Novel Multiple Type Molecular Targeted Antitumor Agents: Preparation and Preclinical Evaluation of Low-Molecular-Weight Phospha Sugar Derivatives

Article abstract of DOI:10.1080/10426507.2014.993758

Phospha sugar derivatives of 2,3-dibromo-3-methyl-1-phenylphospholane 1-oxide and 2,3,4-tribromo-3-methyl-1-phenylphospholane 1-oxide were synthesized from 2-phospholenes and evaluated by in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide method against leukemia cell lines, and then characterized by cell cycle analysis, Western blot analysis, and molecular docking simulation. Phospha sugar derivatives were revealed to be potential antitumor agents against leukemia cell lines such as U937 and K562. Cell cycle analysis indicated that phospha sugar derivatives induced apoptoses. Western blot analyses against U937 showed that phospha sugar derivatives regulate many mitotic regulators in cell cycle. Results of molecular docking simulation suggested that phospha sugar derivatives enter the pockets present in cell cycle and apoptosis-related proteins.

Full text of DOI:10.1080/10426507.2014.993758

Phosphorus, Sulfur, and Silicon and the Related Elements
ISSN: 1042-6507 (Print) 1563-5325 (Online) Journal homepage: http://www.tandfonline.com/loi/gpss20
Novel Multiple Type Molecular Targeted Antitumor
Agents: Preparation and Preclinical Evaluation of
Low-Molecular-Weight Phospha Sugar Derivatives
Reiko Makita, Mitsuji Yamashita, Mayumi Yamaoka, Michio Fujie, Satoki
Nakamura, Tatsuo Oshikawa, Junko Yamashita, Manabu Yamada, Kazuhide
Asai, Takuya Suyama, Mitsuru Kondo, Hiroko Hasegawa, Yoshimitsu Okita,
Kazutaka Hirakawa, Mitsuo Toda, Kazunori Ohnishi & Haruhiko Sugimura
To cite this article: Reiko Makita, Mitsuji Yamashita, Mayumi Yamaoka, Michio Fujie, Satoki
Nakamura, Tatsuo Oshikawa, Junko Yamashita, Manabu Yamada, Kazuhide Asai, Takuya
Suyama, Mitsuru Kondo, Hiroko Hasegawa, Yoshimitsu Okita, Kazutaka Hirakawa, Mitsuo
Toda, Kazunori Ohnishi & Haruhiko Sugimura (2015) Novel Multiple Type Molecular Targeted
Antitumor Agents: Preparation and Preclinical Evaluation of Low-Molecular-Weight Phospha
Sugar Derivatives, Phosphorus, Sulfur, and Silicon and the Related Elements, 190:5-6, 733-740,
DOI: 10.1080/10426507.2014.993758
To link to this article: http://dx.doi.org/10.1080/10426507.2014.993758
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Date: 11 November 2015, At: 06:27

Phosphorus, Sulfur, and Silicon, 190:733–740, 2015
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2014.993758
NOVEL MULTIPLE TYPE MOLECULAR TARGETED
ANTITUMOR AGENTS: PREPARATION AND PRECLINICAL
EVALUATION OF LOW-MOLECULAR-WEIGHT PHOSPHA
SUGAR DERIVATIVES
Reiko Makita,1 Mitsuji Yamashita,1 Mayumi Yamaoka,2
Michio Fujie,3 Satoki Nakamura,3 Tatsuo Oshikawa,4
Junko Yamashita,1 Manabu Yamada,1 Kazuhide Asai,2
Takuya Suyama,2 Mitsuru Kondo,1 Hiroko Hasegawa,1
Yoshimitsu Okita,1 Kazutaka Hirakawa,1 Mitsuo Toda,2
Kazunori Ohnishi,3 and Haruhiko Sugimura3
1Graduate School of Science and Technology, Shizuoka University, Hamamatsu,
Japan
2Graduate School of Engineering, Shizuoka University, Hamamatsu, Japan
3Faculty of Medicine, Hamamatsu University School of Medicine, Hamamatsu,
Japan
4Department of Chemistry and Biochemistry, Numazu National College of
Technology, Numazu, Japan
GRAPHICAL ABSTRACT
Abstract Phospha sugar derivatives of 2,3-dibromo-3-methyl-1-phenylphospholane
1-oxide and 2,3,4-tribromo-3-methyl-1-phenylphospholane 1-oxide were synthesized from
2-phospholenes and evaluated by in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-
Received 28 August 2014; accepted 30 October 2014.
Address correspondence to Mitsuji Yamashita, Department of Nano Materials, Graduate School of Sci-
ence and Technology, Shizuoka University, Johoku 3-5-1, Naka-ku, Hamamatsu 432-8011, Japan. E-mail:
tcmyama@ipc.shizuoka.ac.jp
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/gpss.
733

