Research on phospha sugar analogues to develop novel multiple type molecular targeted antitumor drugs against various types of tumor cells

Article abstract of DOI:10.1080/10426507.2012.744016

The synthesis and antitumor activity evaluation of new branched phospha sugars, especially deoxybromophospha sugar derivatives or bromophospholanes of 2,3-dibromo-3-methyl-1-phenylphospholane 1-oxide (DBMPP: 3) and 2,3,4-tribromo-3-methyl-1- phenylphospho

Full text of DOI:10.1080/10426507.2012.744016

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Phosphorus, Sulfur, and Silicon and the
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Research on Phospha Sugar Analogues to
Develop Novel Multiple Type Molecular
Targeted Antitumor Drugs Against
Various Types of Tumor Cells
Reiko Makita a , Mitsuji Yamashita a , Michio Fujie b , Mayumi
Yamaoka c , Keita Kiyofuji a , Manabu Yamada a , Junko Yamashita
a , Kenji Tsunekawa a , Kazuhide Asai c , Takuya Suyama c , Mitsuo
Toda c , Yasutaka Tanaka c , Haruhiko Sugimura b , Yasuhiro Magata b
, Kazunori Ohnishi b & Satoki Nakamura b
a Department of Nano Materials, Graduate School of Science and
Technology, Shizuoka University , Hamamatsu , Japan
b Faculty of Medicine, Hamamatsu University School of Medicine ,
Hamamatsu , Japan
c Department of Materials Science , Graduate School of Engineering,
Shizuoka University , Hamamatsu , Japan
Accepted author version posted online: 15 Nov 2012.Published
online: 08 May 2013.
To cite this article: Reiko Makita , Mitsuji Yamashita , Michio Fujie , Mayumi Yamaoka , Keita
Kiyofuji , Manabu Yamada , Junko Yamashita , Kenji Tsunekawa , Kazuhide Asai , Takuya Suyama ,
Mitsuo Toda , Yasutaka Tanaka , Haruhiko Sugimura , Yasuhiro Magata , Kazunori Ohnishi & Satoki
Nakamura (2013) Research on Phospha Sugar Analogues to Develop Novel Multiple Type Molecular
Targeted Antitumor Drugs Against Various Types of Tumor Cells, Phosphorus, Sulfur, and Silicon and the
Related Elements, 188:1-3, 213-223, DOI: 10.1080/10426507.2012.744016
To link to this article: http://dx.doi.org/10.1080/10426507.2012.744016
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Phosphorus, Sulfur, and Silicon, 188:213–223, 2013
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2012.744016
RESEARCH ON PHOSPHA SUGAR ANALOGUES TO
DEVELOP NOVEL MULTIPLE TYPE MOLECULAR
TARGETED ANTITUMOR DRUGS AGAINST VARIOUS
TYPES OF TUMOR CELLS
Reiko Makita,1 Mitsuji Yamashita,1 Michio Fujie,2
Mayumi Yamaoka,3 Keita Kiyofuji,1 Manabu Yamada,1
Junko Yamashita,1 Kenji Tsunekawa,1 Kazuhide Asai,3
Takuya Suyama,3 Mitsuo Toda,3 Yasutaka Tanaka,3
Haruhiko Sugimura,2 Yasuhiro Magata,2
Kazunori Ohnishi,2 and Satoki Nakamura2
1Department of Nano Materials, Graduate School of Science and Technology,
Shizuoka University, Hamamatsu, Japan
2Faculty of Medicine, Hamamatsu University School of Medicine, Hamamatsu,
Japan
3Department of Materials Science, Graduate School of Engineering, Shizuoka
University, Hamamatsu, Japan
GRAPHICAL ABSTRACT
Abstract The synthesis and antitumor activity evaluation of new branched phospha
sugars, especially deoxybromophospha sugar derivatives or bromophospholanes of 2,3-
dibromo-3-methyl-1-phenylphospholane 1-oxide (DBMPP: 3) and 2,3,4-tribromo-3-methyl-1-
phenylphospholane 1-oxide (TBMPP: 4), against various types of leukemia cell lines as well
Received 15 September 2012; accepted 23 October 2012.
The authors greatly acknowledge financial supports by Satellite Shizuoka and the Headquarters of Japan
Science and Technology Agency (JST) and Grant-in-Aid for Scientific Research by Japan Society for the Pro-
motion of Science (JSPS) (Grant 19650112, 21650108) and Ministry of Education, Culture, Sports, Science and
Technology (MEXT) (H19-Nano-Ippan-015), and Sugiyama Kohokai Foundation.
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
213

