Novel potent organoselenium compounds as cytotoxic agents in prostate cancer cells

Article abstract of DOI:10.1016/j.bmcl.2007.10.022

A series of 17 symmetrical substituted imidothiocarbamate and imidoselenocarbamate derivatives has been synthesized by reacting appropriately substituted acyl chlorides with alkyl imidothiocarbamates and alkyl imidoselenocarbamates. The antitumoral activities of the compounds were evaluated in vitro by examining their cytotoxic effects against human prostate cancer cells (PC-3). Five compounds showed interesting activity levels and 3p (IC₅₀ = 1.85 μM) was 7.3 times more active than the standard etoposide used in the treatment of prostate cancer and emerges as the most interesting compound.

Full text of DOI:10.1016/j.bmcl.2007.10.022

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6853–6859
Novel potent organoselenium compounds as cytotoxic
agents in prostate cancer cells
a
a
a
b
´
Daniel Plano, Carmen Sanmartın, Esther Moreno, Celia Prior,
Alfonso Calvob and Juan Antonio Palopa,*
aSeccio´n de Sı´ntesis, Departamento de Quı´mica Orga´nica y Farmace´utica, University of Navarra,
Irunlarrea, 1, E-31008 Pamplona, Spain
bOncology Division, Center for Applied Medical Research, CIMA, University of Navarra,
Pı´o XII, 53, E-31008 Pamplona, Spain
Received 31 August 2007; revised 4 October 2007; accepted 5 October 2007
Available online 17 October 2007
Abstract—A series of 17 symmetrical substituted imidothiocarbamate and imidoselenocarbamate derivatives has been synthesized
by reacting appropriately substituted acyl chlorides with alkyl imidothiocarbamates and alkyl imidoselenocarbamates. The antitu-
moral activities of the compounds were evaluated in vitro by examining their cytotoxic effects against human prostate cancer cells
(PC-3). Five compounds showed interesting activity levels and 3p (IC50 = 1.85 lM) was 7.3 times more active than the standard eto-
poside used in the treatment of prostate cancer and emerges as the most interesting compound.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Numerous observations in the epidemiological literature
have linked various dietary, lifestyle, genetic, and non-
traditional factors with the risk of developing prostate
cancer.1 Prostate cancer is the most common cancer in
men and the second highest cause of male cancer deaths
in the United States and the United Kingdom. In recent
years, many epidemiological studies2–5 have suggested
that an essential trace element, such as selenium (Se),
acts to protect against cancer, particularly prostate can-
cer.6 There is a marked geographic variability of Se in
food and this is related to local soil content. Se is also
widely available in over-the-counter supplements and
multivitamins. There is increasing evidence to suggest
that Se works by inhibiting important molecular path-
ways of the early carcinogenesis in a variety of experi-
mental models and that the anticancer activity is
dependent on its chemical form. Se occurs in both or-
ganic and inorganic forms. The organic form is found
predominantly in grains, fish, meat, poultry, eggs, and
dairy products and enters the food chain through plant
consumption.7,8 A number of potential mechanisms
have been proposed for its antitumorigenic effects and
these include antiandrogen activity.9,10 It has also been
suggested that selenium may exert growth inhibitory ef-
fects by regulation of p53,11,12 by antioxidant function,13
DNA damage,14 and numerous pathways involve apop-
tosis15–19 as a critical event. The regulatory mechanisms
of apoptosis are extremely complex and for selenium
compounds they mainly involve mitochondrial path-
way,15,16 protein kinases,17 tumor necrosis factor,18
and reactive oxygen species.19 A survey of the diverse lit-
erature in this field shows that very few organoselenium
compounds have been described, but those that have do
show promising activities. Among them are the seleno-
proteins,20 such as selenomethionine and methylseleno-
cysteine, and a number of synthetic derivatives such as
p-xylylbismethylselenide, sodium selenite,21 and methyl-
seleninic acid.22 In addition, the combination of some of
these derivatives with chemotherapeutic agents shows
synergistic activity in prostate cancer. For instance,
methylselenocysteine enhances the effect of docetaxel,23
whereas methylseleninic acid improves substantially
the therapeutic effect of etoposide in vivo.24
In recent reports, we have described25–28 the synthesis,
cytotoxicity, and apoptotic evaluation of a series of sym-
metrical diaryl derivatives. We observed that symmetry
is a structural property that is frequently present in
cytotoxic and proapoptotic drugs.29 In addition, some
Keywords: Organoselenium; Cytotoxics; Prostate.
*
Corresponding author. Tel.: +34 948 425 600; fax: +34 948 425
649; e-mail: jpalop@unav.es
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.022

6854
D. Plano et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6853–6859
R
N
X
O
O
R
COCl
X
CHCl3
R'
+
NH
pyridine
R'
R'
Y
H2N
NH
Y
Y
1a-d
2a-i
3a-q
1a: X = S; R = CH3
1b: X = Se; R = CH3
2a: R' = 2-Cl; Y = N
2b: R' = H; Y = C
2c: R' = 3,5-diOCH3; Y = C
2d: R' = 4-Cl; Y = C
2e: R' = 4-NO2; Y = C
2f: R' = 4-CF3; Y = C
2g: R' = 4-CN; Y = C
2h: R' = 4-CH3; Y = C
1c: X = Se; R = CH2-CH3
1d: X = Se; R = CH(CH3)2
2i: R' = 4-C(CH3)3; Y = C
Scheme 1. Synthesis of bisacylimidocarbamates 3a–q.
reported selenium derivatives have this property: 1,
4-phenylenebis(methylene)selenocyanate,30
groups. In some examples the ring system is a heteroar-
omatic unit such as pyridine.