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R. MAKITA ET AL.
tetrazolium bromide method against leukemia cell lines, and then characterized by cell cycle
analysis, Western blot analysis, and molecular docking simulation. Phospha sugar derivatives
were revealed to be potential antitumor agents against leukemia cell lines such as U937 and
K562. Cell cycle analysis indicated that phospha sugar derivatives induced apoptoses. Western
blot analyses against U937 showed that phospha sugar derivatives regulate many mitotic
regulators in cell cycle. Results of molecular docking simulation suggested that phospha sugar
derivatives enter the pockets present in cell cycle and apoptosis-related proteins.
Keywords Phospha sugars; molecular targeted antitumor agents; MTT in vitro evaluation; cell
cycle analysis; western blot analysis; molecular docking simulation
INTRODUCTION
Sugar analogues, being replaced an oxygen atom by another atom in the furanose or
pyranose ring of sugar, are known generally as pseudo sugar. Typical pseudo sugars are
carba-, aza-, and thia-sugars,1–4 which have a carbon, nitrogen, or sulfur atom, respectively,
instead of the oxygen atom in the hemiacetal ring of the normal sugars. These pseudo
sugars are synthesized in a general method by sugar chemistry.5 Furthermore, some of
these pseudosugars are known to exist in nature and exert important biological activities.6–9
On the other hand, phospha sugars, being classified into a category of the pseudo sugars
having the phosphorus atom in the hemiacetal ring of sugars, are not yet found in nature
and are difficult to synthesize by the method of sugar chemistry.4,5,10–12
We have been challenging to search biologically active materials of phosphorus
analogues of pseudo sugars, phospha sugars. Synthetic methodology for phospha sugars
containing a phosphorus heterocycle has been developed and many phospha sugar anologs
were prepared by a new methodology from phospholene starting materials.11,12 Some of the
phospha sugar derivatives, such as deoxyhalo, anhydro, branched, and substituted phospha
sugars prepared, were suggested to exert potential antitumor activities against leukemia
cells by in vitro evaluation of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) methods.13 Phospha sugars were also revealed to be potential antitumor
agents against solid tumor cell lines of stomach and skin by handstand phase-contrast
microscope observation.14 In this paper we will deal with the preparation of branched
deoxybromophospha sugar derivatives of 2,3-dibromo-3-methyl-1-phenylphospholane 1-
oxide (DBMPP) and 2,3,4-tribromo-3-methyl-1-phenylphospholane 1-oxide (TBMPP) and
their evaluations by MTT method and the characterizations of DBMPP and TBMPP by cell
cycle analysis, Western blot analysis, and molecular docking simulation.
RESULTS AND DISCUSSION
Branched deoxybromophospha sugar derivatives of DBMPP and TBMPP were pre-
pared from 3-methyl-1-phenyl-2-phospholene 1-oxide and 4-bromo-3-methyl-1-phenyl-2-
phopholene 1-oxide, respectively, as shown in Scheme 1.12 DBMPP and TBMPP were
prepared by addition reaction of bromine to the C C bond of the 2-phospholenes with or
without catalyst.
Antitumor activity of DBMPP and TBMPP were evaluated by MTT in vitro method
against U937 and K562 cell lines, and those results are shown in Figure 1.12 Figure 1
indicates that DBMPP and TBMPP have good antitumor activities and TBMPP has the
higher activity than DBMPP. 50% Cell growth inhibition (IC50) values of DBMPP and
TBMPP against several leukemia cell lines including U937 and K562 were shown in

MOLECULAR TARGETED ANTITUMOR AGENTS
735
Scheme 1 Preparation of phospha sugar derivatives DBMPP and TBMPP.
Figure 2.13 DBMPP and TBMPP indicated similar good activities against several leukemia
cell lines other than U937 and K562.
Cell cycle analysis by flow cytometry for K562 indicated that DBMPP and TBMPP
induced apoptosis similar to Imatinib mesylate (Glivec), a well known molecular targeted
antitumor agent (Figure 3). TBMPP induced more strongly cell cycle arrest and apoptosis
than DBMPP.13
Western blot analysis showed that DBMPP not only suppressed the expression of
tumor accelerators (FoxM1, KIS, Skp2, Cyclin D1, Survivin, and Aurora-B), but also
enhanced the expression of tumor suppressors (p27Kip1 and p21Cip1) against U937 cell lines
(Figure 4).15 Similarly, TBMPP have been reported to suppress the expression of FoxM1,
Cdc25B, KIS, Cyclin D1, Cyclin A, Cyclin B1, Survivin, and Aurora-B, and enhance the
expression of p27Kip1 and p21Cip1 against U937 cell lines.13,16 These results suggest that
DBMPP and TBMPP regulate many mitotic regulators in the cell cycle.13 Furthermore,
we have also reported that TBMPP enhanced the expression of IER5 and then inhibited
leukemia cell proliferation on G2/M cell cycle through the suppression of the expression
of Cdc25B.16 Therefore, branched deoxybromophospha sugar derivatives are expected as
antitumor agents for various kinds of tumor cells and/or new type antitumor agents targeting
IER5 and Cdc25B.
Figure 1 Evaluation of antitumor activity of phospha sugars derivatives DBMPP and TBMPP by MTT in vitro
assay for inhibition from cell proliferation of U937 and K562 cells.

