2.00 mmol) and 7e (429 mg, 1.00 mmol): 8e (181 mg, 44%). M.p. 78–808. IR
(KBr): 1788, 1737, 1703, 1659. 1H-NMR: 8.18 (d, J¼8.4, 2 H); 7.99–7.81 (m, 4 H); 7.60 (d, J¼8.7, 2 H);
4.71 (t, J ¼ 6.3, 2 H,); 4.36 (t, J¼6.3, 2 H); 1.80–1.78 (m, 4 H); 1.60–1.51 (m, 4 H). ESI-MS: 413 ([Mþ
H]þ ). Anal. calc. for C21H20N2O7: C 61.16, H 4.89, N 6.79; found: C 61.32, H 4.81, N 6.69.
2-(Phenylsulfanyl)acetic Acid (10). Benzenethiol (9; 24.2 g, 0.22 mol) and NaOH (8.8 g, 0.22 mol)
were dissolved in 95% EtOH (110 ml) and added to a soln. of ClCH2CO2H (22.7 g, 0.24 mol) and Na2CO3
(12.7 g, 0.12 mol) in H2O (50 ml). The mixture was stirred for ca. 3 h at r.t. and heated to reflux for 1 h,
cooled to r.t., and HCl was added to the soln. until pH 2. After removal of EtOH in vacuo, white crystals
of 10 were formed in a 92% yield (34 g). M.p. 60–628. ESI-MS: 167 ([MꢀH]þ ).

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3,4-Bis(phenylsulfonyl)-1,2,5-oxadizole 2-Oxide (11). To a soln. of 10 (13.4 g, 0.08 mol) in glacial
AcOH (50 ml) was added 30% H2O2 aq. soln. The mixture was stirred for ca. 3 h at r.t., fuming HNO3
(32 ml, 0.172 mol) was added dropwise, and the mixture was stirred for ca. 0.5 h at 908. After cooling to
r.t., white crystals of 11 were formed in 76% yield (22.2 g). M.p. 154–1568. 1H-NMR: 8.19–8.15 (m, 4 H);
7.84–7.63 (m, 6 H).
2-[2-Oxido-3-(phenylsulfonyl)-1,2,5-oxadizol-4-yloxy]ethanol (12a). To a stirred soln. of 11 (732 mg,
2.00 mmol) in THF (20 ml) was first added glycol (248 mg, 4.0 mmol) and then, keeping the temp. at 258,
a 50% NaOH soln. (w/w; 0.35 g, 4.00 mmol). The mixture was kept under stirring for 3 h at r.t. The
solvent was removed in vacuo, and the residue was treated with H2O and extracted with CH2Cl2. The
combined org. layers were dried (Na2SO4) and concentrated in vacuo. The concentrated soln. was treated
with charcoal, filtered, and then evaporated in vacuo to give 12a (372 mg, 65%). White solid. M.p. 118–
1198. 1H-NMR: 8.09 (d, J¼8.7, 2 H); 7.77–7.60 (m, 3 H); 4.72 (t, J¼6.0, 2 H); 3.80 (t, J¼6.9, 2 H); 2.12–
2.07 (br. s, 1 H).
3-[2-Oxido-3-(phenylsulfonyl)-1,2,5-oxadizol-4-yloxy]propan-1-ol (12b). As described for 12a, with
propane-1,3-diol (304 mg, 4.0 mmol) and 11 (732 mg, 2.00 mmol): 12b (432 mg, 72%). M.p. 105–1068.
1H-NMR: 8.01 (d, J¼7.8, 2 H); 7.91–7.75 (m, 3 H); 4.54 (t, J¼5.4, 2 H); 3.86 (t, J¼6.0, 2 H); 2.45–2.36
(m, 2 H); 2.08–2.01 (br. s, 1 H).
1-Methyl-3-[2-oxido-3-(phenylsulfonyl)-1,2,5-oxadizol-4-yloxy]propan-1-ol (12c). As described for
12a, with butane-1,3-diol (360 mg, 4.0 mmol) and 11 (732 mg, 2.00 mmol): 12c (522 mg, 83%). M.p. 104–
1078. 1H-NMR: 8.05 (d, J¼7.5, 2 H); 7.79–7.60 (m, 3 H); 4.52 (t, J¼6.3, 2 H); 4.13–4.07 (m, 1 H); 2.22–
2.13 (m, 2 H); 2.07–1.95 (br. s, 1 H); 1.31 (d, J¼6.0, 3 H).