214
R. MAKITA ET AL.
as the results of the mechanistic studies for characterizing and developing the novel multiple
type molecular targeted antitumor agents are reported in this paper. DBMPP and TBMPP were
prepared from 1-phenyl-3-methyl-2-phospholene 1-oxide (1). The isomer mixture of phospha
sugars prepared were evaluated as novel antitumor agents by MTT in vitro method. DBMPP
and TBMPP were characterized by flow cytometry and Western blot analysis and were revealed
to be potential antitumor agents against leukemia cell lines of K562 (one type of leukemia cell
lines of CML) and U937 (one type of leukemia cell lines of AML) as well as against the various
types of leukemia cell lines and also against solid tumor cell lines of stomach, skin, and lung
cancers by MTT evaluation and observation by a handstand phase-contrast microscope. The
results of the flow cytometry indicated that the mechanism of apoptosis induced by phospha
sugar derivatives not only to tumor cells of leukemia cell lines of U937 but also to tumor cells
of various kinds of leukemia cell lines selectively to decrease the tumor cell viability of various
kinds of leukemia cell lines. The Western blot analyses for phospha sugar DBMPP against
U937 leukemia cell lines showed that the phospha sugar affected on the expressions of the
factors of cell cycles in the manners of suppressing the expression of the accelerator factors of
cell cycles of tumor cells and enhancing the expression of suppressor factors of cell cycles of
tumor cells by the medications of phospha sugars. TBMPP enhanced the expression of IER5
and then suppressed the expression of Cdc25B, which is the common factor to accelerate the
cell cycles of various kinds of tumor cells. Therefore, suppression of the expression of Cdc25B
by TBMPP implies that the branched deoxybromophospha sugar derivatives might be novel
and potential multiple type molecular targeted antitumor agents against various kinds of tumor
cell lines.
Keywords Phosphorus heterocycles; branched deoxybromophospha sugars; molecular tar-
geted antitumor agents; MTT in vitro evaluation; flow cytmetry; Western blot analysis
INTRODUCTION
Well known typical pseudo sugars are carba-, aza-, and thia-sugars,1–4 which have a
carbon–carbon–carbon, carbon–nitrogen–carbon, or carbon–sulfur–carbon linkage, respec-
tively, instead of the carbon–oxygen–carbon linkage in the hemiacetal ring of the normal
sugars. These pseudo sugars are known to exist in nature and are also chemically prepared
by applying methodologies of synthetic sugar chemistry, where common sugars are gener-
ally used as the starting materials, for synthesizing pseudo sugars via a series of reactions of
protection of specific hydroxyl groups, carbon-hetero atom bond formation, deprotection,
and finally formation of the heterocycles by the reconstruction of the hemiacetal ring of
sugars.5 Pseudo sugars are known to exert important biological activities, therefore, many
studies on not only the isolation and synthesis of the pseudo sugars but also on the charac-
terization and evaluation of the biological activities are actively performed and reported.6–9
On the other hand, phospha sugars, being classified into one new category of the pseudo
sugars having the carbon–phosphorus–carbon linkage in the hemiacetal ring of sugars, are
not yet found in nature and the syntheses of them are rather difficult compared with the
typical pseudo sugars such as aza- and thia-sugars.4,5,10–12
We have been searching biologically active phospha sugars and we have first
found new phospha sugar derivatives which provided good antitumor activities against
leukemia cell lines by in vitro evaluation of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
2H-tetrazolium bromide (MTT) methods.13 In this paper we will deal with the suc-
cessful preparation of branched phospha sugars including deoxybromophospha sugars of

RESEARCH ON PHOSPHA SUGAR FOR ANTITUMOR DRUG
215
2,3-dibromo-3-methyl-1-phenylphospholane 1-oxide (DBMPP: 3) and 2,3,4-tribromo-3-
methyl-1-phenylphospholene 1-oxide (TBMPP: 4). The evaluations and characterizations
of the DBMPP and TBMPP by the MTT method and the Western blot analysis revealed
that phospha sugars have excellent characters as antitumor agents with high activities,
wide spectra, good selectivity, and specificity against various kinds of leukemia cell lines.
Western blot analysis for DBMPP and TBMPP against leukemia cell lines showed that
DBMPP suppressed the expression of several tumor cell cycle accelerators and enhanced
the expression of tumor cell cycle suppressors. TBMPP enhanced the expression of IER5
and then suppressed the expression of Cdc25B.
RESULTS AND DISCUSSION
Synthesis of Phospha Sugar Derivatives
Branched deoxypbromophospha sugar derivatives of 2,3-dibromo-3-methyl-1-
phenylphospholane 1-oxide (DBMPP: 3) and 2,3,4-tribromo-3-methyl-1-phenylphos-
pholane 1-oxide (TBMPP: 4) were prepared from the starting material of 3-methyl-1-
phenyl-2-phospholene 1-oxide (1) and 4-bromo-3-methyl-1-phenyl-2-phopholene 1-oxide
(2) as shown in Scheme 1.12 Here, DBMPP (3) and TBMPP (4) were prepared by simple
and efficient synthetic methods by addition of bromine to the C=C double bond of the
2-phospholenes 1 and 2 with or without catalyst in yields of 90% and 69%, respectively.
Deoxybromophospha sugar derivatives with oxygen functionalities OR1 (5 (R1 = H), 6
(R1 = CH2CH2OH), and 7 (R1 = (CH2CH2O)2H)) on the 3-position were prepared by the
reaction of 1 with bromine in the presence of water, ethylene glycol, and diethylene glycol,
respectively, in yields of 55%, 88%, and 58%. 4-Imidazolyl derivative 8 was prepared
from 4-bromo derivative 2 by the substitution reaction of the 4-bromo group by imidazole
in a yield of 93% (Scheme 1).
Scheme 1 Preparation of phospha sugar derivatives 2–8 from 2-phospholene 1.
Antitumor Activity and Cell Cycle Analysis13,14
Antitumor activity for the prepared phospha sugar derivatives 2–8 was evaluated
by MTT in vitro method against K562 and U937 cell lines, whose results of antitumor
activities are summarized in Table 1. Among these branched phospha sugar derivatives,
bromo derivatives were active, especially DBMPP (3) and TBMPP (4) were quite active,

