Design, synthesis and antitumor evaluation of novel thalidomide dithiocarbamate and dithioate analogs against Ehrlich ascites carcinoma-induced solid tumor in Swiss albino mice

Article abstract of DOI:10.1016/j.bmc.2008.09.071

A series of 16 novel thalidomide sulfur analogs containing one and two sulfur atoms 2 and 4-18, respectively, were designed and synthesized. These compounds were screened for in vitro antitumor activity against Ehrlich ascites carcinoma (EAC) cell line and exhibited potent cytotoxic activity. On the bases of the obtained results for in vitro cytotoxic activity, thalidomide sulfur analogs containing two sulfur atoms 8, 9, 13 and 14 were selected and tested in vivo against EAC-induced solid tumor in female mice compared to thalidomide 1 as well as its analog 2 and exhibited a highly significant reduction in tumor volume (TV). Results illustrated the antioxidative activity of these compounds as the level of hepatic lipid peroxidation decreased and levels of antioxidant enzymes like superoxide dismutase (SOD) and catalase were elevated. The histopathological investigations revealed that thalidomide sulfur analogs 2, 8, 9, 13 and 14 have antimitotic, apoptotic and necrotic activities against solid tumor. These compounds lead to increase of Fas-L expression. The immunohistochemical studies showed a decrease in Ki67 and vascular endothelial growth factor (VEGF) staining in tumor cells from treated-animals when compared with non-treated groups, which suggests an inhibition of tumor proliferation rate and angiogenic process associated with tumor growth. Compounds 9 and 13 were the most potent compounds in tumor necrosis without liver necrosis. At the same time, treatment with compound 9 resulted in liver degeneration.

Full text of DOI:10.1016/j.bmc.2008.09.071

Bioorganic & Medicinal Chemistry 16 (2008) 9708–9718
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and antitumor evaluation of novel thalidomide
dithiocarbamate and dithioate analogs against Ehrlich ascites
carcinoma-induced solid tumor in Swiss albino mice
b
c
a
d
Magdy A.-H. Zahran a, , Tarek A.-R. Salem , Rehab M. Samaka , Hussein S. Agwa , Ayman R. Awad
*
a Chemistry Department, Faculty of Science, Menoufiya University, Egypt
b Department of Molecular Biology, Genetic Engineering & Biotechnology Institute, Menoufiya University, Egypt
c Department of Pathology, Faculty of Medicine, Menoufiya University, Egypt
d Arab Drug Company, El-Amiria, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 16 novel thalidomide sulfur analogs containing one and two sulfur atoms 2 and 4–18, respec-
tively, were designed and synthesized. These compounds were screened for in vitro antitumor activity
against Ehrlich ascites carcinoma (EAC) cell line and exhibited potent cytotoxic activity. On the bases
of the obtained results for in vitro cytotoxic activity, thalidomide sulfur analogs containing two sulfur
atoms 8, 9, 13 and 14 were selected and tested in vivo against EAC-induced solid tumor in female mice
compared to thalidomide 1 as well as its analog 2 and exhibited a highly significant reduction in tumor
volume (TV). Results illustrated the antioxidative activity of these compounds as the level of hepatic lipid
peroxidation decreased and levels of antioxidant enzymes like superoxide dismutase (SOD) and catalase
were elevated. The histopathological investigations revealed that thalidomide sulfur analogs 2, 8, 9, 13
and 14 have antimitotic, apoptotic and necrotic activities against solid tumor. These compounds lead
to increase of Fas-L expression. The immunohistochemical studies showed a decrease in Ki67 and vascu-
lar endothelial growth factor (VEGF) staining in tumor cells from treated-animals when compared with
non-treated groups, which suggests an inhibition of tumor proliferation rate and angiogenic process asso-
ciated with tumor growth. Compounds 9 and 13 were the most potent compounds in tumor necrosis
without liver necrosis. At the same time, treatment with compound 9 resulted in liver degeneration.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 16 August 2008
Revised 27 September 2008
Accepted 30 September 2008
Available online 2 October 2008
Keywords:
Thalidomide
Dithiocarbamates
Dithioates
Antitumor
1. Introduction
after this, in 1998, US Food and Drug Administration (FDA) approved
thalidomide in the treatment of ENL.11
Thalidomide [(R,S)-2-(2,6-dioxo-3-piperidinyl)-1H-isoindole-
1,3(2H)-dione 1 was administrated in 1950s as a treatment for
insomnia and as an antiemetic agent. The racemic compound 1
was assumed to be non-toxic compared to the other existing seda-
tive drugs, the barbiturates. Later investigations found that while
the R-enantiomer of thalidomidewas responsible for thesedative ef-
fect the S-enantiomer had teratogenic properties.1–3 This apparent
lack of toxicity led to its use in the treatment of morning sickness
in pregnant women.4 Due to its side effects like teratogenicity5,6
and peripheral neuropathy,7,8 thalidomide was withdrawn from
the worldwide market long back in 1961.9 Sheskin, in 1965, first re-
ported clinical efficacy of thalidomide in the treatment of erythema
nodosum leprosum (ENL), an acute inflammatory state occurring in
lepromatous leprosy.10 This led to the renewal of interest in the clin-
ical development of thalidomide and discovery novel analogs with
improved activities and decreased side effects. Nearly three decades
Thalidomide, once discarded as the worst teratogen, is again
regaining status as a drug in the treatment of various hematologi-
cal malignancies12 as well as variety of inflammatory and autoim-
mune diseases13 and in the treatment of multiple myeloma and
other solid tumors.14 The unique and broad physiological proper-
ties discovered during recent years, prompted a reevaluation of
its therapeutic potential.15–17 Thus, thalidomide is currently ap-
plied for treatment of painful inflammations associated with lep-
rosy,18 rheumatoid arthritis19 and graft-versus host disease
(GVHD).20 Furthermore, promising results in the case of treatment
of AIDS,21 Crohn’s disease,22 behcet’s syndrome23 and cancer re-
lated pathologic angiogenesis24–27 have been disclosed.
Thalidomide teratogenic effect was partially explained by its
anti-angiogenic activity.28 This property appeared to be particu-
larly interesting in the treatment of solid tumors such as brain or
prostate cancers, and clinical trials were realized.21,29
On the other hand, dithiocarbamates have received consider-
able attention due to their numerous biological activities30 and
have found recently application in the treatment of cancer.31–36
* Corresponding author. Tel.: +20 123136252/24198896; fax: +20 24159770.
E-mail address: magdyzahran@gmail.com (M.A.-H. Zahran).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.071

M. A.-H. Zahran et al. / Bioorg. Med. Chem. 16 (2008) 9708–9718
9709
In view of the fact that, cancer is another fatal condition which
O
O
ClCH2SCH3
KOH
is continuing to be one of the largest causes of death in both men
and women, claiming over 6 million lives each year in the world. In
the last decade, basic cancer research has produced remarkable ad-
vances in understanding of cancer biology and cancer genetics.37
To date; many anticancer drugs have been developed and applied
by physicians. However, the resistance to anticancer drugs and side
effects were discovered. Therefore, the research and development
of new and safe drugs have become necessary by the pharmaceu-
tical industry.38 Antioxidants play an important role in inhibiting
and scavenging radicals, thus providing protection against infec-
tion and degenerative diseases.39
From these viewpoints and in continuation of our interest
searching for antitumor active compounds,40–43 the present study
was carried out to evaluate the antitumor activity, lipid peroxida-
tion, antioxidant, apoptotic, necrotic, antimitotic, antiproliferative
and anti-angiogenic effects of thalidomide and its sulfur analogs
against EAC in Swiss albino mice.
N
O
S
N
O
N
NH
O
2
O
O
O
1
CH3
1) CH2O
2) SO2Cl
O
N
O
Amine / CS2 / r.t
CH3CN / 48h
O
N
N
O
S
S
N
O O
Cl
O O
4-18
Amine
3
Scheme 1. Synthesis of thalidomide sulfur analogs 2 and 4–18.
ucts such as urethane in alcoholic media. These intermediates also
require high reaction temperature, gave low or moderate yield of
products and usually entail multistep procedures. These reactions
require very toxic reagents and harmful organic solvent such as
DMF and DMSO in the presence of a catalyst. Due to these disad-
vantages, a simple reaction condition without catalyst or harmful
organic solvent was established.
Chloromethylthalidomide 3, carbonyl sulfide and the appropri-
ate amine (Table 1) were dissolved in acetonitrile and stirred for 48
h at room temperature to afford thalidomide dithiocarbamate and
dithioate analogs 4–18 in good to excellent yields (Table 1).