1,2-[bis(1,
2-benzoiso selenazolone)-3(2H)-ketone]ethane,31 and
alkyl32 and biscyclodextrin33 diselenides.
The synthesis of the bisacylimidocarbamates 3a–q36 was
carried out according to Scheme 1, starting from the
appropriate S-alkyl imidothiocarbamate (1a) or Se-alkyl
imidoselenocarbamate (1b–d) hydroiodides and the cor-
responding acyl chloride (2a–i) in a 1:2.1 molar ratio,
respectively, in chloroform in the presence of pyridine
as a catalyst at room temperature. The compounds were
obtained in yields ranging from 26% to 89%. The
scope and generality of this procedure are illustrated
in Table 1. The purity of the compounds was assessed
by TLC and elemental analyses and their structures were
identified from spectroscopic data.36
Based on these findings we planned to undertake the
synthesis of new symmetrical compounds that contain
selenium. The rationale behind the design of these com-
pounds was to maintain the general structural pattern
described in our previous studies,25–28 which in this pa-
per corresponds to molecules with a central nucleus con-
sisting of an alkyl imidothiocarbamate (alkyl
isothiourea) or alkyl imidoselenocarbamate (alkyl iso-
selenourea) connected by a carbonyl group onto two
identical lateral aromatic or heteroaromatic rings. The
sulfur and selenium substituents were varied between
methyl, ethyl, and isopropyl in order to determine the ef-
fect of the alkyl chain length and its ramifications on the
activity.34 In addition, the differences in the anticarcino-
genic activity between two similar chemical elements,
selenium and sulfur,35 were studied. The lateral rings
in these systems are aromatic rings bearing one or more
electron-donating (methoxy, methyl, tert-butyl) or elec-
tron-withdrawing (nitro, trifluoromethyl, cyano, chloro)
New compounds (3a–q) were evaluated for their in vitro
cytotoxic activity against a human prostate cancer cell
line (PC-3, ATCC, Manassas, VA) using the MTT as-
say.37 Results are tabulated as IC50 values. All experi-
ments were independently performed at least three
times and the values were calculated after 72 h exposure
(compound concentrations of 2, 5, 7, and 10 lM). The
results are shown in Table 2 and in Figure 1. High cyto-
Table 1. Synthesis of title compounds 3a–q
Compound
From
Time (h)
Yield (%)
Mp (ꢁC)
Recrystallization solvent
3a
3b
3c
3d
3e
3f
(1a + 2a)
(1b + 2a)
(1a + 2b)
(1b + 2b)
(1c + 2b)
(1d + 2b)
(1b + 2c)
(1d + 2c)
(1a + 2d)
(1b + 2d)
(1d + 2d)
(1b + 2e)
(1b + 2f)
(1b + 2g)
(1a + 2h)
(1b + 2h)
(1b + 2i)
38
30
48
48
48
48
52
48
15
40
36
15
33
24
48
48
60
38
35
67
86
50
56
45
40
60
35
89
33
26
26
35
77
65
191–192
176–177
147–148
137–138
104–105
97–98
EtOH/N,N-DMF
EtOH/N,N-DMF
EtOH
EtOH
EtOH
EtOH
3g
3h
3i
160–162
149–151
174–175
177–178
155–156
214–215
164–165
179–180
152–153
159–160
141–142
EtOH/N,N-DMF
EtOH/N,N-DMF
EtOH
3j
EtOH
EtOH
3k
3l
EtOH/N,N-DMF
EtOH
EtOH
3m
3n
3o
3p
3q
EtOH/N,N-DMF
EtOH
EtOH

D. Plano et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6853–6859
Table 2. Cytotoxic activity of the compounds 3a–q
6855
R
N
O
X
O
N
H
R'
R'
Y
Y
a
b
Compound
X
Y
R
R0
IC50 (lM)
LD50 (lM)
3a
3b
S
N
N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
—
—
Methyl
Methyl
Methyl
Methyl
Ethyl
2-Cl
2-Cl
H
>10
ndc
>10
nd
4.1
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
>10
nd
Se
S
9.14
>10
3c
3d
Se
Se
Se
Se
Se
S
H
2.50
>10
3e
H
3f
3g
Isopropyl
Methyl
Isopropyl
Methyl
Methyl
Isopropyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
—
H
>10
3,5-diOCH3
3,5-diOCH3
4-Cl
6.50
>10
3h
3i
>10
3j
3k
Se
Se
Se
Se
Se
S
4-Cl
4-Cl
7.60
>10
3l
3m
4-NO2
4-CF3
4-CN
>10
>10
3n
3o
>10
>10
4-CH3
4-CH3
4-C(CH3)3
—
3p
3q
Se
Se
—
—
1.85
>10
8.3837
13.6 2.238
MSAd
Etoposide
—
—
a Cell line PC-3.
b Cell line RWPE-1.
c nd, no data.
d MSA, methylseleninic acid.
toxic activity values were found for five compounds (3b,
3d, 3g, 3j, and 3p) and these ranged from 1.85 to 10 lM.