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R. MAKITA ET AL.
Figure 2 Antitumor activities (IC50) of phospha sugar derivatives DBMPP and TBMPP against leukemia cell
lines.
Figure 3 Effect of TBMPP and DBMPP on cell cycle and apoptosis against K562 cell lines.
Figure 4 Western blot analysis by DBMPP against U937 cell lines.

MOLECULAR TARGETED ANTITUMOR AGENTS
737
Table 1 Docking energy of phospha sugar derivatives and proteins
Proteins
TBMPP Udock (kcal/mol)
DBMPP Udock (kcal/mol)
Aurora-B
Survivin
Bcl-2
−20.4
−14.3
−15.3
−17.3
−14.0
−14.8
Molecular docking simulation of phospha sugars and proteins was performed by
MOE, Molecular Operating Environment. DBMPP and TMBPP were simulated with
Aurora-B, Survivin, and Bcl-2 registered Protein Data Bank among cell cycle and apoptosis
related proteins. Stability of docking simulated is represented by docking energy (Udock
= Uele + Uvdw + Ustrain). The more negative the value is, the more the docking state is
stable. Docking energy for phospha sugars and proteins were low (Table 1). Especially,
docking energy for TBMPP and Aurora-B was very low (Udock = –20.4 kcal/mol). Pharma-
cophore model and two-dimensional diagram of TBMPP-Aurora-B composite were shown
in Figure 5. Figure 5 indicates that TBMPP enters the pocket in Aurora-B. These results
suggest that phospha sugars enter the pockets present in the proteins, and induce cell cycle
arrest and apoptosis.
In conclusion, branched deoxybromophospha sugar derivatives of DBMPP and
TBMPP were prepared from 2-phospholenes. DBMPP and TBMPP were indicated to
have good antitumor activities by in vitro MTT method. Cell cycle analysis, Western
blot analysis, and molecular docking simulation indicated that DBMPP and TBMPP in-
duce cell cycle arrest and apoptosis because DBMPP and TBMPP regulate many mitotic
regulators in the cell cycle and enter the pockets present in the proteins. Phospha sugar
derivatives have potentials for multiple type molecular-targeted antitumor agents and/or
IER5/Cdc25B-targeted antitumor agents.
Figure 5 MOE ASEDock results of TBMPP and Aurora-B.