The amines applied in all entries A–O in this reaction protocol
were selected from various classes of amines such as aliphatic,
alcyclic and aromatic amines (Table 1). In case of using phen-
ylhydrazine (Entry M), the reaction finished after 6 h stirring at
room temperature. Surprisingly, the thalidomide dithiocarbamate
6 showed the introduction of two molecules of the amino ethanol.
The structure of the dithiocarbamate 6 was elucidated on the basis
of IR, MS, 1H NMR, and elemental analysis. Moreover, the single-
crystal and molecular structure of dithiocarbamate 6 was estab-
lished by X-ray crystallographic analysis48 (Fig. 1).
During the course of adjusting an applicable reaction condition,
several trials had been done using different solvents as chloroform,
dichloromethane and diethyl ether. But unfortunately these reac-
tions were failed to proceed properly. In other highly polar solvents
such as dimethylformamide (DMF) and tetrahydrofuran (THF), a
mixture of the desired product beside other byproducts was ob-
tained. Applying 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or tri-
ethylamine as bases to catalyze the reaction afforded hydrolyzed
product of 1-chloromethylthalidomide 3.
The chemical structure of all the synthesized compounds was
elucidated on the basis of their spectral data IR, 1H NMR and elec-
tron impact (EI) mass spectrometry beside the elemental analysis.
The IR spectra of thalidomide sulfur analogs 2 and 4–18 supported
the expected structures and showed absorption bands in the region
1609–1785 cmꢀ1 resulting from the C@O function. The IR spectra
of thalidomide dithiocarbamate and dithioate 4–18 supported
the expected structures and showed absorption bands in the
1108–1183 cmꢀ1 region resulting from C@S function. The 1H
2. Results and discussion
2.1. Chemistry
In a previous investigation on the synthesis of different class of
heterocyclic compounds with promising antitumor activities,
Obvious and clear antitumor activities were recognized for differ-
ent N-substituted thalidomide derivatives which were synthesized
by our group through the alkylation of thalidomide at the nitrogen
of the piperidin-dione moiety with different alkylating agents con-
taining oxygen, nitrogen and sulfur. Most of these alkylated thalid-
omide analogs were screened in vitro, investigated in vivo and
exhibited higher antitumor activity than thalidomide itself. The
more potent and active analog was that contain sulfur atom. Mean-
while of this investigation, a Chinese patent was published44
including some of our synthesized N-alkylated thalidomide deriv-
atives and the published results in the patent were in a complete
accordance with the our data.45
One of our new N-alkylated thalidomide derivatives, to our
knowledge does not reported yet, was methylthiomethylthalido-
mide 2 which showed higher antitumor activity compared to the
other synthesized N-alkylated thalidomide derivatives. By study-
ing this observation, it was found that this activity might be re-
ferred to the presence of sulfur atom.
On the basis of this recognition and the understanding for the
reason of the activity of methylthiomethylthalidomide 2, it was
contemplated to design and synthesize a novel series of thalido-
mide dithiocarbamate and dithioate analogs 4–18 which contain
two sulfur atoms in order to investigate the effect of increasing
the sulfur content in the thalidomide analogs. Thus, on the bases
of the previously mentioned thalidomide activities, the fact that
dithiocarbamate compounds acting as anticancer agents and the
enhanced antitumor activity of methylthiomethylthalidomide 2,
we present in this context a novel synthesis of a new thalidomide
dithiocarbamate and dithioate analogs 4–18 through the introduc-
tion of two sulfur atoms in the side chain at the nitrogen of the pip-
erdin-dione moiety which might enhance the anticancer activity
(Scheme 1).
NMR spectra of methylthiomethylthalidomide
2 showed the
General methods for the synthesis of dithiocarbamate deriva-
tives involve the reaction of an amine with costly and toxic re-
agents, such as thiophosgene and/or an isothiocyanate.46
Furthermore, a one-pot reaction of amines with carbonyl sulfide
and alkyl halides in the presence of a catalyst also has been re-
ported.47 However, there are several disadvantages for these meth-
ods; many isothiocyanates are hazardous, tedious to prepare and
display poor long-term stability with the formation of side prod-
appearance of the methylene protons (N–CH2–S) as multiplet at d
5.28–5.34 ppm. The 1H NMR spectra of thalidomide dithiocarba-
mate and dithioate analogs 4–18 showed the appearance of each
proton of the methylene protons (N–CH2–S) as doublet at d 4.96
and 5.10 ppm. The 1H NMR spectra of compound 2 and 4–18 dis-
play signals at d 5.21–5.31 (m, H-30), d 2.10–2.15, 2.83–2.88 (m, H-
40) and d 2.53–2.63, 3.01–3.12 ppm (m, H-50) were associated with
the piperidone ring. EI mass spectra of thalidomide sulfur analogs 2

9710
M. A.-H. Zahran et al. / Bioorg. Med. Chem. 16 (2008) 9708–9718
Table 1
Appropriate amine entries (A–O) and the corresponding thalidomide sulfur analogs 4–18
Entry
Amine
Product
Structure
Yield (%)/(mp°C)
O
N
O
O
S
S
A
CH3NH2
4
89/(140–142)
N
N
O
NHCH3
NH
O
N
O
S
S
B
CH3CH2CH2NH2
5
73/(146–148)
O O
CH3
O
N
O
S
S
N
N
OH
OH
C
D
E
HOCH2CH2NH2
(CH3CH2)2NH
6
7
8
83/(175–177)
58/(140–142)
72/(175–177)
O O
N
N
O
N
O
S
S
O
O
CH3
CH3
O
N
O
S
S
NH2
N
N
O
O
O
O
NH
NH
N
O
S
S
F
9
57/(170–172)
88/(160–162)
O
N CH2CH2NH2
N
N
O
N
O
S
S
N
N
O
O
O
G
10
HN N CH2CH2NH2
NH
NH
O
NH
N
O
S
S
O
H
I
11
12
58/(115–117)
58/(120–122)
O
O
N CH2CH2NH2
N
O
O
N
O
S
S
N
N
N
N NH2
O
O
O
O
NH N
O
O
N
O
S
S
J
13
14
15
16
91/(110–112)
81/(167–169)
71/(248–250)
67/(165–167)
H3C N
N NH2
O
NH N N CH3
O
N
O
S
S
K
L
NH
O
N
O
N
O
S
S
N
N
O
NH
O
O
O
N
O
O
N
O
S
S
M
NHNH2
O
NH NH

M. A.-H. Zahran et al. / Bioorg. Med. Chem. 16 (2008) 9708–9718
9711
Table 1 (continued)
Entry
Amine
Product
Structure
Yield (%)/(mp°C)
O
N
O
O
S
S
N
O
17
N
N
75/(110–112)
91/(120–122)
CH2NH2
O
O
NH
NH
O
N
O
O
S
S
18
CH2CH2NH2
Figure 1. X-ray crystal structure of derivative 6.
and 4–18 displayed molecular ions (M+) which confirmed their
molecular weights. The microanalysis data confirmed the structure
of thalidomide sulfur analogs 2 and 4–18. All the analytical data
were in complete accordance with the proposed structures. Re-
agents and analytical data are presented in Section 4.
say. Results shown in Table 2 indicated that thalidomide sulfur
analogs 2 and 4–18 exhibit dose-dependent significant increases
in cytotoxicity when compared to those of thalidomide 1. The max-
imal cytotoxic activity of tested compounds was observed at dose
30 lL. Meanwhile, it is noted that most of compounds containing
two sulfur atoms 4–18 have higher cytototoxic effect rather than
that of compound 2 which contains one sulfur atom. Furthermore,
results illustrated that compounds 8, 9, 13 and 14 exhibited the
highest antiproliferative activities against EAC cells in vitro
(95.2 3.3%, 100 0.0%, 100 0.0% and 95.3 2.7%, respectively),
and can therefore be candidates for further stages of screening
in vivo.