Comparison of these results with the standards used
showed that five of the compounds are more active than
methylseleninic acid and all of them have IC50 values
lower than that of etoposide. Compound 3p was the
most potent (IC50: 1.85 lM) and was 4.5 times more ac-
methyl > ethyl > isopropyl (3d > 3e > 3f, 3g > 3h, and
3j > 3k). The cytotoxic activity assay for four pairs of
sulfur and selenium analogs (3a–3b, 3c–3d, 3i–3j, and
3o–3p) was also investigated. Replacement of sulfur with
selenium in 3a, 3c, 3i, and 3o had a positive effect on the
activity. S and Se are similar in some aspects but differ-
ent in others. In essence, the trends for oxidized and re-
duced Se and S species are similar, but the proportions
differ quite significantly, suggesting important differ-
ences in the biochemistry of S and Se.40 Ip35 reported
comparative studies between analogous sulfur and sele-
nium compounds and demonstrated that selenium is
much more active than sulfur in inhibiting cancer cell
growth. Furthermore, selenium may have a multi-modal
mechanism in preventing cellular transformation. El-
Bayoumi41 later found evidence to support this idea by
extending the studies to other analogous sulfur and sele-
nium derivatives.
tive
than
standard
methylseleninic
acid
(IC50 = 8.38 lM38) and 7.3 times more active than eto-
poside (IC50 = 13.6 lM39), a drug currently used in the
treatment of prostate cancer.
In the present study a precise structure–activity relation-
ship cannot be defined, although it is possible to high-
light some general trends. A relationship seems to
exist between the presence of substituents with strongly
deactivating electron-withdrawing groups (e.g., nitro
and trifluoromethyl) in the phenyl ring and reduced
activity (3l, 3m) and this is followed closely by the cyano
group (3n). However, electron-donating groups like
methoxy and methyl appear to increase the activity
(3g, 3p). On the other hand, the biological results for
the tert-butyl derivative (3q), an electron-donating but
voluminous substituent, showed this to be inactive and
this result suggests that the size of R0 is also important
for activity.
Some of the compounds (3b, 3d, and 3p) that showed
good in vitro activity were examined in more detail for
toxicity with regard to selectivity and, as an orientative
measure, in a cell culture of one non-tumoral prostate
line (RWPE-1). Drug concentrations studied ranged
from 2 to 10 lM. The results are reported in Table 2
and in Figure 2 and expressed as DL50. All the com-
pounds showed low toxicity with DL50 > 10 lM except
for 3d, whose DL50 was only 1.6 times higher than its
IC50. For this reason, and for its low solubility it has
The effect of changing the alkyl chain length was also
investigated and the order of activity was found to be

6856
D. Plano et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6853–6859
Figure 1. Effects of compounds 3a–q on growth of cells. Dose-dependent effect on PC-3 cells. Data are expressed as percent survival and error bars
show SD (n = 4).
not been selected for studying the mechanism of
action.
proved activity. Compound 3p can be highlighted as a
candidate as an antitumoral agent for prostate cancer.
However, further biological work is urgently needed to
elucidate the mechanism of action. Our efforts are now
focused on evaluating its effects on apoptosis, oxidative
stress, and cell cycle. These results have encouraged us
In conclusion, it has been shown that the potency and
selectivity of these compounds make them valid leads
for the synthesis of new compounds that possess im-

D. Plano et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6853–6859
6857
Chan, T. C.; Chou, K. M.; Chang, J. Y. Mol. Cancer Ther.
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18. Yamaguchi, K.; Uzzo, R. G.; Pimkina, J.; Makhov, P.;
Golovine, K.; Crispen, P.; Kolenko, V. M. Oncogene 2005,
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19. Li, G. X.; Hu, H. B.; Jiang, C.; Schuster, T.; Lu, J. X. Int.
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20. Pinto, J. T.; Sinha, R.; Papp, K.; Facompore, N. D.;
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21. Husbeck, B.; Nonn, L.; Peehl, D. M.; Knox, S. J. Prostate
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J. D. Mol. Biol. Cell 2004, 15, 506.
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Z.; Foster, B. A.; Rustum, Y. M. Mol. Cancer Ther. 2006,
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Figure 2. Effects of compounds 3b, 3d, and 3p on non-tumoral
prostate cells (RWPE-1). Data are expressed as percent survival and
error bars show SD (n = 4).
24. Gonzalez-Moreno, O.; Segura, V.; Serrano, D.; Nguewa,
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to carry out further work in the general area of
selenocompounds.
´
25. Sanmartın, C.; Echeverrıa, M.; Mendıvil, B.; Cordeu, L.;
Cubedo, E.; Garcıa-Foncillas, J.; Font, M.; Palop, J. A.
