2
042
D. Desai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2038–2043
1
1
1. El-Bayoumy, K. E.; Sinha, R.; Pinto, J. T.; Rivlin, R. S. J. Nutri. 2006, 864S.
2. Clark, L. C.; Combs, G. F., Jr.; Tumbull, B. W.; Slate, E. H.; Chalker, D. K.; Chow, J.;
Davis, L. S.; Glover, R. A.; Graham, G. F.; Gross, E. G.; Krongrad, A.; Lesher, J. L.,
Jr.; Park, H. K.; Sanders, B. B., Jr.; Smith, C. L.; Taylor, J. R. J. Am. Med. Assoc. 1996,
and dried to yield 1 (1.19 g, 78%) as white powder, mp, 234–236 °C;54 1H NMR
(D O): 7.25 (s, 4H, aromatic), 3.37 (t, 4H, Ph-CH2, J = 7.2 Hz), 3.00 (t, 4H, C–CH2,
J = 6.86 Hz); MS m/z (intensity) 283 (M , 100), 266 (40), 207 (30), 142 (70),
2
+
1
131 (100), 104 (10), 77 (30).
0
276, 1957.
56. Synthesis of Se,Se -1,4-phenylene-bis(1,2-ethanediyl)bis-isoselenourea dihydrobr-
1
3. Clark, L. C.; Dalkin, B.; Krongrad, G. F.; Combs, G. F., Jr.; Tumbull, B. W.; Slate, E.
H.; Witherington, R.; Herlong, J. H.; Janosko, E.; Carpenter, D.; Borosso, C.; Falk,
S.; Rounder, J. Br. J. Urol. 1998, 81, 730.
omide (PBISe, 2): compound 2 was prepared in a similar manner as reported for
compound 1 by reacting 1,4-di(2-bromoethyl)benzene (7)(1.0 g, 3.43 mmol)
1
with selenourea (0.84 g, 6.86 mmol) to yield 2 (1.3 g, 69%), mp, 247–249 °C;
H
1
4. Narisawa, T.; Sato, M.; Tani, M.; Kudo, T.; Takahashi, T.; Goto, A. Cancer Res.
NMR (D
2
O): 7.25 (s, 4H, aromatic), 3.37 (t, 4H, Ph-CH2, J = 7.2 Hz), 3.21 (t, 4H,
+
1
981, 41, 1954.
C–CH2, J = 6.86 Hz); MS m/z; ion intensity 377 and 379 (M , 50), 335 and 337
(80), 253 and 255 (70), 188 and 190 (100), 131 (50), 73 (10). ESI-MS: m/z, calcd:
378.9934 for C12H N Se ; found: 378.9935.
19 4 2
1
1
1
1
1
2
2
2
5. Wattenberg, L. W. Cancer Res. 1983, 43, 2448s.
6. Rao, C. V.; Tokumo, K.; Kelloff, G.; Reddy, B. S. Carcinogenesis 1991, 12, 1051.
7. Kudo, T.; Narisawa, T.; Abo, S. Gann 1980, 71, 260.
8. Reddy, B. S.; Maruyama, H.; Kelloff, G. J. Cancer Res. 1987, 47, 5340.
9. Reddy, B. S.; Rao, C. V.; Rivenson, A.; Kelloff, G. J. Cancer Res. 1993, 53, 3493.
0. Pollard, M.; Luckert, P. H. J. Natl. Cancer Inst. 1983, 70, 1103.
1. Nigro, N.; Bull, A. W.; Boyd, M. E. J. Natl. Cancer Inst. 1986, 77, 1309.
2. Kingsworth, A. N.; King, W. K.; Diekema, K. A.; McCann, P. P.; Ross, J. S.; Malt, R.
A. Cancer Res. 1983, 43, 2545.
3. Nayini, J. R.; Sugie, S.; El-Bayoumy, K. E.; Rao, C. V.; Rigotty, J.; Sohn, O. S.;
Reddy, B. S. Nutr. Cancer 1991, 15, 129.
4. Wattenberg, L. W. In Cancer Chemoprevention; Wattenberg, L. W., Lipkin, M.,
Boone, C. W., Kelloff, G. J., Eds.; CRC press: Boca Raton, FL, 1990; pp 19–39.