In Table 3, the in vivo results presented that treatment of solid-
tumor bearing mice with thalidomide analogs containing one sul-
fur atom 2 as well as with two sulfur atom 8, 9, 13 and 14 resulted
in a highly significant reduction in TV (p < 0.001) as compared with
that of tumor control group of animals. Compounds 9 and 13
exhibited the most potent inhibitory action on the TV (99.6% and
98.9%, respectively) as compared to that of the compound 2 that
contains one sulfur atom. Meanwhile, compound 2 exhibited con-
sequently higher potent inhibitory action on TV (90.6%) rather than
thalidomide 1 (80.6%). From these findings, we can conclude that
2.2. Antitumor and antioxidative examination
The present study evaluated the antitumor activity of new sen-
sitized derivatives of well-known drug thalidomide. Screening for
antitumor activity included measurement of% cell death of EAC
by Trypan blue exclusion assay.49 Three serial concentrations of
thalidomide and its derivatives were tested in vitro cytotoxicity as-
Table 2
In vitro cytotoxic activities of thalidomide 1 and its sulfur analogs 2 and 4–18
Compound
% Cytotoxicity
20
10
lL
l
L
30 lL
Control media
1
2
4
5
6
7
8
0.0 0.0
20.5 2.5
62.5 6.5
76.5 3.4
10.6 2.1
20.6 3.2
10.2 5.6
20.6 3.6
80.5 7.6
45.5 4.3
20.6 2.1
30.5 3.2
80.4 4.6
10.3 2.1
80.0 6.5
80.0 5.3
56.7 1.5
45.0 2.6
1.0 0.0
20.5 1.8
71.5 7.8
80.0 8.6
75.5 7.6
30.6 7.5
20.7 3.5
60.8 7.1
80.8 5.6
60.6 8.6
75.7 6.0
70.6 2.3
80.7 5.1
80.4 3.6
80.5 5.4
81.6 4.1
59.0 1.0
47.3 2.1
1.1 0.2
56.4 2.1
82.8 3.2
85.7 3.2
88.6 2.5
87.8 2.7
90.6 1.8
95.2 3.3
100 0.0
68.8 1.6
94.7 4.3
91.3 2.1
100 0.0
95.3 2.7
89.7 1.6
92.0 1.7
61.3 0.6
52.0 1.0
Table 3
Effect of thalidomide and its sulfur analogs on the volume of solid tumor
Groups
TV ꢁ 10ꢀ3 (mm3)
Inhibition %
9
Control
1
2
8
9
13
14
188.6 2.5
36.4 0.8*
17.7 0.7*
13.6 0.1*
0.7 0.1*
2.0 0.1*
7.7 0.1*
—
10
11
12
13
14
15
16
17
18
80.6
90.6
92.7
99.6
98.9
95.9
*
Statistical significant reduction of tumor volume as compared with a tumor
control group (p < 0.001).

9712
M. A.-H. Zahran et al. / Bioorg. Med. Chem. 16 (2008) 9708–9718
Table 6
the degree of inhibitory activity may be attributed to the presence
of sulfur atoms in the compound in the order of 2S > 1S > 0.
The level of malondialdehyde (MDA) as the end product of lipid
peroxidation and antioxidant enzymes activities in solid-tumor
bearing mice after treatment with 1.25 mM/kg body weights of
thalidomide and its sulfur analogs 2, 8, 9, 13 and 14 for five consti-
tutive days was demonstrated (Table 4). Results showed a signifi-
cant decrease (p < 0.001) in the level of hepatic lipid peroxidation
in the tumor-bearing mice treated with these compounds as com-
pared with that of tumor control group. On the other hand, the
activities of SOD and catalase measured in the liver homogenate
were significantly elevated (p < 0.001) in tumor bearing mice after
treatment with thalidomide sulfur analogs 2, 8, 9, 13 and 14 as
compared with that of tumor control group. In Table 4, treatment
of solid tumor with thalidomide sulfur analogs 9 and 13 resulted
in reasonable significant changes in lipid peroxidation and catalase
that were closed to the normal control group as compared to that
of the rest of compounds 1, 2, 8 and 14. Similarly, level of SOD
activity was the most significant value which close to the normal
control after treatment of solid-tumor bearing mice with com-
pound 13 compared to that of the rest of compounds 1, 2, 8, 9
and 14. Taken together, these findings suggest that these thalido-
mide sulfur analogs 2, 8, 9, 13 and 14 might exert its antitumor
activity through the scavenging of free radicals resulting in a
reduction of lipid oxidation and augmentation the level of antiox-
idant enzymes.
Effect of thalidomide and its sulfur analogs on liver
Groups
Liver degeneration
Liver necrosis
Normal control
Tumor control
1
2
8
9
13
14
0
0
1
0
1
2
1
1
0
0
1
0
0
0
0
0
(p < 0.001). On the other hand, thalidomide analogs 9 and 13
showed the highest statistical significance in necrosis percentage
and H score compared with that of the rest compounds 1, 2, 8
and 14.
The thalidomide sulfur analogs 2, 8, 9, 13 and 14 had no necro-
tic effect on the liver, while thalidomide 1 exhibited toxic effect on
the liver of treated mice (Table 6). As regard with the statistical sig-
nificance, Tables 5 and 6 revealed that compound 9 is the most po-
tent compound in tumor necrosis without liver necrosis as
compared with the other compounds (Fig. 2A). Moreover, liver
degeneration has been remarkably observed after treatment with
compound 9 (Fig. 2H).
Apoptosis is one of the most important mechanisms in the anti-
tumor drugs. Fas-L belongs to the tumor necrosis factor (TNF) fam-
ily. Fas (also known as APO-1 or CD95) are the receptor for Fas-L, a
member of TNF receptors nerve growth factor receptor superfam-
ily. Fas-L and Fas pathway is one of the famous extrinsic apoptotic
pathways.50 The histopathological examination by using H & E
staining showed a statistically significant increase in apoptotic in-
dex (AI) after treatment of tumor-bearing mice with thalidomide 1
and its sulfur analogs 2, 8, 9, 13 and 14 as compared with that of
the tumor control group. As shown in Figure 2B and Table 7, treat-
ment with compound 13 exhibited the most potent apoptotic
activity (p < 0.001) rather than the rest of the compounds 1, 2, 8,
9 and 14. On the other hand, the mitotic index (MI) was signifi-
cantly decreased (p < 0.001) in the studied analogs 1, 2, 8, 9, 13
and 14 as compared to that of the tumor control group (Table 7).
Compound 13 exhibited the most potent antimitotic activity
(p < 0.001).
2.3. The histopathological and immunohistochemical
examinations
In addition to the antitumor and antioxidative activities ob-
served for thalidomide sulfur analogs 1, 2, 8, 9, 13 and 14, the his-
topathological and immunohistochemical examinations of these
compounds were investigated to explore more details on the
mechanism of their action. Data presented in Table 5 illustrated
a massive necrosis of the solid tumor after treatment with com-
pound 9 as compared with that of compounds 1, 2, 8, 13 and 14
Table 4
Effect of thalidomide and its sulfur analogs on the level of lipid peroxidation and
antioxidant enzymes activity in solid-tumor bearing mice
Groups
Lipid peroxidation
(nmol MDA/g tissue)
Catalase
(kU/mg tissue)
SOD
(U/g tissue)
Thus, immunohistochemical staining for Fas-L, as the one of the
famous extrinsic apoptotic pathways, showed a highly statistical
significant increase (p < 0.001) in the intensity, percentage and H
score of Fas-L after treatment with thalidomide 1 and its sulfur
analogs 2, 8, 9, 13 and 14 as compared to those of tumor control
group (Table 8 and Fig. 2C). These data showed that compound
14 which contain two sulfur atoms exhibited the most potent stim-
ulatory effect on the Fas-L expression (120.0 5.0%) as compared
to that of the rest of compounds 1, 2, 8, 9 and 13. These results
were in agreement with the previous findings reported that thalid-
omide has a potent apoptotic effect in cancer cells by enhancement
of cell sensitivity to Fas-induced apoptosis.51,52
Normal control
Tumor control
1
2
8
9
13
14
246.6 5.4
405.2 8.7
6.9 1.4
5.1 1.0
5.8 1.5
6.7 0.9*
6.4 1.4*
7.2 2.0*
8.2 1.0*
7.2 0.9*
156.2 6.8
100.4 8.3
108.4 10.2
139.8 5.9*
128.6 10.5*
145.8 8.6*
162.2 8.8*
152.4 8.0*
330.2 8.1*
289.6 6.3*
257.8 10.0*
247.6 12.4*
248.4 11.9*
276.2 10.8*
*
Statistical significant change as compared with a tumor control group (p < 0.001).
Angiogenesis is the process of generating new capillary blood
vessels which plays an important role in the proliferation, invasion
and metastasis of malignant tumor. Blocking tumor induced angi-
ogenesis continues to be an attractive strategy for cancer ther-
apy.53 Also, VEGF is the prototypical pro-angiogenic molecule
that has been implicated in several steps throughout the angio-
genic process because it disrupts the endothelial barrier function
that might contribute to tumor cell extravasations and metasta-
sis.54 The effect of these compounds on the angiogenesis process
by immunohistochemical staining for VEGF was examined. Figure
2D and E shows strong and weak expressions of VEGF in tumor
control and after treatment with compound 13, respectively. As
Table 5
Effect of thalidomide and its sulfur analogs on the grade, percentage of tumor necrosis
and its H score
Groups
Tumor necrosis grade
Tumor necrosis %
Tumor necrosis H score
Control
1
2
8
9
13
14
—
2
2
2
3
3
2
—
—
44.0 4.2
49.0 4.2
41.0 4.2
100.0 0.0*
61.0 4.2*
50.0 3.5
69.0 20.1
87.0 18.6
82.0 8.4
300.0 0.0*
110.0 23.5*
109.0 24.6*
*
Statistical significant change as compared with thalidomide group (p < 0.001).

