3
526
H. Schott et al. / Bioorg. Med. Chem. 19 (2011) 3520–3526
1
3
H5), 7.64–7.75 (m, 1H, H6); C NMR (100 MHz, D
2
O): d = 32.4
3. Widler, L.; Jaeggi, K. A.; Glatt, M.; Muller, K.; Bachmann, R.; Bisping, M.; Born, A.
R.; Cortesi, R.; Guiglia, G.; Jeker, H.; Klein, R.; Ramseier, U.; Schmid, J.; Schreiber,
G.; Seltenmeyer, Y.; Green, J. R. J. Med. Chem. 2002, 45, 3721.
0
0
00
0
0
0
0
(
(
(
C1 ), 38.4 (C2 ), 60.8 (C5 ), 75.4 (C2 ), 75.5 (C3 ), 83.6 (C4 ), 85.6
C1 ), 96.5 (C5), 140.7 (C6), 157.2 (C2), 163.2 (C4). P NMR
0
31
4
.
Brown, J. E.; Cook, R. J.; Major, P.; Lipton, A.; Saad, F.; Smith, M.; Lee, K. A.;
Zheng, M.; Hei, Y. J.; Coleman, R. E. J. Natl. Cancer Inst. 2005, 97, 59.
Coleman, R. E. Br. J. Cancer 2008, 98, 1736.
161 MHz, D
2
O) d = 17.8 ppm.
5
6
.
.
Adzamli, I. K.; Blau, M.; Pfeffer, M. A.; Davis, M. A. Magn. Reson. Med. 1993, 29,
4
3
.2.9. Synthesis of N -[propyl-(3-hydroxy-3-phosphono)-
phosphonate]-5-methyl-3 -azido-2 ,3 -dideoxycytidine
505.
0
0
0
7. Adzamli, I. K.; Johnson, D.; Blau, M. Invest. Radiol. 1991, 26, 143.
8
9
.
.
Bhushan, K. R.; Tanaka, E.; Frangioni, J. V. Angew. Chem., Int. Ed. 2007, 46, 7969.
Kubicek, V.; Rudovsky, J.; Kotek, J.; Hermann, P.; Vander Elst, L.; Muller, R. N.;
Kolar, Z. I.; Wolterbeek, H. T.; Peters, J. A.; Lukes, I. J. Am. Chem. Soc. 2005, 127,
16477.
(
AZT-pamidronate) (7c)
By substitution of 3c (1.8 g, 5 mmol) with 5 (1.4 g, 5 mmol) in
analogy to the preparation of 6a the BPs derivat 7c was obtained.
ꢀ
ꢀ
ꢀ
10. Zhang, S.; Gangal, G.; Uludag, H. Chem. Soc. Rev. 2007, 36, 507.
11. Vitha, T.; Kubicek, V.; Hermann, P.; Kolar, Z. I.; Wolterbeek, H. T.; Peters, J. A.;
Lukes, I. Langmuir 2008, 24, 1952.
12. Ghosh, S.; Chan, J. M.; Lea, C. R.; Meints, G. A.; Lewis, J. C.; Tovian, Z. S.; Flessner,
R. M.; Loftus, T. C.; Bruchhaus, I.; Kendrick, H.; Croft, S. L.; Kemp, R. G.;
Kobayashi, S.; Nozaki, T.; Oldfield, E. J. Med. Chem. 2004, 47, 175.
Yield 1.2 g (41%). MS (FAB ) 483.0 [MꢀH ]; 505.1 [M+Na ],
ꢀ
5
27.1 [M+2Na ]. Anal. Calcd C13
H
20
N
6
Na
2
O
10
2
P ⁄3.5H
2
O (591.34).
1
C, 26.41, H, 4.60, N, 14.21. Found: C, 25.98, H, 4.46, N, 14.75.
O): d = 1.93 (s, 3H, CH
H2 ), 2.35–2.52 (m, 2H, H1 ), 3.69–3.89 (m, 4H, H2 + H5 ), 3.97–
.03 (m, 1H, H3 ), 4.28–4.35 (m, 1H, H4 ), 4.8 (s, br, OH + NH + D O),
2
.16–6.22 (m, 1H, H1 ), 7.49 (s, 1H, H6). C NMR (100 MHz, D
H
NMR (400 MHz, D
2
3
), 2.17–2.31 (m, 2H,
0
0
00
0
0
1
3. Sanders, J. M.; Gomez, A. O.; Mao, J.; Meints, G. A.; Van Brussel, E. M.;
Burzynska, A.; Kafarski, P.; Gonzalez-Pacanowska, D.; Oldfield, E. J. Med. Chem.
2003, 46, 5171.