´
´
´
Bioorg. Med. Chem. 2005, 13, 2031.
´
26. Sanmartın, C.; Ardaiz, E.; Cordeu, L.; Cubedo, E.;
Garcıa-Foncillas, J.; Font, M.; Palop, J. A. Lett. Drug
Acknowledgments
´
Design Discov. 2005, 2, 341.
27. Font, M.; Ardaiz, E.; Cordeu, L.; Cubedo, E.; Garcıa-
´
Foncillas, J.; Sanmartın, C.; Palop, J. A. Bioorg. Med.
Chem. 2006, 14, 1942.
The authors wish to express their gratitude to the Uni-
´
de la Universidad de Navarra, PIUNA) for financial sup-
port for the project and thank the Department of Indus-
try of the Navarra Government for the award of two
grants (D. P. and E. M.).
´
versity of Navarra Research Plan (Plan de Investigacion
´
28. Echeverrıa, M.; Mendıvil, B.; Cordeu, L.; Cubedo, E.;
Garcıa-Foncillas, J.; Font, M.; Sanmartın, C.; Palop, J. A.
´
´
´
Arch. Pharm. 2006, 339, 182.
´
29. Sanmartın, C.; Font, M.; Palop, J. A. Mini-Rev. Med.
Chem. 2006, 6, 639.
´
30. Rao, C. V.; Wang, C. Q.; Simi, B.; Rodrıguez, J. G.;
References and notes
Cooma, I.; El-Bayoumi, K.; Reddy, B. S. Cancer Res.
2001, 61, 3647.
1. Sonn, G. A.; Aronson, W.; Litwin, M. S. Prostate Cancer
Prostatic Dis. 2005, 8, 304.
31. Shi, C.; Yu, L.; Yang, F.; Yan, J.; Zeng, H. Biochem.
Biophys. Res. Commun. 2003, 309, 578.
2. Brinkman, M.; Reulen, R. C.; Kellen, E.; Buntinx, F.;
Zeegers, M. P. Eur. J. Cancer 2006, 42, 2463.
3. Menter, D. G.; Sabichi, A. L.; Lippman, S. M. Cancer
Epidem. Biomar. 2000, 9, 1171.
32. Meotti, F. C.; Stangherlin, E. C.; Zeni, G.; Nogueira, C.
W.; Rocha, J. B. T. Environ. Res. 2004, 94, 276.
33. Liu, J. Q.; Luo, G. M.; Ren, X. J.; Mu, Y.; Bai, Y.; Shen,
J. C. Biochim. Biophys. Acta 2000, 1481, 222.
4. Drake, E. N. Med. Hypotheses 2006, 67, 318.
5. Whanger, P. D. Brit. J. Nutr. 2004, 91, 11.
6. Klein, E. A. Annu. Rev. Med. 2006, 57, 49.
7. Silvera, S. A. N.; Rohan, T. E. Cancer Cause Control 2007,
18, 7.
34. Perrey, D. A.; Scannell, M. P.; Narla, R. K.; Uckun, F. M.
Bioorg. Med. Chem. Lett. 2000, 10, 551.
35. Ip, C.; Ganther, H. E. Carcinogenesis 1992, 13, 1167.
36. General procedure for the synthesis of compounds (1a–
d):To a cooled (0 ꢁC), stirred mixture of thiourea (2.47 g,
32.5 mmol) or selenourea (4.0 g, 32.5 mmol) in dry ethanol
(25 mL) was added dropwise the respective alkyl iodide
(45.0 mmol). The mixture was heated under reflux for
90 min. The solvent was removed in vacuo and the
product recrystallized from ethanol.
´
8. Gonzalez, C. A.; Salas-Salvado, J. Brit. J. Nutr. 2006, 96,
587.
9. Lee, S. O.; Chun, J. Y.; Nadiminty, N.; Trump, D. L.; Ip,
C.; Dong, Y.; Gao, A. C. Prostate 2006, 66, 1070.
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W.; Gao, A. C. Mol. Cancer Ther. 2006, 5, 913.
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R. Cancer Biol. Ther. 2006, 5, 529.
Methyl imidothiocarbamate
(KBr):3311–3102, 1638 cmꢀ1
;
hydroiodide,
1a:IR
1H NMR (400 MHz,
DMSO-d6, d):2.57 (s, 3H, S–CH3), 8.97 (br s, 4H, NHÆHI,
NH2). Anal. Calcd for C2H6N2SÆHI (%):C, 15.52; H, 3.88;
N, 12.07. Found:C, 15.65; H, 3.86; N, 12.05.
12. Smith, M. L.; Lancia, J. K.; Mercer, T. I.; Ip, C.
Anticancer Res. 2004, 24, 1401.
13. Rebsch, C. M.; Penna, F. J.; Copeland, P. R. Cell Res.
2006, 16, 940.
Methyl imidoselenocarbamate
(KBr):3319–3102, 1635 cmꢀ1
;
hydroiodide,
1b:IR
1H NMR (400 MHz,
14. Juang, S. H.; Lung, C. C.; Hsu, P. C.; Hsu, K. S.; Li, Y.
C.; Hong, P. C.; Shiah, H. S.; Kuo, C. C.; Huang, C. W.;
Wang, Y. C.; Huang, L.; Chen, T. S.; Chen, S. F.; Fu, K.