M. A.-H. Zahran et al. / Bioorg. Med. Chem. 16 (2008) 9708–9718
9713
Figure 2. Histopathological and immunohistochemical examination of the study (A) showed extensive necrosis in solid-tumor bearing mice treated with compound 9; (B)
showed apoptotic bodies (arrows) in tumor after treatment with compound 13; (C) showed strong immunoreactivity staining for Fas-L in the membrano-cytoplasmic of the
cells of compound 14; (D and E) showed strong and weak expression of VEGF in tumor control and after treatment with compound 13, respectively; (F and G) showed strong
nuclear positivity for Ki67 staining in tumor control and negative nuclear staining in treated mice with compound 2, respectively and (H) illustrated the H & E stained section
from the liver of treated mice with compound 9 showing a diffuse degeneration (grade +++).
Table 7
Table 8
Effect of thalidomide and its sulfur analogs on apoptotic and mitotic indices (AI and
MI)
Intensity, H score and percentage of Fas-L expression to thalidomide and its sulfur
analogs
Groups
AI
MI
Groups
Fas-L intensity
2
Fas-L%
Fas-L (H score)
Control
1
2
8
9
13
14
0.001 0.001
0.003 0.002*
0.002 0.001
0.002 0.001
0.004 0.002*
0.005 0.001*
0.002 0.001
0.018 0.0
1
3
0.006 0.0*
0.004 0.0*
0.005 0.001*
0.004 0.002*
0.002 0.0*
0.004 0.0*
Control
1
2
8
9
13
14
5
0
0
0
0
0
0
0
3
1
1
1
4
2
0
2
4
4
2
1
3
16.0 4.2
66.0 4.2*
45.0 5.0*
44.0 4.2*
55.0 5.0*
60.0 5.0*
120.0 5.0*
16.0 4.2
159.0 40.1*
125.0 18.7*
116.0 33.4*
129.0 17.7*
131.0 20.7*
337.0 61.9*
*
Statistical significant change as compared with tumor control group (p < 0.001).
*
Statistical significant change as compared with tumor control group (p < 0.001).
Test of significance v2 (Chi square) = 88.83.
shown in Table 9,
a highly statistical significant decrease
(p < 0.001) in the intensity, percentage and H score of VEGF for
the treated tumor-bearing mice with thalidomide 1 and its sulfur
analogs 2, 8, 9, 13 and 14 compared to that of untreated group.
These data showed that compound 13 which contain two sulfur
atoms exhibited the most potent inhibitory effect on the VEGF
expression (2.0 0.0%).
This finding was compatible with the previous report which
concluded that thalidomide exhibit potent anti-angiogenic proper-
ties that were thought to contribute to the teratogenic effects of
limb bud malformations observed in offspring of thalidomide ex-
posed gravid mothers.28 Other studies displayed that thalidomide
has been shown to suppress the induction of VEGF in co-cultures
of multiple myeloma cell lines and bone marrow stromal cells.55
Proliferation index is an important prognostic factor in cancer.
The Ki67 protein is expressed in all phases of the cell cycle except
G0 and serves as a good marker for proliferation.56 The prolifera-
tion index was investigated by immunohistochemical staining for
Ki67 on solid tumors removed from the untreated and treated ani-

9714
M. A.-H. Zahran et al. / Bioorg. Med. Chem. 16 (2008) 9708–9718
Table 9
time, treatment with compound 9 resulted in liver degeneration.
Moreover, this study shed light on the mechanism of action of
these newly sensitized compounds. As obtained from the results,
it can be concluded that thalidomide sulfur analogs may exert their
anticancer activity via antioxidative pathway resulting in dimin-
ishing of free radicals and augmentation of antioxidant enzymes.
These compounds have the ability to interfere with the expression
of VEGF and Ki67 resulting in their significant reduction and sub-
sequently tumor growth inhibition. Induction of Fas-L expression
was significantly shown with the treatment of thalidomide sulfur
analogs suggesting the apoptotic activity of these compounds.
Intensity, H score and percentage of VEGF expression to thalidomide and its sulfur
analogs
Groups
VEGF Intensity
2
VEGF%
VEGF (H score)
1
3
Control
1
2
8
9
13
14
1
1
0
0
0
0
0
4
4
1
2
1
2
4
0
0
4
3
2
3
1
20.0 5.0
8.0 5.0*
3.4 1.5*
7.0 2.7*
3.2 1.0*
2.0 0.0*
7.0 4.2*
55.0 4.5
25.0 18.7*
9.2 3.7*
16.0 8.6*
8.3 2.7*
5.2 1.9*
20.0 12.5*
*
Statistical significant change as compared with tumor control group (p < 0.001).
Test of significance v2 (Chi square) = 76.65.
4. Experimental
4.1. General information
Table 10
Intensity, percentage and H score of Ki67 expression to thalidomide and its sulfur
analogs
The synthesized product and each reaction were monitored on
Merck silica gel 60 F254 (type E; Merck) plates and spots were lo-
cated by UV light. All 1H and 13C NMR spectra were recorded on a
Varian Gemini 300 MHz Spectrometer, Cairo University. Chemical
shifts are reported in parts per million (ppm) relative to the respec-
tive solvent or tetramethylsilane (TMS) and standard abbreviations
were used (a = apparent; b = broad; s = singlet; d = doublet;
t = triplet; q = quartet; m = multiple). Coupling constant are given
in Hz. Elemental analyses were determined on a Yanaca CHN Cor-
der MT-3 elemental analyzer in microanalysis laboratory at Cairo
University. IR spectra were recorded (KBr) on a Pye-Unicam Sp-
883 Perkins- Elmer spectrophotometer, Microanalytical Labora-
tory, Faculty of Science, Cairo University. MS spectra were run on
GC MS-QP 1000 EX (SHIMADZU) Mass Spectrometer, Microanalyt-
ical Laboratory, Faculty of Science, Cairo University. Melting points
were recorded on Stuart scientific melting point apparatus. All dia-
grams and calculations were performed using maXus (Bruker Non-
ius, Delft & MacScience, Japan). Scoring was carried out using an
Olympus CH2 light microscope, Tokyo, Japan with wide angle (field
size of 0.274 mm2, and field diameter of 0.59 mm). The starting
material, 2-(1-Chloromethyl-2,6-dioxo-piperidine-3-yl)-1,3-dihy-
dro-2H-isoindole-1,3-dione 3 was prepared according to the re-
ported procedure.58 All chemicals and solvents were purchased
from E. Merck (Darmstadt, Germany) and Sigma–Aldrich.
Groups
Ki67 Intensity
2
Ki67%
Ki67 (H score)
1
3
Control
1
2
8
9
13
14
0
0
3
3
3
3
4
0
3
2
1
1
2
1
5
2
0
1
1
0
0
78 5.7
7.4 2.5*
3.2 1.6*
7.2 2.6*
3.4 1.4*
3.4 1.5*
24 4.2*
234 17.1
16.8 3.0*
4.6 3.3*
8.2 2.5*
4.2 1.1*
4.4 1.5*
72 12.6*
*
Statistical significant change as compared with tumor control group (p < 0.001).
Test of significance v2 = 81.2.
mals with thalidomide 1 and its sulfur analogs 2, 8, 9, 13 and 14.
Nuclear staining and good preservation of morphological details
were observed in tumor section immunostained with Ki67 anti-
body. Figure 2F and G shows the amount of Ki67 negative and po-
sitive cells in analyzed slides, respectively. In Table 10, the relative
number of Ki67 positive tumor cells was substantially smaller in
tumors from mice treated thalidomide and its sulfur analogs
(p < 0.001). These data showed that compound 2 which contain
one sulfur atom exhibited the most potent inhibitory effect on
the Ki67 expression (3.2 1.6%) as compared to that of the rest
of compounds 1, 8, 9, 13 and 14. These findings were in accordance
with the previous report which concluded that thalidomide has a
direct anti-myeloma activity mediated through inhibition of DNA
synthesis resulting in cell proliferation arrest in the G1 phase.57
Further investigations are now under consideration on thalido-
mide 1 and its sulfur analogs 9 and 13 to assess their antitumor
activity on female albino mice-bearing solid tumor by studying
the differential display of the expressed proteins in healthy, can-
cerous and treated tissues as well as the determination of NO con-
centration in homogenate liver tissues. Moreover, following up the
biochemical analysis of serum in treated and untreated mice and
studying the level of methylation in the genomic DNA during the
development of cancerous tissues in treated and healthy mice. Fi-
nally, using the RT-PCR technique to study two of transcribed
genes involved in treated and untreated cancerous tissues.