4-[2-Oxido-3-(phenylsulfonyl)-1,2,5-oxadizol-4-yloxy]butan-1-ol (12d). As described for 12a, with
butane-1,4-diol (360 mg, 4.0 mmol) and 11 (732 mg, 2.00 mmol): 12d (428 mg, 68%). M.p. 61–648.
1H-NMR: 8.04 (d, J¼7.8, 2 H); 7.59–7.44 (m, 3 H); 4.48 (t, J¼6.0, 2 H); 3.82 (t, J¼6.0, 2 H); 2.11–2.05
(br. s, 1 H); 2.03–1.98 (m, 2 H); 1.79–1.74 (m, 2 H).
4-[2-Oxido-3-(phenylsulfonyl)-1,2,5-oxadizol-4-yloxy]but-2-en-1-ol (12e). As described for 12a,
with but-2-ene-1,4-diol (352 mg, 4.0 mmol) and 11 (732 mg, 2.00 mmol): 12e (456 mg, 73%). M.p. 72–
748. 1H-NMR: 8.06 (d, J¼7.5, 2 H); 7.66–7.60 (m, 3 H); 6.05–5.99 (m, 1 H); 5.86–5.78 (m, 1 H); 5.13 (t,
J¼5.1, 2 H); 4.32 (t, J¼6.3, 2 H); 2.07–2.03 (br. s, 1 H).
4-[2-Oxido-4-(phenylsulfonyl)-1,2,5-oxadizol-4-yloxy]but-2-yn-1-ol (12f). As described for 12a, with
but-2-yne-1,4-diol (344 mg, 4.0 mmol) and 11 (732 mg, 2.00 mmol): 12f (372 mg, 60%). M.p. 110–1128.
1H-NMR: 8.03 (d, J¼7.8, 2 H); 7.73–7.65 (m, 3 H); 5.11 (s, 2 H); 4.35 (s, 2 H); 2.05–2.01 (br. s, 1 H).
5-[2-Oxido-3-(phenylsulfonyl)-1,2,5-oxadizol-4-yloxy]pentan-1-ol (12g). As described for 12a, with
pentane-1,5-diol (416 mg, 4.0 mmol) and 11 (732 mg, 2.00 mmol): 12g (460 mg, 71%). M.p. 63–678.
1H-NMR: 8.05 (d, J¼7.8, 2 H); 7.78–7.64 (m, 3 H); 4.44 (t, J¼6.3, 2 H); 3.70 (t, J¼6.0, 2 H); 2.13–2.09
(br. s, 1 H); 1.96–1.88 (m, 2 H); 1.70–1.63 (m, 4 H).
2-{2-[2-Oxido-4-(phenylsulfonyl)-1,2,5-oxadizol-2-yloxy]ethoxy}ethanol (12h). As described for
12a, with diethylene glycol (424 mg, 4.0 mmol) and 11 (732 mg, 2.00 mmol): 12g (342 mg, 52%). M.p.
1
58–608. H-NMR: 8.09 (d, J¼7.8, 2 H); 7.79–7.60 (m, 3 H); 4.59 (t, J¼4.4, 2 H); 3.95 (t, J¼4.4, 2 H);
3.79 (t, J¼4.4, 2 H); 3.70 (t, J¼7.8, 2 H); 2.08–2.03 (br. s, 1 H).
2-[2-Oxido-3-(phenylsulfonyl)-1,2,5-oxadizol-4-yloxy]ethyl 4-(2,3-Dihydro-1,3-dioxo-1H-isoindol-2-
yl)benzoate (13a). To a stirred soln. of 5 (265 mg, 1.00 mmol) in THF (8.0 ml) was added EDCI (183 mg,
1.00 mmol) and DMAP (306 mg, 3 mmol). The mixture was stirred for 1 h in ice-water bath. Then, 12a
(286 mg, 1.00 mmol) was added, and the mixture was stirred at r.t. overnight. Solvent was put into ice-
H2O, and the H2O layer was extracted with AcOEt. The combined org. layers were dried (Na2SO4) and
evaporated in vacuo. The product was purified by CC (SiO2; AcOEt/PE 1:2): 13a (380 mg, 71%). M.p.
