Novel Molecular Targeted and Wide Spectrum Antitumor Agents : Preparation and Preclinical Evaluation of IER 5 / Cdc 25 B Targeted Low-molecular-weight Phospha Sugar Derivatives

Hiroko Hasegawa, Mitsuji Yamashita,* Reiko Makita, Mayumi Yamaoka, Michio Fujie, Satoki Nakamura, Tatsuo Oshikawa, Junko Yamashita, Manabu Yamada, Mitsuru Kondo, Kazutaka Hirakawa, Mitsuo Toda, Yasuo Takehara, Kazunori Ohnishi, Haruhiko Sugimura, Sophie Laurent, and Robert N. Muller 1 Graduate School of Science and Technology, Shizuoka University, Hamamatsu 432-8561, Japan 2 Faculty of Medicine, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan 3 Department of Chemistry and Biochemistry, Numazu National College of Technology, Numazu 410-8501, Japan 4 Faculty of Medicine, University of Mons, Mons, Hainaut, Belgium


Introduction
It is important to treat as well as to diagnose cancer more efficiently and safely than the present clinical technologies for both medical reasons and societal needs at the earlier stage of tumors by R and D on novel medical materials.We have been very much interested in chemistry and biological activities of carbohydrates and phosphorus compounds not only on the basis of medicinal chemistry but also as an opportunity for improved treatments for cancer patients as well as diagnosing cancer tissues.In this research we will deal with phospha sugar antitumor agents to provide innovative cancer therapy technologies.

Biology (MTT in vitro evaluation)
Phospha sugar derivatives (MBMPP, DBMPP, TBMPP, etc.) in the compound library were prepared and tested by the MTT method for antitumor activity against the K562 (human chronic myelogenous leukemia), U937 (human acute myelogenous leukemia), etc. cell lines.Cells were seeded in 24-well flat-bottomed microplates at a density of 3 × 10 4 per well and incubated at various concentrations of phospha sugar derivatives for 5 days.The cells were then washed with phosphate-buffered saline (PBS), harvested, and suspended in a 0.4% trypan blue solution for trypan blue exclusion assay, in which viable cells were counted with a hemocytometer at the indicated incubation day.For the MTT assay, the cells were seeded in 96-well flat-bottomed microplates at a density of 5 × 10 5 per well.The cells were incubated at various concentration of phospha sugar derivatives for 24 h.After incubation, 10 μL 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) solution (Sigma) was added to each well at a final concentration of 1 mg/mL.After incubation at 37 o C for 4 h, absorbance was measured at a wavelength of 560 nm using a microplate reader.

Results and Discussion
Sugar derivatives, whose oxygen atom in the hemiacetal ring of Haworth projection is replaced by a carbon, nitrogen, or sulfur atom are called pseudo sugars (Fig. 1).Some pseudo sugars, such as carba, aza, and thia sugars, respectively, are analogues of naturally occurring or synthesized products, and are well investigated, and many of them are known to be biologically active compounds.
Phospha sugars are also one category of the pseudo sugar analogues (Fig. 2), which have a phosphorus residue instead of the oxygen atom in the hemiacetal ring.However, phospha sugars are not yet found in nature, and the synthesis and the characterization of them are not yet so well studied in spite of their potentially important biological activities [1].We have successfully synthesized various kinds of phospha sugar derivatives from heterocycles (using new synthetic methods) and/or carbohydrates (using more traditional synthetic methods for preparation of pseudo sugars) to construct a compound library [2], and then evaluated their biological activities [3].We have found that deoxybromophospha sugar derivatives (Fig. 3 and Scheme), as well as some substituted phospha sugar analogues such as anhydrophospha sugar derivatives, exerted strong, selective, and tumor cell specific antitumor activities by MTT in vitro evaluation against various kinds of leukemia cells via apoptosis induction, with little or no damage to normal cells [4].The phospha sugars DBMPP and TBMPP have excellent antitumor activities against various kinds of leukemia cells such as the K562 (Fig. 4) and U937 cell lines, as well as solid cancer cells such as stomach cancer, lung cancer, etc. Mechanistic studies of the phospha sugar DBMPP against leukemia cells U937 by Western blotting showed that the phospha sugar suppresses the expression of "Accelerator Factors of Tumor Cells" such as FoxM1, KIS, Skp2, Aurora-B, etc. and accelerates the expression of "Suppressor Factors of Tumor Cells" such as p27 Kip1 and p21 Cip1 .Together these effects, control tumor cell viability (Fig. 5).Further mechanistic studies of the phospha sugar TBMPP against leukemia cells by Western blotting showed that the phospha sugar enhanced the expression of IER5, and then suppressed the expression of Cdc25B.Cdc25B is an essential and common factor for the mitosis of tumor cells.Thus the present observations of mechanism imply that phospha sugar induces apoptosis at G2/M stage and inhibits the proliferation of various kinds of cancer cells (Fig. 6) [4,[5][6][7].
The observed results regarding enhanced expression of IER5 and suppressed expression of Cdc25B by TBMPP as shown by Fig. 6 can explain why this phospha sugar exerts wide spectrum and selective as well as tumor cell specific antitumor activities against various kinds of tumor cells yet does not damage normal cells.The in vivo evaluation with phospha sugar TBMPP was also performed successfully using a nude mouse transplanted K562 cells on the skin (Fig. 7).

Conclusion
Low-molecular-weight phospha sugar derivatives, e.g., DBMPP, TBMPP, etc., should be developed to be clinically useful.They provide novel and innovative, wide spectrum antitumor agents that target IER5/Cdc25B.With these innovative novel antitumor agents, as well as improved sugar dendritic Gd-DTPA complex MRI contrast agents [8], medical treatments for cancer patients will be improved.They may also be used to treat various kinds of tumors successfully with a single antitumor agent.

Fig. 5 .Fig. 6 .
Fig. 5. Phospha sugar (DBMPP) suppresses the expression of "Accelerator Factors of Tumor Cells" and accelerates the expression of "Suppressor Factors of Tumor Cells", and thus controls tumor cell viability.