4.2. Chemistry
4.2.1. The procedure for the synthesis of methylthiomethyl
thalidomide (2)
Powdered KOH (15 mmol) and tris[2-(2-methoxy)eth-
oxy)ethyl]amine (TDA-1, 0.31 mmol) were added to a solution of
thalidomide 1 (6 mmol) in anhydrous acetonitrile (50 mL). The
mixture was stirred for 1 h at room temperature. After that,
ClCH2SCH3 (6.3 mmol) was added and stirring was continued for
48 h. Insoluble materials were filtered off and the filtrate was evap-
orated under reduced pressure. The residue was purified by pre-
parative TLC with chloroform.
4.2.1.1. 2-{1-[(Methylthio)methyl]-2,6-dioxopiperidin-3-yl}-1H-
isoindole-1,3(2H)-dione (2). White powder; IR (KBr) 3039, 2977,
2932, 2876, 1787, 1772, 1690, 1680, 1513 cmꢀ1; 1H NMR (DMSO-d6)
d 2.10–2.13 (m, 1H, H-40), 2.14 (s, 3H, CH3), 2.48–2.59 (m, 1H, H-50),
2.84–2.87 (m, 1H, H-40), 2.95–3.11 (m, 1H, H-50CO), 4.81–4.86 (m,
3. Conclusion
A series of novel thalidomide dithiocarbamate and dithioate
analogs were designed and synthesized as potential antitumor
agents. Compounds containing two sulfur atoms 8, 9, 13 and 14
displayed the most potent antitumor activity higher than com-
pound 2 that contain one sulfur atom.
1H, H-30), 5.28–5.34 (m, 2H, NCH2S), 7.90–7.97 (m, 4H, Harom); 13C
0
0
NMR (DMSO-d6) d 15.82 (CH3), 21.11 (C4 ), 26.59 (C5 ), 41.37 (NCH2S),
0
50.11 (C3 ), 123.12 (C4, C7), 130.12 (C3a, C7a), 132.44 (C5, C6), 167.14,
+
0
0
167.92, 169.02 (C1, C3, C2 , C6 ); EIMS m/z = 318 (M ). Anal. Calcd for
C15H14N2O4S: C, 56.59; H, 4.43; N, 8.80; S, 10.07. Found: C, 56.34;
H, 4.22; N, 8.68; S, 10.10.
Compound 9 and 13 were commonly exhibited the most potent
tumor necrotic activity with no toxicity to the liver. At the same

M. A.-H. Zahran et al. / Bioorg. Med. Chem. 16 (2008) 9708–9718
9715
4.2.2. General procedure for the synthesis of thalidomide
ditiocarbamate and dithioate analogs (4–18)
4.2.2.5. [3-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-2,6-diox-
opiperidin-1-yl]methyl cyclohexyldithiocarbamate (8). White
powder; IR (KBr) 3219, 2939, 2882, 2859, 2835, 2781, 1719, 1685,
A
mixture of chloromethylthalidomide 3 (1 mmol), CS2
(2 mmol) and the appropriate amine (2 mmol) (Table 1) in acetoni-
trile (50 mL) was stirred for 48 h at room temperature. The solvent
was removed in vacuo; the residue was coevaported two times
with dichloromethane and crystallized from ethanol to afford tha-
lidomide dithiocarbamates and dithioate analogs 4–18.
1676, 1619, 1507, 1391, 1126 cmꢀ1 1H NMR (DMSO-d6) d 1.09–
;
1.92 (m, 10H, cyclohex. C2,3,4,5,6-H), 2.11–2.16 (m, 1H, H-40), 2.56–
2.60 (m, 1H, H-50), 2.84–2.88 (m, 1H, H-40), 2.90–2.91 (m, 1H,
cyclohex. C1-H), 3.01–3.04 (m, 1H, H-50CO), 4.91–4.92 (m, 1H,
NH), 5.28–5.32 (m, 1H, H-30), 5.33 (d, J = 11.7 Hz, 1H, NCH2S),
5.47 (d, J = 11.7 Hz, 1H, NCH2S), 7.89–8.06 (m, 4H, Harom); 13C
0
4.2.2.1. [3-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-2,6-dioxopi-
peridin-1-yl] methyl methyldithiocarbamate (4). White pow-
der; IR (KBr) 3235, 3046, 2977, 2948, 2923, 1768, 1728, 1710,
NMR (DMSO-d6) d 21.29 (C4 ), 24.82 (cyclohex. C3, C5), 25.71
0
(cyclohex. C4), 29.06 (C5 ), 31.64 (cyclohex. C2, C6), 42.10 (NCH2S),
50.09 (C3 ), 51.31 (cyclohex. C1), 123.55 (C4, C7), 131.06 (C3a, C7a),
133.10 (C5, C6), 167.06, 168.11, 169.02 (C1, C3, C2 , C6 ), 199.71
(C@S); EIMS m/z = 445 (M+). Anal. Calcd for C21H23N3O4S2: C,
56.61; H, 5.20; N, 9.43; S, 14.39. Found: C, 56.32; H, 4.99; N,
9.50; S, 14.40.
0
1669, 1549, 1389, 1161 cmꢀ1
;
1H NMR (DMSO-d6) d 2.11–2.17 (m,
0
0
1H, H-40), 2.48–2.59 (m, 1H, H-50), 2.84–2.87 (m, 1H, H-40), 2.95–
3.11 (m, 1H, H-50CO), 3.01 (d, 3H, CH3), 4.90–4.92 (m, 1H, NH),
5.28-5.32 (m, 1H, H-30), 5.34 (d, J = 12.6 Hz, 1H, NCH2S), 5.54 (d,
J = 12.6 Hz, 1H, NCH2S), 7.89–7.96 (m, 4H, Harom); 13C NMR
0
0
(DMSO-d6) d 21.34 (C4 ), 29.13 (C5 ), 30.12 (CH3), 42.18 (NCH2S),
4.2.2.6. [3-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-2,6-dioxopi-
peridin-1-yl]methyl 2-piperidin-1-ylethyldithiocarbamate
(9). White powder; IR (KBr) 3429, 3028, 2955, 2920, 1785,
0
50.17 (C3 ), 123.60 (C4, C7), 131.15 (C3a, C7a), 133.15 (C5, C6),
0
0
167.22, 168.11, 169.22 (C1, C3, C2 , C6 ), 200.61 (C@S); EIMS m/
z = 377 (M+). Anal. Calcd for C16H15N3O4S2: C, 50.91; H, 4.01; N,
11.13; S, 16.99. Found: C, 51.12; H, 3.90; N, 10.96; S, 17.10.
1770, 1717, 1691, 1467, 1390, 1153 cmꢀ1 1H NMR (DMSO-d6)
;
d 1.53–1.57 (m, 6H, pip. C3,4,5-H), 2.10–2.14 (m, 1H, H-40),
2.57–2.61 (m, 1H, H-50), 2.63-2.69 (m, 6H, CH2–N–C2,6–H pip.),
2.82–2.91 (m, 1H, H-40), 3.04–3.08 (m, 1H, H-50CO), 3.25–3.38
(m, 2H, –HN–CH2–), 4.90–4.92 (m, 1H, NH), 5.08–5.22 (m, 1H,
H-30), 5.24 (d, J = 11.7 Hz, 1H, NCH2S), 5.33 (d, J = 11.7 Hz, 1H,
NCH2S), 7.88–7.95 (m, 4H, Harom); 13C NMR (DMSO-d6) d 21.63
4.2.2.2. [3-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-2,6-dioxopi-
peridin-1-yl]methyl propyldithiocarbamate (5). White crystals;
IR (KBr) 3267, 3055, 2965, 2932, 2876, 1786, 1770, 1716, 1679,
1529, 1393, 1156 cmꢀ1 1H NMR (DMSO-d6) d 1.05 (t, 3H, CH3),
;
1.50–1.66 (m, 2H, CH2), 2.08–2.14 (m, 1H, H-40), 2.55–2.65 (m, 1H,
H-50), 2.79–2.88 (m, 1H, H-40), 2.97–3.04 (m, 1H, H-50CO), 3.61 (t,
2H, NCH2), 4.91–4.92 (m, 1H, NH), 5.27–5.34 (m, 1H, H-30), 5.33
(C4 ), 24.50 (pip. C4), 25.94 (pip. C3, C5), 29.19 (C5 ), 41.31
0
0
0
(NHCH2), 44.98 (NCH2S), 50.74 (C3 ), 50.88 (CH2N), 54.31 (pip.