0
0
4
6
0
13
2
O):
0
0
00
0
0
0
14. Song, Y.; Chan, J. M.; Tovian, Z.; Secrest, A.; Nagy, E.; Krysiak, K.; Bergan, K.;
d = 12.3 (C7), 32.6 (C2 ), 36.4 (C1 ), 37.2 (C2 ), 59.9 (C5 ), 61.0 (C3 ),
Parniak, M. A.; Oldfield, E. Bioorg. Med. Chem. 2008, 16, 8959.
0
0
7
1
3.2 (t, JCP = 130 Hz), 83.9 (C1 ), 85.3 (C4 ), 105.6 (C5), 136.3 (C6),
15. Budman, D. R.; Calabro, A. Oncology 2006, 70, 147.
3
1
2
57.5 (C2), 163.1 (C4). P NMR (161 MHz, D O) d = 18.0 ppm.
16. Clezardin, P. Cancer Treat. Rev. 2005, 31, 1.
1
1
7. Coleman, R.; Gnant, M. Curr. Opin. Support. Palliat. Care 2009, 3, 213.
8. Neville-Webbe, H. L.; Rostami-Hodjegan, A.; Evans, C. A.; Coleman, R. E.; Holen,
I. Int. J. Cancer 2005, 113, 364.
3
3
.3. Hydroxyapatite binding
19. Roelofs, A. J.; Thompson, K.; Gordon, S.; Rogers, M. J. Clin. Cancer Res. 2006, 12,
6222s.
.3.1. 5-FdU and untreated 5-FdU-alendronate
-FdU (246 mg, 1 mmol) or 5-FdU-ale (475 mg) were dissolved
20. Santini, D.; Vespasiani Gentilucci, U.; Vincenzi, B.; Picardi, A.; Vasaturo, F.; La
5
Cesa, A.; Onori, N.; Scarpa, S.; Tonini, G. Ann. Oncol. 2003, 14, 1468.
in distilled water (100 ml). The A260-units of the obtained solution
and the absorbance ratios of 250/260 and 280/260 were deter-
mined using an UV-spectrophotometer. After addition of solid
hydroxyapatite as listed in Table 1 the obtained suspension was
stirred at room temperature for 24 h. Then, an aliquot (1 ml) of
21. Vogt, U.; Bielawski, K. P.; Bosse, U.; Schlotter, C. M. Oncol. Rep. 2004, 12, 1109.
22. Winter, M. C.; Coleman, R. E. Curr. Opin. Oncol. 2009, 21, 499.
23. Winter, M. C.; Holen, I.; Coleman, R. E. Cancer Treat. Rev. 2008, 34, 453.
24. Hirabayashi, H.; Fujisaki, J. Clin. Pharmacokinet. 2003, 42, 1319.
25. Sturtz, G.; Appéré, G.; Breistol, K.; Fodstad, O.; Schwartsmann, G.; Hendriks, H.
R. Eur. J. Med. Chem. 1992, 27, 825.
26. Sturtz, G.; Couthon, H.; Fabulet, O.; Mian, M.; Rosini, S. Eur. J. Med. Chem. 1993,
the suspension was filtered through a sterile filter and the A260
-
2
8, 899.
units of the filtrate were measured. The amount of drug bound to
hydroxyapatite was calculated on the basis of the decreased
27. Fabulet, O.; Sturtz, G. Phosphorus, Sulfur Silicon 1995, 101, 225.
28. Morioka, M.; Kamizono, A.; Takikawa, H.; Mori, A.; Ueno, H.; Kadowaki, S.;
Nakao, Y.; Kato, K.; Umezawa, K. Bioorg. Med. Chem. 2010, 18, 1143.
A260-units of the suspension.
29. Gil, L.; Han, Y.; Opas, E. E.; Rodan, G. A.; Ruel, R.; Seedor, J. G.; Tyler, P. C.;
Young, R. N. Bioorg. Med. Chem. 1999, 7, 901.
3
.3.2. Acidified pretreatment 5-FdU-alendronate
A solution of 5-FdU-ale (475 mg) in 100 ml H O was acidified to
30. Klenner, T.; Wingen, F.; Keppler, B.; Valenzuela-Paz, P.; Amelung, F.; Schmahl,
D. Clin. Exp. Metastasis 1990, 8, 345.
2
31. Klenner, T.; Valenzuela-Paz, P.; Keppler, B. K.; Angres, G.; Scherf, H. R.; Wingen,
pH 2 by adding HCl and stirred at 50 °C for 24 h, followed by neu-
tralization with solid sodium carbonate. UV-active degradation
products were not detectable at 254 nm by TLC-analysis with
CHCL /MeOH (80:20) using silica gel plates. The hydroxyapatite
3
binding of the pretreated 5-FdU-ale was analyzed as described
above.
F.; Amelung, F.; Schmahl, D. Cancer Treat. Rev. 1990, 17, 253.