C.; Hsu, C. L.; Lin, M. J.; Chang, C. J.; Ashendel, C. L.;
DMSO-d6, d):2.47 (s, 3H, Se–CH3); 6.57 (br s, 1H, NH);
9.00 (br s, 2H, NH2). Anal. Calcd for C2H6N2SeÆ0.8-
HIÆ0.3NH3 (%):C, 9.81; H, 3.14; N, 13.16. Found:C, 10.04;
H, 2.66; N, 13.44.

6858
D. Plano et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6853–6859
Ethyl imidoselenocarbamate
(KBr):3266–3101, 1636 cmꢀ1
;
hydroiodide,
1c:IR
1.65 [d, 6H, J = 7.0 Hz, Se–CH–(CH3)2]; 4.26 [sept, 1H,
J = 7.0 Hz, Se–CH–(CH3)2]; 7.51 (m, 2H, H3 + H5); 7.58
1H NMR (400 MHz,
0
0
0
DMSO-d6, d):1.42 (t, 3H, Se–CH2–CH3); 3.18 (q, 2H,
Se–CH2-CH3); 6.58 (br s, 1H, NH); 9.06 (br s, 2H, NH2).
Anal. Calcd for C3H8N2SeÆ0.85HIÆ0.55NH3 (%):C, 13.37;
H, 3.90; N, 13.26. Found:C. 13.61; H, 3.59; N, 13.00.
Isopropyl imidoselenocarbamate hydroiodide, 1d:IR
(m, 3H, H3 þ H5 þ H4); 7.67 (m, 1H, H4 ); 8.07 (d, 2H,
0
0
0
0
0
0
J2 –3 ¼ J6 ꢀ5 ¼ 7:2 Hz, H2 þ H6 ); 8.36 (d, 2H, J2–3 = J6–
5 = 7.2 Hz, H2 + H6). Anal. Calcd for C18H18N2O2Se (%):
C, 57.91; H, 4.83; N, 7.51. Found: C, 57.70; H, 4.86; N,
7.51.
(KBr):3262–3108, 1643 cmꢀ1
;
1H NMR (400 MHz,
Methyl N,N0-bis(3,5-dimethoxybenzoyl)imidoselenocarba-
mate, 3g: IR (KBr): 3417, 1688 cmꢀ1; 1H NMR (400 MHz,
CDCl3,d): 2.51 (s, 3H, Se–CH3); 3.89 (s, 12H, OCH3); 6.70
DMSO-d6, d):1.46 [d, 6H, Se–CH–(CH3)2]; 4.07 [m, 1H,
Se–CH–(CH3)2]; 6.58 (br s, 1H, NH); 9.14 (br s, 2H, NH2).
Anal. Calcd for C4H10N2SeÆ0.9HIÆ0.5NH3 (%):C, 16.62;
H, 3.94; N, 12.12. Found:C, 16.84; H, 3.80; N, 12.36.
General procedure for the synthesis of compounds (3a–
q):A solution of the corresponding acyl chloride 2a–i
(6.87 mmol) in chloroform (10 mL) was slowly added
0
0
0
(s, 1H, H4); 6.72 (s, 1H, H4 ); 7.17 (s, 2H, H2 þ H6 ); 7.75
(s, 2H, H2 + H6); 13C NMR (100 MHz, DMSO-d6): d 8.9
0
(Se–CH3); 56.0 (4[OCH3]); 106.0 ðC5 Þ; 108.1 (C5); 129.2
0
0
0
ðC3 þ C7 Þ; 131.3 (C3 + C7); 133.8 ðC2 Þ; 138.9 (C2); 161.0
0
0
ðC4 þ C6 þ C4 þ C6 Þ; 166.2 (C1); 172.5 (C–Se); 176.0
0
dropwise to
a
stirred solution of compounds 1a–d
ðC1 Þ. Anal. Calcd for C20H22N2O6Se (%): C, 51.61; H,
(3.27 mmol) in dry chloroform (15 mL) and pyridine
(5 mL). The mixture was stirred for 15–60 h at room
temperature. Solvents were removed under vacuum by
rotatory evaporation and the residue was treated with
water (50 mL) and purified as indicated in Table 1.
4.73; N, 6.02. Found (%): C, 51.29; H, 4.48; N, 5.78.
Isopropyl N,N0-bis(3,5-dimethoxybenzoyl)imidoselenocar-
bamate, 3h: IR (KBr): 3414, 1689 cmꢀ1
;
1H NMR
(400 MHz, CDCl3, d): 1.64 [s, 6H, Se–CH–(CH3)2]; 3.89
(s, 12H, O–CH3); 4.21 [m, 1H, Se–CH–(CH3)2]; 6.69 (d,
Methyl N,N0-bis(2-chloropyridine-3-carbonyl)-imidothioc-
1H,
J4–2 = J4–6 = 2 Hz,
H4);
6.72
(d,
1H,
arbamate, 3a:IR (KBr):3426, 1697 cmꢀ1
;
1H NMR
J4 –2 ¼ J4 ꢀ6 ¼ 2 Hz;H4 ); 7.17 (d, 2H, H2 þ H6 ); 7.52
(d, 2H, H2 + H6). Anal. Calcd for C22H26N2O6Se (%): C,
53.55; H, 5.27; N, 5.68. Found: C, 53.23; H, 4.98; N, 5.58.