C2, C6), 123.70 (C4, C7), 131.65 (C3a, C7a), 133.30 (C5, C6),
0
0
(d, J = 12.9 Hz, 1H, NCH2S), 5.52 (d, J = 12.9 Hz, 1H, NCH2S), 7.88–
167.48, 168.37, 170.12 (C1, C3, C2 , C6 ), 200.10 (C@S); EIMS m/
z = 474 (M+). Anal. Calcd for C22H26N4O4S2: C, 55.68; H, 5.52;
N, 11.80; S, 13.51. Found: C, 55.44; H, 5.33; N, 11.96; S, 13.50.
13
0
7.95 (m, 4H, Harom); C NMR (DMSO-d6) d 11.22 (CH3), 21.33 (C4 ),
0
0
22.43 (CH2), 29.10 (C5 ), 42.15 (NCH2S), 46.44 (NHCH2), 50.14 (C3 ),
123.58 (C4, C7), 131.12 (C3a, C7a), 133.14 (C5, C6), 167.19, 168.11,
+
0
0
169.11 (C1, C3, C2 , C6 ), 200.11 (C = S); EIMS m/z = 405 (M ). Anal.
Calcd for C18H19N3O4S2: C, 53.32; H, 4.72; N, 10.36; S, 15.82. Found:
C, 52.87; H, 5.32; N, 9.96; S, 15.80.
4.2.2.7. [3-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-2,6-dioxopiperi-
din-1-yl]methyl 2-piperazin-1-ylethyldithiocarbamate (10). White
powder; IR (KBr) 3418, 2944, 2831,1780, 1770, 1716, 1621, 1514, 1318,
1108 cmꢀ1 1H NMR (DMSO-d6) d 2.10–2.14 (m, 1H, H-40), 2.40–2.41
;
4.2.2.3. [3-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-2,6-dioxopiperi-
din-1-yl]methyl bis(2-hydroxyethyl)dithiocarbamate (6). White
crystals; IR (KBr) 3520, 3038, 2957, 2923, 2885, 1769, 1736, 1713,
(m, 4H, pip. C2,6–H), 2.47–2.50 (m, 2H, CH2–N–), 2.52–2.61 (m, 1H, H-
50), 2.63–2.67 (m, 4H, pip. C3,5–H), 2.87–2.91 (m, 1H, H-40), 3.04–3.07
(m, 1H, H-50CO), 3.61–3.75 (m, 2H, –NH–CH2–), 4.90–4.92 (m, 1H,
NH), 5.28–5.32 (m, 1H, H-30), 5.33 (d, J = 11.7 Hz, 1H, NCH2S), 5.47 (d,
J = 11.7 Hz, 1H, NCH2S), 7.62–5.92 (m, 4H, Harom); 13C NMR (DMSO-
1689, 1513, 1485, 1145 cmꢀ1 1H NMR (DMSO-d6) d 2.11–2.17 (m,
;
1H, H-40), 2.57–2.64 (m, 1H, H-50), 2.84–2.86 (m, 1H, H-40), 3.01–3.02
(m, 1H, H-50CO), 3.64–3.69 (m, 4H, CH2NCH2), 3.82–3.86 (m, 2H,
CH2OH), 4.04–4.08 (m, 2H, CH2OH), 4.86 (t, 1H, OH), 5.02 (t, 1H, OH),
5.28–5.32 (m, 1H, H-30), 5.33 (d, J = 11.7 Hz, 1H, NCH2S), 5.47 (d,
J = 11.7 Hz, 1H, NCH2S), 7.88–7.96 (m, 4H, Harom); 13C NMR (DMSO-
0
0
d6) d 21.65 (C4 ), 29.21 (C5 ), 41.33 (NHCH2), 45.12 (NCH2S), 46.24 (pip.
0
C3, C5), 50.76 (C3 ), 53.10 (CH2N), 57.32 (pip. C2, C6), 123.71 (C4, C7),
0
131.69 (C3a, C7a), 133.32 (C5, C6), 167.51, 168.40, 170.45 (C1, C3, C2 ,
+
0
C6 ), 200.12 (C@S); EIMS m/z = 475 (M ). Anal. Calcd for C21H25N5O4S2:
0
0
0
d6) d 21.40 (C4 ), 29.15 (C5 ), 42.43 (NCH2S), 50.18 (C3 ), 58.13 (CH2OH),
C, 53.03; H, 5.30; N, 14.73; S, 13.48. Found: C, 52.96; H, 5.44; N,
14.66; S, 13.50.
59.72 (CH2N), 123.61 (C4, C7), 131.24 (C3a, C7a), 133.16 (C5, C6),
0
0
167.23, 168.12, 169.40 (C1, C3, C2 , C6 ), 196.31 (C@S); EIMS m/z = 451
(M+). Anal. Calcd for C19H21N3O6S2: C, 50.54; H, 4.69; N, 9.31; S,
14.20. Found: C, 50.30; H, 4.44; N, 9.54; S, 14.21.
4.2.2.8. [3-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-2,6-diox-
opiperidin-1-yl]methyl 2-morpholin-4-ylethyldithiocarbamate
(11). White powder; IR (KBr) 3472, 3028, 2955, 2917, 1785,
4.2.2.4. [3-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-2,6-dioxopi-
peridin-1-yl]methyl diethyldithiocarbamate (7). colorless crys-
tals; IR (KBr) 3072, 2977, 2959, 2930, 2871, 1783, 1769, 1716,
1770, 1717, 1690, 1467, 1390, 1151 cmꢀ1 1H NMR (DMSO-d6) d
;
2.10–2.14 (m, 1H, H-40), 2.57–2.61 (m, 1H, H-50), 2.63–2.69 (m,
6H, –CH2–N–C3,5–H morph.), 2.82–2.87 (m, 1H, H-40), 3.00–3.04
(m, 1H, H-50CO), 3.24–3.39 (m, 2H, –HN–CH2–), 3.56–3.60 (m,
4H, morph. C2,6–H), 4.90–4.92 (m, 1H, NH), 5.13–5.18 (m, 1H, H-
1693, 1554, 1488, 1137 cmꢀ1 1H NMR (DMSO-d6) d 1.14 (m, 3H,
;
CH3), 2.11–2.17 (m, 1H, H-40), 2.57–2.62 (m, 1H, H-50), 2.82–2.89
(m, 1H, H-40), 3.01–3.04 (m, 1H, H-50CO), 3.69 (q, 2H, CH2), 3.94 (q,
2H, CH2), 5.28–5.33 (m, 1H, H-30), 5.34 (d, J = 11.7 Hz, 1H, NCH2S),
5.49 (d, J = 11.7 Hz, 1H, NCH2S), 7.88–7.94 (m, 4H, Harom); 13C NMR
30), 5.24 (d, J = 11.7 Hz, 1H, NCH2S), 5.34 (d, J = 11.7 Hz, 1H, NCH2S),
13
0
7.88–7.96 (m, 4H, Harom); C NMR (DMSO-d6) d 21.69 (C4 ), 29.22
0
0
(C5 ), 41.34 (NHCH2), 45.40 (NCH2S), 50.77 (C3 ), 53.35 (CH2N),
55.81 (morph. C3, C5), 66.76 (morph. C2, C6), 123.73 (C4, C7),
0
0
(DMSO-d6) d 12.13 (CH3), 21.31 (C4 ), 29.09 (C5 ), 42.11 (NCH2S),
0
48.60 (CH2), 50.11 (C3 ), 123.57 (C4, C7), 131.12 (C3a, C7a), 133.11
131.70 (C3a, C7a), 133.34 (C5, C6), 167.52, 168.42, 170.55 (C1, C3,
+
0
0
0
0
(C5, C6), 167.09, 168.09, 169.11 (C1, C3, C2 , C6 ), 196.31 (C@S); EIMS
m/z = 419 (M+). Anal. Calcd for C19H21N3O4S2: C, 54.40; H, 5.05; N,
10.02; S, 15.29. Found: C, 54.33; H, 5.20; N, 9.90; S, 15.30.
C2 , C6 ), 200.16 (C@S); EIMS m/z = 476 (M ). Anal. Calcd for
C21H24N4O5S2: C, 52.92; H, 5.08; N, 11.76; S, 13.46. Found: C,
52.80; H, 4.91; N, 11.82; S, 13.50.