1
195–1968. IR (KBr): 1780, 1744, 1705, 1370, 1158. H-NMR: 8.09–8.00 (m, 6 H); 7.82–7.62 (m, 7 H);
4.87 (t, J¼6.6, 2 H); 4.76 (t, J¼6.0, 2 H). ESI-MS: 536 ([MþH]þ ). Anal. calc. for C25H17N3O9S: C 56.07,
H 3.20, N 7.85; found: C 56.19, H 3.12, N 7.73.
3-[2-Oxido-3-(phenylsulfonyl)-1,2,5-oxadizol-4-yloxy]propyl 4-(2,3-Dihydro-1,3-dioxo-1H-isoindol-
2-yl)benzoate (13b). As described for 13a, with 5 (265 mg, 1.00 mmol) and 12b (286 mg, 1.00 mmol): 13b
(307 mg, 56%). M.p. 185–1878. IR (KBr): 1771, 1745, 1714, 1365, 1156. 1H-NMR: 8.09–8.03 (m, 6 H);

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
473
7.78–7.59 (m, 7 H); 4.65(t, J¼6.0, 2 H); 4.25 (t, J¼6.6, 2 H); 2.52–2.45 (m, 2 H). ESI-MS: 550 ([Mþ
H]þ ). Anal. calc. for C26H19N3O9S: C 56.83, H 3.49, N 7.65; found: C 57.11, H 3.57, N 7.39.
1-Methyl-3-[2-oxido-3-(phenylsulfonyl)-1,2,5-oxadizol-4-yloxy]propyl 4-(2,3-Dihydro-1,3-dioxo-1H-
isoindol-2-yl)benzoate (13c). As described for 13a, with 5 (265 mg, 1.00 mmol) and 12c (300 mg,
1.00 mmol): 13c (366 mg, 65%). M.p. 163–1658. IR (KBr): 1786, 1739, 1700, 1311, 1169. 1H-NMR: 8.11–
7.99 (m, 6 H); 7.82–7.59 (m, 7 H); 5.46–5.39 (m, 1 H); 4.58 (t, J¼6.6, 2 H); 2.34–2.27 (m, 2 H); 1.57 (d,
J¼6.3, 3 H). ESI-MS: 564 ([MþH]þ ). Anal. calc. for C27H21N3O9S: C 57.55, H 3.76, N 7.46; found: C
57.43, H 3.60, N 7.50.
4-[2-Oxido-3-(phenylsulfonyl)-1,2,5-oxadizol-4-yloxy]butyl 4-(2,3-Dihydro-1,3-dioxo-1H-isoindol-
2-yl)benzoate (13d). As described for 13a, with 5 (265 mg, 1.00 mmol) and 12d (300 mg, 1.00 mmol):
1
13d (333 mg, 59%). M.p. 152–1548. IR (KBr): 1780, 1728, 1705, 1366, 1189. H-NMR: 8.08–7.97 (m,
6 H); 7.85–7.61 (m, 7 H); 4.52 (t, J¼6.0, 2 H); 4.46 (m, J¼6.0, 2 H); 2.07–1.99 (m, 4 H). ESI-MS: 564
([MþH]þ ). Anal. calc. for C27H21N3O9S: C 57.55, H 3.76, N 7.46; found: C 57.39, H 3.92, N 7.32.
4-[2-Oxido-3-(phenylsulfonyl)-1,2,5-oxadizol-4-yloxy]but-2-enyl 4-(2,3-Dihydro-1,3-dioxo-1H-iso-
indol-2-yl)benzoate (13e). As described for 13a, with 5 (265 mg, 1.00 mmol) and 12e (298 mg,
1.00 mmol): 13e (269 mg, 48%). M.p. 149–1508. IR (KBr): 1782, 1743, 1687, 1368, 1142. 1H-NMR:
7.99–7.83 (m, 6 H); 7.82–7.60 (m, 7 H); 6.09–5.94 (m, 2 H); 5.15 (d, J¼5.7, 2 H); 4.35 (d, J¼5.4, 2 H).