32. Klenner, T.; Wingen, F.; Keppler, B. K.; Krempien, B.; Schmahl, D. J. Cancer Res.
Clin. Oncol. 1990, 116, 341.
3
3
3. Uludag, H.; Kousinioris, N.; Gao, T.; Kantoci, D. Biotechnol. Prog. 2000, 16, 258.
4. Ora, M.; Lonnberg, T.; Florea-Wang, D.; Zinnen, S.; Karpeisky, A.; Lonnberg, H. J.
Org. Chem. 2008, 73, 4123.
3
5. Reinholz, M. M.; Zinnen, S. P.; Dueck, A. C.; Dingli, D.; Reinholz, G. G.; Jonart, L.
A.; Kitzmann, K. A.; Bruzek, A. K.; Negron, V.; Abdalla, A. K.; Arendt, B. K.;
Croatt, A. J.; Sanchez-Perez, L.; Sebesta, D. P.; Lonnberg, H.; Yoneda, T.; Nath, K.
A.; Jelinek, D. F.; Russell, S. J.; Ingle, J. N.; Spelsberg, T. C.; Dixon, H. B.;
Karpeisky, A.; Lingle, W. L. Bone 2010, 47, 12.
3
3
.4. In vitro cytotoxicity testing
36. Papapoulos, S. E. Bone 2006, 38, 613.
.4.1. Cell viability assay
37. Migianu, E.; Monteil, M.; Even, P.; Lecouvey, M. Nucleosides Nucleotides Nucleic
Acids 2005, 24, 121.
Lewis Lung carcinoma cells (LLC) were cultivated in RPMI-140
with
in DMEM (Dulbecco’s Modified Eagle’s Medium) containing
.5 mg/L glucose. Both media were supplemented with 10% foetal
bovine serum (FBS) and with 1% antibiotics (10,000 U/ml penicillin,
0,000 g/ml streptomycin).
For the cell viability tests the cells were seeded at 5–10,000 cells
L
-glutamine and RAW 264.7 macrophages were cultivated
38. Kalek, M.; Jemielity, J.; Stepinski, J.; Stolarski, R.; Darzynkiewicz, E. Tetrahedron
Lett. 2005, 46, 2417.
3
4
9. Chen, H.; Wu, L.-Y.; Zeng, J.-C.; Wu, Y. West China J. Pharm. Sci. 2005, 2, 10.
0. Divakar, K. J.; Reese, C. B. J. Chem. Soc., Perkin Trans. 1 1982, 1171.
4
41. Gao, Y.; Zhang, P.; Wu, L.; Matsuura, T.; Meng, J. Synth. Commun. 2003, 33, 2635.
4
4
2. Schott, H.; Häussler, M. P.; Schwendener, R. A. Liebigs Ann. Chem. 1994, 465.
3. Schott, H. In Preparative Scale-Chromatography; Grushka, E., Ed.; Marcel
Dekker: New York and Basel, 1989; Vol. 46, p 269.
1
l
per well in 96- or 48-well plates, respectively. Cell viability was
44. Pan, H.; Sima, M.; Kopeckova, P.; Wu, K.; Gao, S.; Liu, J.; Wang, D.; Miller, S. C.;
determined with a resazurin assay.52
Kopecek, J. Mol. Pharmacol. 2008, 5, 548.
45. Leu, C. T.; Luegmayr, E.; Freedman, L. P.; Rodan, G. A.; Reszka, A. A. Bone 2006,
3
8, 628.
Acknowledgment
46. Nancollas, G. H.; Tang, R.; Phipps, R. J.; Henneman, Z.; Gulde, S.; Wu, W.;
Mangood, A.; Russell, R. G.; Ebetino, F. H. Bone 2006, 38, 617.
4
7. Wingen, F.; Sterz, H.; Blum, H.; Moller, H.; Pittermann, W.; Pool, B. L.; Sinn, H.
The authors wish to thank the National Cancer Institute
Bethesda, USA) for the in vitro antitumor screening of 5-FdU-ale.
J.; Spring, H.; Schmahl, D. J. Cancer Res. Clin. Oncol. 1986, 111, 209.
(
48. Chhikara, B. S.; Parang, K. Expert Opin. Drug Delivery 2010, 7, 1399.
4
9. Monkkonen, H.; Kuokkanen, J.; Holen, I.; Evans, A.; Lefley, D. V.; Jauhiainen, M.;
Auriola, S.; Monkkonen, J. Anticancer Drugs 2008, 19, 391.
0. Schott, S. Pharm. Ztg. 2001, 146, 24.
1. Kieczykowski, G. R.; Jobson, R. B.; Melillo, D. G.; Reinhold, D. F.; Grenda, V. J.;
Shinkai, I. J. Org. Chem. 1995, 60, 8310.
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