9716
M. A.-H. Zahran et al. / Bioorg. Med. Chem. 16 (2008) 9708–9718
4.2.2.9. [3-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-2,6-dioxopi-
peridin-1-yl]methyl morpholin-4-yldithiocarbamate(12). White
powder; IR (KBr) 3430, 2959, 2917, 2853, 1785, 1771, 1717, 1691, 1518,
1330, 1151 cmꢀ1; 1H NMR (DMSO-d6) d 2.10–2.14 (m, 1H, H-40), 2.52–
2.61 (m, 1H, H-50), 2.79–2.84 (m, 1H, H-40), 3.01–3.04 (m, 1H, H-50CO),
3.15–3.36 (m, 4H, morph. C3,5–H), 3.65–3.69 (m, 4H, morph. C2,6–H),
4.90–4.92 (m, 1H, NH), 5.11–5.22 (m, 1H, H-30), 5.24 (d, J = 11.7 Hz,
1H, H-40), 2.51–2.69 (m, 1H, H-50), 2.88–2.91 (m, 1H, H-40), 3.01–3.04
(m, 1H, H-50CO), 4.90–4.92 (m, 1H, NH), 5.27–5.32 (m, 1H, H-30), 5.33
(d, J = 11.7 Hz, 1H, NCH2S), 5.47 (d, J = 11.7 Hz, 1H, NCH2S), 6.90–7.55
(m, 9H, Harom), 10.31 (s, 1H, NH aromatic); 13C NMR (DMSO-d6) d
0
0
0
21.55 (C4 ), 29.16 (C5 ), 44.88 (NCH2S), 50.38 (C3 ), 113.22 (ph. C2, C6),
122.81 (ph. C4), 123.79 (C4, C7), 129.23 (ph. C3, C5), 131.55 (C3a, C7a),
0
133.27 (C5, C6), 149.10 (ph. C1), 167.45, 168.26, 169.98 (C1, C3, C2 ,
+
0
1H, NCH2S), 5.34 (d, J = 11.7 Hz, 1H, NCH2S), 7.89–7.96 (m, 4H, Harom);
C6 ), 203.10 (C@S); EIMS m/z = 454 (M ). Anal. Calcd for C21H18N4O4S2:
13
0
0
C NMR (DMSO-d6) d 21.90 (C4 ), 29.27 (C5 ), 45.55 (NCH2S), 50.88
C, 55.49; H, 3.99; N, 12.33; S, 14.11. Found: C, 55.30; H, 4.12; N, 12.20;
S, 14.12.
0
(C3 ), 55.11 (morph. C3, C5), 65.22 (morph. C2, C6), 123.79 (C4, C7),
0
131.76 (C3a, C7a), 133.70 (C5, C6), 167.61, 168.52, 171.12 (C1, C3, C2 ,
+
0
C6 ), 203.16 (C@S); EIMS m/z = 448 (M ). Anal. Calcd for C19H20N4O5S2:
4.2.2.14. [3-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-2,6-diox-
opiperidin-1-yl]methyl benzyldithiocarbamate (17). White
powder; IR (KBr) 3219, 3004, 2965, 2932, 2893, 1784, 1770,
C, 50.88; H, 4.49; N, 12.49; S, 14.30. Found: C, 51.10; H, 4.32; N, 12.25;
S, 14.31.
1714, 1690, 1586, 1343, 1124 cmꢀ1 1H NMR (DMSO-d6) d 2.10–
;
4.2.2.10. [3-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-2,6-diox-
opiperidin-1-yl]methyl 4-methylpiperazin-1-yldithiocarbamate
(13). White powder; IR (KBr) 3418, 2941, 2831, 2772, 2686, 1773,
2.14 (m, 1H, H-40), 2.51–2.61 (m, 1H, H-50), 2.84–2.88 (m, 1H, H-
40), 3.04–3.31 (m, 1H, H-50CO), 4.82 (d, 2H, –NH–CH2–), 4.91–
4.93 (m, 1H, NH), 5.08–5.34 (m, 1H, H-30), 5.31 (d, J = 12.6 Hz, 1H,
1735, 1700, 1654, 1534, 1280, 1183 cmꢀ1
;
1H NMR (DMSO-d6) d
NCH2S), 5.57 (d, J = 12.6 Hz, 1H, NCH2S), 7.28–7.96 (m, 9H, Harom);
13
2.11–2.16 (m, 1H, H-40), 2.22 (s, 3H, CH3), 2.34–2.40 (m, 4H, pip.
C3,5–H), 2.49–2.63 (m, 1H, H-50), 2.75–2.85 (m, 1H, H-40), 2.97 (br s,
4H, pip. C2,6–H), 3.01–3.22 (m, 1H, H-50CO), 4.90 (m, 1H, NH),
5.28–5.32 (m, 1H, H-30), 5.33 (d, J = 11.7 Hz, 1H, NCH2S), 5.47 (d,
J = 11.7 Hz, 1H, NCH2S), 7.91–7.92 (m, 4H, Harom); 13C NMR
C NMR (DMSO-d6) d 21.50 (C4 ), 29.14 (C5 ), 44.43 (NCH2S),
0
0
0
50.34 (C3 ), 52.34 (NHCH2), 123.66 (C4, C7), 126.72 (ph. C4),
126.91 (ph. C2, C6), 128.56 (ph. C3, C5), 131.54 (C3a, C7a), 133.23
0
0
(C5, C6), 137.93 (ph. C1), 167.44, 168.23, 169.66 (C1, C3, C2 , C6 ),
200.62 (C@S); EIMS m/z = 453 (M+). Anal. Calcd for C22H19N3O4S2:
C, 58.26; H, 4.22; N, 9.27; S, 14.14. Found: C, 58.10; H, 4.30; N,
9.14; S, 14.12.
0
0
(DMSO-d6) d 21.82 (C4 ), 29.26 (C5 ), 45.51 (CH3), 45.55 (NCH2S),
50.84 (C3 ), 52.22 (pip. C3, C5), 56.22 (pip. C2, C6), 123.78 (C4, C7),
0
0
131.75 (C3a, C7a), 133.46 (C5, C6), 167.59, 168.48, 171.10 (C1, C3, C2 ,
C6 ), 203.11 (C@S); EIMS m/z = 461 (M+). Anal. Calcd for
4.2.2.15. [3-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-2,6-diox-
opiperidin-1-yl]methyl phenethyldithiocarbamate (18). White
powder; IR (KBr) 3267, 3004, 2942, 2876, 2697, 1786, 1769, 1716,
0
C20H23N5O4S2: C, 52.04; H, 5.02; N, 15.17; S, 13.89. Found: C,
51.96; H, 5.12; N, 15.10; S, 13.90.
1679, 1554, 1390, 1151 cmꢀ1 1H NMR (DMSO-d6) d 2.09–2.14 (m,
;
4.2.2.11. [3-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-2,6-diox-
opiperidin-1-yl]methyl piperidin-1-carbodithioate (14). White
powder; IR (KBr) 2948, 2843, 2808, 2762, 2737, 1785, 1772, 1717,
1H, H-40), 2.55–2.65 (m, 1H, H-50), 2.80–2.90 (m, 1H, H-40), 2.93–
3.10 (m, 1H, H-50CO), 3.72–3.79 (m, 2H, –CH2-phenyl), 3.88–3.93 (t,
2H, –NH–CH2–), 4.90–4.92 (m, 1H, NH), 5.15–5.36 (m, 1H, H-30),
1695, 1592, 1469, 1141 cmꢀ1
;
1H NMR (DMSO-d6) d 1.54–1.69 (m,
5.33 (d, J = 12.6 Hz, 1H, NCH2S), 5.55 (d, J = 12.6 Hz, 1H, NCH2S),
13
6H, pip. C3,4,5–H), 2.11–2.14 (m, 1H, H-40), 2.51–2.69 (m, 1H, H-50),
2.85–2.91 (m, 1H, H-40), 3.00–3.11 (m, 1H, H-50CO), 3.65–3.85 (m,
4H, pip. C2,6–H), 4.15–4.22 (m, 4H, pip. C2,6–H), 5.27–5.33 (m, 1H, H-
7.20–7.96 (m, 9H, Harom); C NMR (DMSO-d6) d 21.44 (C4 ), 29.11
0
0
0
(C5 ), 34.43 (CH2-phenyl), 44.14 (NCH2S), 44.52 (NHCH2), 50.31 (C3 ),
123.65 (C4, C7), 125.92 (ph. C4), 127.70 (ph. C2, C6), 128.64 (ph. C3,
30), 5.34 (d, J = 11.7 Hz, 1H, NCH2S), 5.49 (d, J = 11.7 Hz, 1H, NCH2S),
C5), 131.52 (C3a, C7a), 133.20 (C5, C6), 139.40 (ph. C1), 167.40, 168.21,
13
+
0
0
0
7.88–7.95 (m, 4H, Harom); C NMR (DMSO-d6) d 21.70 (C4 ), 24.24
(pip. C4), 24.71 (pip. C3, C5), 29.24 (C5 ), 45.43 (NCH2S), 48.63 (pip. C2,
C6), 50.79 (C3 ), 123.75 (C4, C7), 131.72 (C3a, C7a), 133.40 (C5, C6),
169.59 (C1, C3, C2 , C6 ), 200.14 (C@S); EIMS m/z = 467 (M ). Anal. Calcd
for C23H21N3O4S2: C, 59.08; H, 4.53; N, 8.99; S, 13.72. Found: C, 58.94;
H, 4.44; N, 9.14; S, 13.73.