ESI-MS: 562 ([MþH]þ ). Anal. calc. for C27H19N3O9S: C 57.75, H 3.41, N 7.48; found: C 57.82, H 3.47, N
7.48.
4-[2-Oxido-3-(phenylsulfonyl)-1,2,5-oxadizol-4-yloxy]but-2-ynyl 4-(2,3-Dihydro-1,3-dioxo-1H-iso-
indol-2-yl)benzoate (13f). As described for 13a, with 5 (265 mg, 1.00 mmol) and 12f (296 mg,
1.00 mmol): 13f (314 mg, 56%). M.p. 134–1368. IR (KBr): 1767, 1735, 1782, 1392, 1188. 1H-NMR:
8.15–8.05 (m, 6 H); 7.84–7.63 (m, 6 H); 5.14 (s, 2 H); 4.88 (s, 2 H). ESI-MS: 560 ([MþH]þ ). Anal. calc.
for C27H17N3O9S: C 57.96, H 3.06, N 7.51; found: C 57.75, H 3.30, N 7.29.
5-[2-Oxido-3-(phenylsulfonyl)-1,2,5-oxadizol-4-yloxy]pentyl 4-(2,3-Dihydro-1,3-dioxo-1H-isoindol-
2-yl)benzoate (13g). As described for 13a, with 5 (265 mg, 1.00 mmol) and 12g (314 mg, 1.00 mmol): 13g
1
(381 mg, 66%). M.p. 114 1168. IR (KBr): 1779, 1746, 1695, 1348, 1167. H-NMR: 8.10–8.01 (m, 6 H);
7.84–7.61 (m, 7 H); 4.56 (t, J¼6.3, 2 H); 4.35 (t, J¼6.2, 2 H); 1.98–1.92 (m, 4 H); 1.58–1.52 (m, 2 H).
ESI-MS: 578 ([MþH]þ ). Anal. calc. for C28H23N3O9S: C 58.23, H 4.01, N 7.28; found: C 58.21, H 4.14, N
7.27.
2-{2-[2-Oxido-3-(phenylsulfonyl)-1,2,5-oxadizol-4-yloxy]ethoxy}ethyl 4-(2,3-Dihydro-1,3-dioxo-1H-
isoindol-2-yl)benzoate (13h). As described for 13a, with 5 (265 mg, 1.00 mmol) and 12h (316 mg,
1.00 mmol): 13h (353 mg, 61%). M.p. 85–878. IR (KBr): 1782, 1738, 1700, 1373, 1163. 1H-NMR: 8.18–
7.96 (m, 6 H); 7.84–7.56 (m, 7 H); 4.60 (t, J¼4.5, 2 H); 4.55 (t, J¼4.2, 2 H); 4.00–3.93 (m, 4 H). ESI-MS:
580 ([MþH]þ ). Anal. calc. for C27H21N3O10S: C 55.96, H 3.65, N 7.25; found: C 56.04, H 3.81, N 7.11.
2. Cytotoxicity Assays. All of the prepared compounds were tested for their in vitro anticancer
activities against HepG2, A549, and PC-3 cells by MTT-based assay [21] with some modifications.
Briefly, cells were cultured in RPMI 1640 medium, supplemented with 10% fetal calf serum at 378
with 5% CO2. For experiments, cells were plated in 96-well plates (105 cells/well for adherent cells or
0.3ꢂ106 cells/well for suspended cells in 100 ml medium). After 24 h, the compounds (10ꢀ4 –10ꢀ8 m)
dissolved in DMSO (5%) were added to each well and incubated for 3 d (72 h). Control groups received
the same amount of DMSO. 5-Fluorouracil (5-FU) was used as positive control. At the end of 72-h
incubation, the medium in each well was replaced by fresh medium (200 ml) containing 0.5 mg/ml of
MTT. Three h later, the formazan product of MTT reduction was dissolved in DMSO, and absorbance
was measured using an ELISA micro-plate reader. Growth of tumoral cells was quantitated by the ability
of living cells to reduce the yellow dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
hydrobromide (MTT) to a purple formazan product. Drug effect was quantified as the percentage of
control absorbance of reduced dye at 590 nm. In all experiments, three replicate wells were used for each
drug concentration, and each assay was carried out at least three times. The results of this experiment
were summarized in the Table.

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Received January 7, 2008