Methyl N,N0-bis(4-chlorobenzoyl)imidothiocarbamate, 3i:
0
0
0
0
0
0
0
(400 MHz, CDCl3, d):2.61 (s, 3H, S–CH3); 7.37 (dd, 1H,
J5–4 = 6.9 Hz, J5–6 = 4 Hz, H5); 7.45 (dd, 1H,
0
0
0
0
0
J5 –4 ¼ 6:9 Hz, J5 –6 ¼ 4 Hz, H5 ); 8.11 (d, 1H, H4); 8.40
0
0
(d, 1H, H4 ); 8.52 (d, 1H, H6 ); 8.61 (d, 1H, H6); 13.72 (br
s, 1H, NH). Anal. Calcd for C14H10Cl2N4O2S (%):C,
45.54; H, 2.73; N, 15.17. Found:C, 45.26; H, 2.78; N,
14.96.
IR (KBr): 1703 cmꢀ1 1H NMR (400 MHz, CDCl3, d):
;
2.67 (s, 3H, S–CH3); 7.47 (d,2H, H3 + H5J3–4 = 8.6 Hz);
0
0
0
0
7.55 (d, 2H, H3 þ H5 ); 7.99 (d, 2H, H2 þ H6 ); 8.28 (d,
2H, H2 + H6). Anal Calcd for C16H12Cl2N2O2S (%): C,
52.32; H, 3.27; N, 7.63. Found: C, 52.41; H, 3.26; N, 7.78.
Methyl N,N0-bis(4-chlorobenzoyl)imidoselenocarbamate,
Methyl N,N0-bis(2-chloropyridine-3-carbonyl)-imidosele-
nocarbamate, 3b:IR (KBr):3419, 1688 cmꢀ1 1H NMR
;
(400 MHz, CDCl3, d):2.46 (s, 3H, Se–CH3); 7.37 (dd, 1H,
J5–4 = 7.4 Hz, J5–6 = 4.1 Hz, H5); 7.46 (dd, 1H,
3j: IR (KBr): 3413, 1691 cmꢀ1 1H NMR (400 MHz,
;
CDCl3, d): 2.51 (s, 3H, Se–CH3); 7.47 (br s, 2H, H3 + H5);
0
0
0
0
0
0
0
0
0
J5 –4 ¼ 7:5 Hz, J5 –6 ¼ 4:1 Hz, H5 ); 8.14 (d, 1H, H4);
7.54 (br s, 2H, H3 þ H5 ); 7.99 (br s, 2H, H2 þ H6 ); 8.27
0
0
8.43 (d, 1H, H4 ); 8.53 (d, 1H, H6 ); 8.62 (d, 1H, H6); 13.88
(br s, 1H, NH). Anal. Calcd for C14H10Cl2N4O2Se (%):C,
40.41; H, 2.42; N, 13.46. Found:C, 40.23; H, 2.34; N,
13.22.
Methyl
(KBr):3430, 1700 cmꢀ1
(br s, 2H, H2 + H6); 13C NMR (100 MHz, CDCl3, d): 9.0
0
0
0
0
(Se–CH3); 129.3 ðC3 þ C7 þ C4 þ C6 þ C4 þ C6 Þ; 129.9
0
0
(C3 + C7); 132.2 (C2); 135.2 ðC2 Þ; 140.0 (C5); 140.8 ðC5 Þ;
0
165.2 (C1); 173.6 (C–Se); 175.7 ðC1 Þ. Anal. Calcd for
N,N0-bisbenzoylimidothiocarbamate,
3c:IR
C16H12Cl2N2O2Se (%): C, 46.38; H, 2.90; N, 6.76. Found:
C, 46.06; H, 2.81; N, 6.72.
;
1H NMR (400 MHz, CDCl3,
d):2.69 (s, 3H, S–CH3); 7.51 (m, 2H, H3 + H5); 7.58 (m,
Isopropyl N,N0-bis(4-chlorobenzoyl)imidoselenocarbamate,
0
0
0
3H, H3 þ H5 þ H4); 7.67 (m, 1H, H4 ); 8.07 (d,
3k: IR (KBr): 3414, 1692 cmꢀ1 1H NMR (400 MHz,
;
0
0
0
0
0
0
2H,J2 –3 ¼ J6 ꢀ5 ¼ 8:4 Hz, H2 þ H6 ); 8.37 (d, 2H, J2–
3 = J6–5 = 8.4 Hz, H2 + H6). Anal. Calcd for C16H14N2O2S
(%): C, 62.54; H, 4.72; N, 9.12. Found: C, 62.54; H, 4.48;
N, 8.99.