0
0
0
0
167.55, 168.44, 170.66 (C1, C3, C2 , C6 ), 201.71 (C@S); EIMS m/
z = 431 (M+). Anal. Calcd for C20H21N3O4S2: C, 55.67; H, 4.91; N, 9.74;
S, 14.86. Found: C, 55.95; H, 4.77; N, 9.60; S, 14.89.
4.3. Cytotoxic activity
4.3.1. Tumor cells
4.2.2.12.[3-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-2,6-dioxopi-
peridin-1-yl]methyl morpholin-4-carbodithioate (15). White
powder; IR (KBr) 3061, 2946, 2893, 2852, 2759, 1784, 1770, 1714,
EAC cells were supplied by National Cancer institute, Cairo,
Egypt. The cells were maintained in vivo in Swiss albino female
mice by intraperitoneal transplantation (0.2 mL of 2 ꢁ 106 cells/
mL). EAC cells were aspirated from the peritoneal cavity of mice,
washed with saline and given intraperitoneally to develop ascites
tumor.
1690, 1586, 1469, 1110 cmꢀ1 1H NMR (DMSO-d6) d 2.13–2.16 (m,
;
1H, H-40), 2.49–2.61 (m, 1H, H-50), 2.85–2.86 (m, 1H, H-40), 2.97–
3.13 (m, 1H, H-50CO), 3.49–3.52 (m, 4H, morph. C2,6–H), 3.63–3.66
(m, 4H, morph. C2,6–H), 3.75–3.79 (m, 4H, morph. C3,5–H), 4.27–
4.30 (m, 4H, morph. C3,5–H), 5.27–5.34 (m, 1H, H-30), 5.39 (d,
4.3.2. Preparation of suspensions and solutions and
J = 11.7 Hz, 1H, NCH2S), 5.54 (d, J = 11.7 Hz, 1H, NCH2S), 7.90–7.94
methodology of the in vitro cancer screen
13
0
0
(m, 4H, Harom); C NMR (DMSO-d6) d 21.77 (C4 ), 29.24 (C5 ), 45.48
For the cytotoxicity assay against the EAC cell line, thalidomide
and its sulfur analogs were dissolved in DMSO/H2O (0.7:0.3) to ob-
tain the concentration of 10 mmol. These stock solutions were
stored at 4 °C until further use.
Cytotoxic activity of these compounds was assayed by deter-
mining the percentage viability of EAC cells using the Trypan blue
dye exclusion technique.59 Briefly, EAC cells were aspirated asepti-
cally from the peritoneal cavity of the mice and washed with
Hank’s balanced salt solution (HBSS) and centrifuged for 15 min
at 1500 rpm in a cooling centrifuge. The pellet was re-suspended
with HBSS and the process was repeated three times. Finally, the
0
(NCH2S), 49.44 (morph. C3, C5), 50.81 (C3 ), 65.51 (morph. C2, C6),
123.76 (C4, C7), 131.74 (C3a, C7a), 133.41 (C5, C6), 167.56, 168.46,
+
0
0
170.98 (C1, C3, C2 , C6 ), 201.79 (C@S); EIMS m/z = 433 (M ). Anal.
Calcd for C19H19N3O5S2: C, 52.64; H, 4.42; N, 9.69; S, 14.79. Found:
C, 52.51; H, 4.39; N, 9.88; S, 14.80.
4.2.2.13. [3-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-2,6-dioxopi-
peridin-1-yl]methyl 2-phenylhydrazinecarbodithioate (16). White
powder; IR (KBr) 3430, 3004, 2942, 2847, 2697, 1769, 1716, 1691,
1609, 1499, 1343, 1167 cmꢀ1 1H NMR (DMSO-d6) d 2.11–2.16 (m,
;

M. A.-H. Zahran et al. / Bioorg. Med. Chem. 16 (2008) 9708–9718
9717
cells were suspended in known volume of RPMI1640, supple-
mented with 10% fatal bovine serum, 10 g/mL streptomycin and
4.7. Immunohistochemistry (IHC) examination specimens
l
100 U/mL penicillin, and the cell count was adjusted to
2 ꢁ 106 cells/mL. Then, 0.2 mL of this diluted cell suspension was
titrated in 96 flat-bottomed tissue culture plates. Thirty microliters
of each tested compound was added in triplicate and incubated at
37 °C for 24 h in 5% CO2 atmosphere. The Trypan blue dye exclu-
sion test was performed to determine the percentage viability
and cytotoxicity were calculated against control EAC cells.49
IHC staining was performed on formalin fixed, paraffin embed-
ded material that were sectioned at 5 lm thickness and placed onto
positive charged slides. Fas-L, VEGF and Ki67 IHC staining were per-
formed using the Universal Dako cytomation Labelled stretavidin–
BiotinÒ2 system, Horseradish Peroxidase (LSABÒ2 System, HRP Kit,
Cat. No. k0679). All slides were de-paraffinized using xylene and
then dehydrated in decreasing concentrations of ethanol. Antigen
retrieval using microwave heating (20 min; 10 mmol/citrate buffer,
pH 6.0) after inhibition of endogenous peroxidase activity (0.3
hydrogen peroxidase for 15 min) were used. The primary antibodies
were applied to the slides. The slides were incubated overnight with
the primary antibody at room temperature, and washed by using
phosphate buffered solution (PBS) then incubated with secondary
antibody for 15 min followed by PBS wash. Finally the detection of
bound antibody was accomplished using a modified labeled avi-
din–biotin (LAB) reagent for 20 min then PBS wash. A 0.1% solution
of diaminobenzidine (DAB) was used for 5 min as a chromogen.
Slides were counterstained with Mayer’s hematoxylin for 5–
10 min Fas-L, VEGF and Ki67 immunoreactivities were evaluated
using a light microscope by three methods for each marker (inten-
sity, % and H score).66,72–75 Unintentional bias was prevented by cod-
ing tissue samplesso that, IHC study was done withoutknowledge of
the used component characteristics.
4.4. Antitumor activity
The antitumor activity of thalidomide and its sulfur analogs
was evaluated in vivo on mice-bearing solid tumor. Solid tumor
was induced in Swiss albino female mice by implementation of
0.2 mL of EAC cells (2 ꢁ 106 cell/mL) subcutaneously (S.C) be-
tween thighs of the lower limb.60 Animals were divided into 8
groups (n = 8). Seven days post-tumor induction; all groups were
treated with 1.25 mM/kg of thalidomide derivatives by subcuta-
neous daily for 5 constitutive days except positive control group
which treated with DMSO/H2O. Mice were sacrificed, dissected
and change in TV was assessed on day 12 after implantation
of EAC cells. The following formula was used to calculate the
volume of the developed tumor mass61
:
2
Tumor volume ðmm3Þ ¼ 0:52 ꢁ length ꢁ ðwidthÞ :
4.8. Statistical analysis
Data are presented as means SD. The differences between
experimental groups were compared by ANOVA followed by t-Stu-
dent test of significance (p < 0.05).
4.5. Estimation of catalase, SOD and lipid peroxidation levels in
liver homogenate
The livers were excised, rinsed in ice-cold normal saline fol-
lowed by cold 0.15 mol/L Tris–HCl buffer (pH 7.4), blotted dry,
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A 10% w/v homogenate was prepared in
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10 randomly selected microscopic fields under the same magnifi-
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48. Crystal data of derivative 6. C19H21N3O6S2, M = 451.520, Triclinic, a = 9.8839(4),
b = 10.4674(5), c = 11.6383(6) Å, = 92.628(2)°, b = 106.602(2)°,
= 115.725(4)°, V = 1019.21(8) Å3, T = 298 K, Z = 2, k = 0.71073, h = 2.910–
25.028°, (Mo-K 4368 measured
) = 0.30 mmꢀ1 Dx = 1.471 Mg/m3,
reflections, (Rint = 0.037), which were used in all calculation. Full
crystallographic details of have been deposited at the Cambridge
a
c
l
a
,
6
30. Caladas, E. D.; Hosana Conceicua, M.; Miranda, M. C. C.; Souzana, L.; Lima, J. F. J.
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Crystallographic Data Center and allocated the deposition number CCDC
697786.
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