5. Newmark, H. L. Nutr. Cancer 1984, 6, 58.
57. Synthesis of Se-2-phenylethylisoselenourea hydrobromide (PEISe, 4): compound 4
was prepared in similar manner as reported above for compound 2 by reacting
2-bromoethyl benzene (1.0 g, 5.4 mmol) with selenourea (0.67 g, 5.4 mmol) to
yield 4 (1.26 g, 80%); mp, 140–142 °C; H NMR (D
aromatic), 3.02 (t, 2H, CH
(m/z, intensity): 229 (M , 100), 181 (80), 159 (10), 105 (10).
1
2
O): 7.29–7.20 (m, 5H,
-Se, J = 7.0 Hz); EMS
2
-Ph, J = 7.0 Hz), 3.48 (t, 2H, CH
2
+
58. Cell lines and culture conditions: colon adenocarcinoma cell line (Caco-2; ATCC
No. HTB-37, HT-29; ATCC No. HTB-38, HCT-116; ATCC No. CCL-247, and SW-
480; CCL-228) were grown in Advanced DMEM (2 Mm) supplemented with
10% heat treated (56 °C for 30 min) FBS (Hyclone, Logan, UT) and L-glutamine
or RPMI-1640 containing 10% FBS. Lung adenocarcinoma (A549; ATCC No. CCL-
185), fibrosarcoma (HT-1080; ATCC No. CCL-121), prostate adenocarcinoma
(PC-3; ATCC No. CRL-1435), ovarian adenocarcinoma (NIH: OVCAR-3; ATCC No.
HTB-161), and a breast adenocarcinoma cell line (MDA-MB-231; ATCC No.
HTB-26) were grown in DMEM supplemented with 10% FBS. The human
vertical growth phase (VGP) melanoma cell line WM115 was maintained in
Tu2% medium lacking calcium chloride, but supplemented with 2% heat
2
2
2
2
2
2
2
3
6. Sporn, M. B. Cancer Res. 1976, 36, 2699.
7. Reddy, B. S.; Rao, C. V.; Seibert, K. Cancer Res. 1996, 56, 4566.
8. Kawamori, T.; Rao, C. V.; Seibert, K.; Reddy, B. S. Carcinogenesis 1998, 58, 409.
9. Rao, C. V.; Kawamori, T.; Hamid, R.; Reddy, B. S. Carcinogenesis 1999, 20, 641.
0. Reddy, B. S.; Hirsoe, Y.; Lubet, R.; Steele, V. E.; Kelloff, G. J.; Paulson, S.; Seibert,
K.; Rao, C. V. Cancer Res. 2000, 60, 293.
treated (56 °C for 30 min) FBS and L-glutamine as described previously.
3
1. Rao, C. V.; Indranie, C.; Simi, B.; Manning, P. T.; Connor, J. R.; Reddy, B. S. Cancer
Res. 2002, 62, 165.
59. To investigate the structural requirements, we first compared the cell viability
of colon adenocarcinoma cells (Caco-2, HT-29, HCT-116, SW-480) upon
treating with PBIT (1), PBISe (2), and their corresponding monosubstituted
compounds PEIT (3), and PEISe (4) (Table 1). The IC50 values were calculated by
3
2. Chen, T.; Nines, R. G.; Peschke, S. M.; Kresty, L. A.; Stoner, G. D. Cancer Res. 2004,
64, 3714.
3
3
3. Southan, G. J.; Salzman, A. L.; Szabo, C. Life Sci. 1996, 58, 1139.
4. Reinhold, U.; Biltz, H.; Bayer, W.; Schmidt, K. H. Acta Derm. Venereol. 1989, 69,
treating with increasing concentrations of PBIT, PEIT, PEISe (10–100
lM) or
PBISe (1.6–50 M) for 72 h and the number of viable cells quantified using 3-
l
1
32.
(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium (MTS) (Promega, WI).