CDCl3, d): 1.64 [d, 6H, Se–CH–(CH3)2, J = 7.0 Hz]; 4.21
[m, 1H, Se–CH–(CH3)2]; 7.48 (d, 2H, J3–2 = 6.6 Hz,
0
0
0
0
H3 + H5);7.55 (d, 2H, J3 –2 ¼ 6:6 Hz, H3 þ H5 ); 7.99 (d,
0
0
2H, H2 þ H6 ); 8.26 (d, 2H, H2 + H6). Anal. Calcd for
C18H16Cl2N2O2Se (%): C, 48.87; H, 3.62; N, 6.33. Found:
C, 48.60; H, 3.47; N, 6.21.
Methyl N,N0-bisbenzoylimidoselenocarbamate, 3d: IR
(KBr): 3446, 1691 cmꢀ1; H NMR (400 MHz, CDCl3, d):
2.53 (s, 3H, Se–CH3); 7.51 (m, 2H, H3 + H5); 7.58 (m, 3H,
1
Methyl N,N0-bis(4-nitrobenzoyl)imidoselenocarbamate, 3l:
0
0
0
H3 þ H5 þ H4Þ; 7.67 (m, 1H, H4 Þ; 8.07 (d, 2H,
IR (KBr): 3414, 1691, 1529 cmꢀ1 1H NMR (400 MHz,
:
0
0
0
0
0
0
J2 –3 ¼ J6 ꢀ5 ¼ 7:1 Hz, H2 þ H6 ); 8.38 (d, 2H, J2–
DMSO-d6, d): 2.55 (s, 3H, Se–CH3); 8.11 (m, 4H,
3 = J6–5 = 7.1 Hz, H2 + H6); 13C NMR (100 MHz, CDCl3,
H3 þ H5 þ H3 þ H5 );
8.36
(br
s,
4H,
0
0
0
0
0
0
d): 8.9 (Se–CH3); 128.5 (C4 + C6); 128.8 ðC4 þ C6 Þ; 129.6
H2 þ H6 þ H2 þ H6 ). Anal. Calcd for C16H12N4O6-
SeÆHCl (%): C, 40.72; H, 2.76; N, 11.88. Found: C,
40.73; H, 2.57; N, 11.78.
0
0
0
ðV3 þ C3 þ C7 þ C7 Þ; 130.9 (C5); 131.8 ðC5 Þ; 134.2 (C2);
0
0
136.9 ðC2 Þ; 166.3 (C1); 172.8 (C–Se); 176.6 ðC1 Þ. Anal.
Calcd for C16H14N2O2Se (%): C, 55.65; H, 4.06; N, 8.12.
Found: C, 55.57; H, 4.19; N, 8.03.
Methyl N,N0-bis(4-trifluoromethylbenzoyl)imidoselenocar-
bamate, 3m: IR (KBr): 3451, 1692 cmꢀ1
;
1H NMR
Ethyl N,N0-bisbenzoylimidoselenocarbamate, 3e: IR (KBr):
(400 MHz, CDCl3, d): 2.54 (s, 3H, Se–CH3); 7.76 (d, 2H,
3452,1694 cmꢀ1; H NMR (400 MHz, CDCl3, d): 1.62 (t,
3H, J = 7.5 Hz, Se–CH2-CH3); 3.21 (q, 2H, J = 7.5 Hz,
J3–2 = J5–6 = 7.3 Hz,
0
H3 + H5);
0
7.86
(d,
2H,
1
0
0
0
0
J3 –2 ¼ J5 ꢀ6 ¼ 7:5 Hz, H3 þ H5 ); 8.17 (d, 2H,
0
0
Se–CH2-CH3); 7.51 (m, 2H, H3 + H5); 7.58 (m, 3H,
0
H2 þ H6 ); 8.45 (d, 2H, H2 + H6). Anal. Calcd for
C18H12F6N2O2Se (%): C, 44.92; H, 2.51; N, 5.82. Found:
C, 44.96; H, 2.56; N, 5.92.
0
0
H3 þ H5 þ H4); 7.67 (m, 1H, H4 ); 8.07 (d, 2H,
0
0
0
0
0
0
J2 –3 ¼ J6 ꢀ5 ¼ 7:4 Hz, H2 þ H6 ); 8.36 (d, 2H, J2–
3 = J6–5 = 7.4 Hz, H2 + H6). Anal. Calcd for
C17H16N2O2Se (%): C, 56.82; H, 4.46; N, 7.80. Found:
C, 56.64; H, 4.46; N, 7.85.