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R. MAKITA ET AL.
EXPERIMENTAL
Instruments
TLC (Silica gel: Wako Chromato sheet and/or Merk Kieselgel 60; Eluent : CHCl3
: MeOH = 20 : 1, in Rf value); MS (MALDI-TOF-MS: GL Science, Voyager-DE
1
Porimerix; Matrix: α-Cyano-4-hydroxycinnamic acid, in m/z); H-NMR (JEOL JNM-
AL300 (300 MHz); Solvent: CDCl3, in δ (ppm) from TMS) were used to record and
collect the data for analyzing the products.
Materials
3-Methyl-1-phenyl-2-phospholene 1-oxide and 4-bromo-3-methyl-1-phenyl-2-
phospholene 1-oxide were prepared as starting materials.12
Synthesis of 2,3-dibromo-3-methyl-1-phenylphospholane
1-oxide (DBMPP)
3-Methyl-1-phenyl-2-phospholene 1-oxide (0.27 g, 1.4 mmol; 1.0 eq.) was dissolved
in CH2Cl2 (5 mL), and added MnO2 (0.24 g, 2.8 mmol; 2.0 eq.). To the mixture was added
CH2Cl2 (10 mL) solution of bromine (0.4 mL; 7.8 mmol; 5.7 eq.) dropwisely and stirred
for 12 h at room temperature. The reaction mixture was treated with saturated sodium
hydrogensulfite solution and was extracted with CHCl3. The CHCl3 extract was washed
with saturated sodium hydrogen carbonate, brine, and water, and then dried over anhydrous
Na2SO4. Evaporation of the solvent from the solution of the product followed by silica gel
column chromatography with the mixed eluent of chloroform and methanol (30:1) afforded
2,3-dibromo-3-methyl-1-phenylphospholane 1-oxide (DBMPP; 0.38 g, 1.1 mmol) in 78%
yield; TLC: Rf = 0.54 (CHCl3 :MeOH = 30 : 1); MS (m/z), 351.5 (MH+, 60), 353.5 (MH+,
100), 355.5 (MH+, 40); 1H-NMR (CDCl3, 300 MHz), δ = 2.09 (s, 3H, C3-CH3), 2.44–2.58
(m, 2H, C5), 2.60–2.85 (m, 2H, C4), 4.66–4.68 (dd, 1H, C2), 7.49–7.80 (m, 5H, Ph).
Synthesis of 2,3,4-tribromo-3-methyl-1-phenylphospholane
1-oxide (TBMPP)
Similarly, the reaction of 4-bromo-3-methyl-1-phenyl-2-phospholene 1-oxide (1.6 g,
6.0 mmol; 1.0 eq.) with bromine (1.2 mL; 24 mmol; 4.0 eq.) in CCl4 (10 mL) under reflux
temperature for 24 h and workup procedure afforded TBMPP (0.48 g, 1.1 mmol) in 19%
yield; TLC: Rf = 0.62 (CHCl3:MeOH = 20:1); MS (m/z): 429.0 (MH+, 45), 431.1 (MH+,
100), 433.1 (MH+, 95), 435.1 (MH+, 30); 1H-NMR (CDCl3, 300 MHz), δ = 2.14 (s, 3H,
C3-CH3), 2.73–3.14 (m, 2H C5), 4.26 (d, 1H, C4), 4.68–4.86 (m, 1H, C2), 7.52–7.79 (m,
5H, Ph).
MTT in vitro Evaluation
Human leukemia cell lines (U937, K562, HL60, NB4, NOMO-1, CEM, MOLT4,
and MEG-01) were purchased from American Type Culture Collection. Phospha sugars
were dissolved in dimethylsulfoxide (Sigma Chemical Company) and diluted in culture
medium immediately before use. Human leukemia cell lines (U937, K562, HL60, NB4,
NOMO-1, CEM, MOLT4, and MEG-01) (American Type Culture Collection) were seeded

MOLECULAR TARGETED ANTITUMOR AGENTS
739
in 96-well flat-bottomed microplates at a density of 5 × 104 per well and incubated with
various concentrations of phospha sugar derivatives for antitumor activity assay or without
any phospha sugars for control experiments, for 48 or 24 h at 37◦C, and then 10 μL solution
of MTT (Sigma) was added to each well at the final concentration of 1.0 μg/mL/well. After
incubation for 4 h at 37◦C, absorbance was measured at a wavelength of 560 nm by using
a microplate reader.12,13
Cell Cycle Analyses
Propidium iodide (PI) (Sigma) staining was used to analyze cellular DNA content.
Cells (K562) treated with Imatinib mesylate (Glivec) (10 μM) or phospha sugar DBMPP
(20 μM) or TBMPP (10 μM) were cultured at 37◦C in 2 mL of complete medium containing
1 × 106 cells. After incubation for 48 h, the cells were washed twice with cold PBS, fixed
with 70% ethanol overnight before treatment with 100 μg/mL RNase A, and then stained
with 50 μg/mL of PI. The relative DNA content per cell was measured by flow cytometry
using an Epics Elite flow cytometer (Coulter Immunotech). The percentage of cells in
the apoptotic sub-G1 phase, as well as G1, S, and G2/M phases, was calculated using the
ModFit program (Becton Dickinson).13,14
Western Blot Analyses
Leukemia cells treated with phospha sugars (DBMPP or TBMPP) were harvested.
Western blot analysis were performed using the following antibodies: rabbit polyclonal
anti-FoxM1 (MPP2 K-19), anti-Skp2, anti-p27Kip1 (Santa Cruz); rabbit polyclonal anti-KIS,
anti-Aurora-B (ABGENT); mouse monoclonal anti-Cyclin D1, anti-Survivin, anti-p21Cip1
(Santa Cruz). Western blotting of actin, used as an internal control, was carried out using a
mouse monoclonal anti-Actin antibody (C-4; ICN).13,15
Molecular Docking Simulation
Molecular docking simulation was performed with Molecular Operating Environment
(MOE; Chemical Computing Group Inc.) of molecular design software. Ducking energy
was obtained by using MMFF94x for the force field.
FUNDING
The authors greatly acknowledge financial supports by Satellite Shizuoka and the
Headquarters of Japan Science and Technology Agency (JST) (Grant No. AS242Z01963Q)
and Grant-in-Aid for Scientific Research by Japan Society for the Promotion of Science
(JSPS) (Grant 20591115, 21650108) and Ministry of Education, Culture, Sports, Science
and Technology (MEXT) (H19-Nano-Ippan-015), and Sugiyama Kohokai Foundation.
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