3
3
3
5. Reid, M. E.; Duffield-Lillico, A. J.; Slate, E.; Natarajan, N.; Turnbull, B.; Jacobs, E.;
Combs, G. F., Jr.; Alberts, D. S.; Clark, L. C.; Marshall, J. R. Nutr. Cancer 2008, 60,
60. Representative example of inhibitory effect of PBISe and PBIT against Caco-2
3
1
55.
cells is shown in Figure 2. In brief, the assay was carried out by plating 5 ꢁ 10
6. Camargo, M. C.; Burk, R. F.; Bravo, L. E.; Piazuelo, M. B.; Hill, K. E.; Fontham, E.
T.; Motley, A. K.; Yepez, M. C.; Mora, Y.; Schneider, B. G.; Correa, P. Arch. Med.
Res. 2008, 39, 443.
7. Reddy, B. S.; Rivenson, A.; El-Bayoumy, K. E.; Upadhyaya, P.; Pittman, B.; Rao, C.
V. J. Natl. Cancer Inst. 1997, 89, 506.
cells per well in 100 lL media for 24 h followed by treatment with either
vehicle control PBS or with increasing concentrations of PBIT (0–120
lM) or
PBISe (0–10 M) for 72 h. The percentages of viable cells compared to control
l
PBS treated cells were determined using MTS assay and IC50 values calculated
using GraphPad Prism version 4.01 (GraphPad software, San Diego, CA). IC50
value for each compound was determined from at least three independent
experiments and represented with a standard error (Fig. 2).
3
3
4
4
4
4
4
4
8. El-Bayoumy, K. E. Nutr. Cancer 2001, 40, 4.
9. El-Bayoumy, K. E.; Sinha, R.; Pinto, J. T.; Rivlin, R. S. J. Nutr. 2006, 136, 864S.
0. Rayman, M. P. Proc. Nutr. Soc. 2005, 64, 527.
61. In vitro inhibitory effect of PBIT and PBISe on various cancer cell lines: by following
similar procedure as reported above against colon cancer cells;60 growth
inhibitory effects on normal fibroblast cells (FF2441), and cultured cancer cells
from human vertical growth phase (VGP) melanoma (WM115), Lung
adenocarcinoma (A549), fibrosarcoma (HT-1080), prostate adenocarcinoma
(PC-3), ovarian carcinoma (OVCAR-3), and breast adenocarcinoma (MDA-MB-
1. Letavayova, L.; Vlckova, V.; Brozmanova, J. Toxicology 2006, 227, 1.
2. Aboul-Fadl, T. Curr. Med. Chem. Anticancer Agents 2005, 5, 637.
3. Yan, L.; Yee, J. A.; McGuire, M. H.; Graef, G. L. Nutr. Cancer 1997, 28, 165.
4. Davis, C. D.; Zeng, H.; Finley, J. W. J. Nutr. 2002, 132, 307.
5. Higdon, J. V.; Delage, B.; Williams, D. E.; Dashwood, R. H. Pharmacol Res. 2007,
5
5, 224.
231) were tested with increasing concentrations of PBIT (10–100
(1–40 M) (Table 2, Fig. 3).
lM) or PBISe
4
4
4
4
6. Finley, J. W.; Davis, C. D. Biofactors 2001, 14, 191.
l
7. Zeng, H.; Davis, C. D.; Finley, J. W. J. Nutr. Biochem. 2003, 14, 227.
8. Wu, Y.; Zu, K.; Warren, M. A.; Wallace, P. K.; Ip, C. Mol. Cancer Ther. 2006, 5, 246.
9. Roy, H. K.; Olusola, B. F.; Clemens, D. L.; Karolski, W. J.; Ratashak, A.; Lynch, H.
T.; Smyrk, T. C. Carcinogenesis 2002, 23, 201.
0. Parsons, D. W.; Wang, T. L.; Samuels, Y. Nature 2005, 436, 792.
1. Ju, J.; Hong, J.; Zhou, J.-N.; Pan, Z.; Bose, M.; Liao, J.; Yang, G.-Y.; Liu, Y. Y.; Hou,
Z.; Lin, Y.; Ma, J.; Shih, W. J.; Carothers, A. M.; Yang, C. S. Cancer Res. 2005, 65,
62. Inhibition of nitrite production by PBISe in Caco-2 cells: to demonstrate that
PBISe and PBIT similarly inhibited nitrite production in Caco-2 cells, the iNOS
activity in terms of total nitrite (nitrate + nitrite) produced was measured
using
a colorimetric assay (Cayman chemical company, Ann Arbor, MI)
6
5
5
(Fig. 4). Caco-2 cells (1 ꢁ 10 ) were plated in 60 mm culture dishes in
advanced DMEM containing 10% heat treated FBS. Twelve hours later,
growing cells were conditioned (ꢀ12 h) with phenol-red free DMEM
containing 0.5% FBS and then treated with increasing concentrations of
1
0623.