Methyl N,N0-bis(4-cyanobenzoyl)imidoselenocarbamate,
3n: IR (KBr): 3416, 2230, 1697 cmꢀ1 1H NMR
;
(400 MHz, CDCl3, d): 2.56 (s, 3H, Se–CH3); 7.81 (d, 2H,
Isopropyl N,N0-bisbenzoylimidoselenocarbamate, 3f: IR
J3–2 = J5–6 = 8.0 Hz,
0
H3 + H5);
0
7.90
(d,
2H,
(KBr): 3430, 1694 cmꢀ1; H NMR (400 MHz, CDCl3, d):
J3 –2 ¼ J5 ꢀ6 ¼ 8:0 Hz, H3 þ H5 ); 8.16 (d, 2H,
1
0
0
0
0

D. Plano et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6853–6859
6859
0
0
H2 þ H6 ); 8.45 (d, 2H, H2 + H6). Anal. Calcd for
C18H12N4O2Se (%): C, 54.70; H, 3.06; N, 14.18. Found:
C, 54.74; H, 3.24; N, 14.13.
compounds. After 72 h of incubation with the compounds,
10 lL MTT solution (5 mg/mL in PBS) was added to each
well and these were stored for an additional 4 h at 37 ꢁC, 5%
CO2. The absorbance of formazan at k = 570 nm was
measured on a Polarstar Galaxy plate reader (BMG
LabTechnologies GmbH). The percentage of viable cells
was calculated to obtain IC50-values in comparison to
untreated control cells. PC-3 cells (human tumorigenic and
metastatic prostate cancer cells) were obtained from Amer-
icanTypeCultureCollection(ATCC), Manassas, USA, and
cultured under standard conditions (Dulbecco’s RPMI
1640 medium, with GlutamaxTM 1, Invitrogen) supple-
mented with 10% fetal bovine serum (Fetalclone III,
SH30109.03, HYCLONE) and 1% Penicillin–Streptomycin
(Invitrogen). RWPE-1 cells (non-tumorigenic human pros-
tate cells) were also obtained from ATCC and cultured in
keratinocyte-SFM Kit (Invitrogen) supplemented with 1%
fetal bovine serum, Fetalclone III, SH30109.03,
HYCLONE, and 1% Penicillin–Streptomycin (Invitrogen).
38. Dong, Y.; Zhang, H.; Hawthorn, L.; Ganther, H. E.; Ip,
C. Cancer Res. 2003, 63, 52.
Methyl N,N0-bis(4-methylbenzoyl)imidothiocarbamate, 3o:
IR (KBr): 3450, 1688 cmꢀ1
;
1H NMR (400 MHz,
CDCl3,d): 2.46 (s, 6H, Ph–CH3), 2.66 (s, 3H, S–CH3),
7.29 (d, 2H, J3–2 = 8.2 Hz, H3 + H5), 7.36 (d, 2H,
0
0
0
0
0
0
J3 –2 ¼ 8:2 Hz, H3 þ H5 ), 7.95 (d, 2H, H2 þ H6 ), 8.25
(d, 2H, H2 + H6). Anal. Calcd for C18H18N2O2S (%): C,
66.26; H, 5.52; N, 8.59. Found: C, 66.01; H, 5.64; N, 8.37.
Methyl N,N0-bis(4-methylbenzoyl)imidoselenocarbamate,
3p: IR (KBr): 3446, 1679 cmꢀ1 1H NMR (400 MHz,
;
CDCl3, d): 2.46 (s, 6H, Ph–CH3); 2.51 (s, 3H, Se–CH3);
7.30 (d, 2H, J3–2 = 7.4 Hz, H3 + H5); 7.36 (d, 2H,
J3 –2 ¼ 7:4 Hz, H3 þ H5 ); 7.96 (d, 2H, H2 þ H6 ); 8.27
(d, 2H, H2 + H6). Anal. Calcd for C18H18N2O2Se (%): C,
57.92; H, 4.86; N, 7.51. Found: C, 58.33; H, 4.64; N, 7.56.
Methyl
mate, 3q: IR (KBr): 3448, 2957–2867, 1692 cmꢀ1
NMR (400 MHz, CDCl3, d): 1.38 [s, 18H, C-(CH3)3]; 2.51
(s, 3H, Se–CH3); 7.53 (d, 2H, J3–2 = 8.1 Hz, H3 + H5); 7.59
0
0
0
0
0
0
N,N0-bis(4-tert-butylbenzoyl)imidoselenocarba-
.
1H
0
0
0
0
0
0
(d, 2H, J3 –2 ¼ 8:1 Hz, H3 þ H5 ); 8.00 (d, 2H, H2 þ H6 );
8.31 (d, 2H, H2 + H6). Anal. Calcd for C24H30N2O2Se (%):
C, 63.02; H, 6.61; N, 6.12. Found: C, 62.97; H, 6.74; N, 6.07.
37. Materials and methods for cytotoxicity assay (MTT assay):
PC-3cellswereseededin96-wellplates(Millipore, Eschborn,
Germany) at a density of 5 · 103 cells per well in 200 lL
of complete medium and the samples were incubated at
37 ꢁC under 5% CO2 overnight prior to the addition of the
39. Van Brussel, J. P.; Oomen, M. A.; Vosselbeld, P. J. M.;
Wiemer, E. A. C.; Sonneveld, P.; Mickisch, G. H. J. BJU
Int. 2004, 93, 1333.
40. Pickering, I. J.; Wright, C.; Bubner, B.; Ellis, D.; Persans,
M. W.; Yu, E. Y.; George, G. N.; Prince, R. C.; Salt, D. E.
Plant Physiol. 2003, 131, 1460.
41. El-Bayoumi, K.; Sinha, R.; Pinto, J. T.; Rivlin, R. S. J.
Nutr. 2006, 136, 864S.