5
5
2. Altomare, D. A.; Testa, J. R. Oncogene 2005, 24, 7455.
3. General synthesis: melting points were recorded on a Fisher–Johnson melting
point apparatus and are uncorrected. Unless stated otherwise, proton NMR
PBIT (40
containing 0.5% FBS for 72 h. Culture supernatants were collected, centrifuged
(500g) and total nitrite (nitrate + nitrite) measured by incubating 80
supernatant with enzyme cofactor mixture (10 L) and nitrate reductase
(10 L) for 2 h. The addition of Griess reagent-I and II (50 L each) produced
characteristic color that was measured at 540 nm using a SPECTRAmax M2
plate reader (Molecular Devices, Sunnyvale, CA). A nitrate standard curve (5–
35 lM) was simultaneously prepared and total nitrite present in samples was
lM) or PBISe (2 lM) dissolved in phenol-red free DMEM (2 mL)
lL
spectra were recorded in CDCl
chemical shifts are reported in ppm downfield from TMS. MS were run on
000 Q trap hybrid triple quadrupole/linear ion trap instrument (Applied
3
using a Bruker 500 MHz instruments. The
l
l
l
4
Biosystems/MDS Sciex) at the proteomic facility in Penn State Cancer Institute
at Penn State College of Medicine, Hershey, PA. High-resolution MS were
determined at the Instrument Center, University of Buffalo, Buffalo, NY. Thin-
layer chromatography (TLC) was on aluminum-supported, pre-coated silica gel
plates (EM Industries, Gibbstown, NJ). All starting materials and reagents were
obtained from Aldrich Chemical Co. (Milwaukee, WI) and used without further
purification.
measured.
63. Western blot analysis: cell lysates were harvested by addition of lyses buffer
containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM EDTA, 10% glycerol,
1% Triton X-100, 1 mM sodium orthovanadate, 0.1 mM sodium molybdate,
1 mM phenylmethylsulfonyl fluoride, 20 lg/mL aprotinin, and 5 lg/mL
5
5
4. Methyl 1,4-phenylenediacetic acid (5), 1,4-phenylene diethanol (6), and 1,4-
di(2-bromoethyl)benzene (7) were prepared as reported by E. P. Garvey, J. A.
Oplinger, G. J. Tanoury, P. A. Sherman, M. Fowler, S. Marshall, M. F. Harmon, J. E.
Paith, E. S. Furfine, J. Biol. Chem., 1994, 269, 26669.
leupeptin. Whole cell lysates were centrifuged (P10,000g) for 10 min at 4 °C
to remove cell debris. Protein concentrations were quantitated using the BCA
assay (Pierce; Rockford, IL), and 30 lg of lysate loaded per lane onto NuPAGE
Gels (Life Technologies, Inc. Carlsbad, CA). Following electrophoresis, samples
were transferred to polyvinylidene difluoride (PVDF) membrane (Pall
Corporation, Pensacola, FL) and the blots probed with antibodies according
to each supplier’s recommendations: antibodies to PRAS40 and
phosphorylated PRAS40 from Invitrogen (Invitrogen Corporation, Carlsbad,
CA); antibodies to cyclin D1, p27, and Erk2 from Santa Cruz Biotechnology
0
5. Synthesis of S,S -1,4-phenylene-bis(1,2-ethanediyl)bis-isothiourea dihydrobromide
(
PBIT, 1): to a mixture of 1,4-di(2-bromoethyl)benzene (7) (1.0 g, 3.43 mmol)
in ethanol (50 mL) was added thiourea (0.52 g, 6.86 mmol). The reaction
mixture was heated to reflux for 2 h. After concentration of ethanol (20 mL),
the precipitated solid was filtered, washed with hexane, methylene chloride,