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R. MAKITA ET AL.
Table 1 Antitumor activities (IC50) of phospha sugar derivatives against leukemia cell lines of K562 and U937
for 48 h at 37◦C)
Phospha sugars
K562 IC50 (μM)
U937 IC50 (μM)
1
2
>200
48
>900
83
3 (DBMPP)
23
24
4 (TBMPP)
5
6
7
3.2
34
>100
63
>100
0.48
2.3
NDa
NDa
NDa
NDa
>500
8
(Imatinib mesylate)
aND = not yet determined.
against leukemia cell lines. DBMPP and TBMPP were more active against U937 cell lines
than imatinib mesylate (Glivec, Gleevec), however, they did not give any damages against
healthy or normal leukocytes (Table 1). Therefore, here we have focused on DBMPP and
TBMPP for further investigation of phospha sugar derivatives to develop novel antitumor
agents.
DBMPP and TBMPP have wide spectral antitumor activities against various kinds of
leukema cell lines (Table 2). The cell cycle analysis revealed that DBMPP induced apoptosis
to stop the progress of the cell cycle of K562 and U937 cell lines likewise the manner of
imatinib mesylate at the Sub G1 stage (Figure 1: against K562 cell lines). Figure 1 shows
that the apoptosis of 78% for the cell cycles was induced by DBMPP (20 μM) against
K562 cell lines. TBMPP (20 μM) also induced the apoptosis against various leukemia cell
lines about 80% (Figure 2). Branched deoxybromophospha sugars DBMPP and TBMPP
have good to excellent antitumor activities against wide spectral leukemia cell lines.
Western Blot Analysis13
Western blot analysis for phospha sugars was performed against leukema cell lines.
DBMPP enhanced the expression of tumor accelerator factors of FoxM1, KIS, Skp2,
Table 2 Antitumor activities (IC50) of phospha sugar derivatives DBMPP and TBMPP against various kinds of
leukemia cell lines (for 48 h at 37◦C)
Cell lines
TBMPP (4) IC50 (μM)
DBMPP (3) IC50 (μM)
HL60
NB4
YRK2
NOMO-1
CEM
MOLT4
SUP-B15
MEG-01
4.8 1.0
3.2 0.9
5.3 1.3
5.5 0.8
6.9 0.3
6.7 1.2
7.1 1.0
8.6 1.4
5.4 0.6
>>200
18 1.5
15 1.4
28 2.6
18 2.1
29 2.4
26 1.8
24 2.8
27 1.9
26 2.1
>>200
SHG3
(Healthy or normal leukocyte)a
aBlast 0% (no leukemia cells).

RESEARCH ON PHOSPHA SUGAR FOR ANTITUMOR DRUG
217
Figure 1 Results of the flow cytometry observed by DBMPP and Imatinib mesylate (Glivec, Gleevec) against
K562 cell lines for 24 h at 37◦C showing apoptosis (%) induced.
Cyclin D1, Survivin, Aurora-B, Actin against U937 cell lines. On the other hand, DBMPP
suppressed the expression of tumor suppressor factors of p27Kip1 and p21Cip1 against U937
cell lines, and then the phospha sugar induced apoptosis and stops the cell cycle progress
(Figure 3). Similarly, DBMPP enhanced the expression of tumor accelerator factors of
Aurora-A, Aurora-B, Survivin, FoxM1, Skp2, hKIS, KPC1, and Pirh1 against K562 cell
lines, and then affected on the cell cycle progression (Figure 4).13
The Western blot analysis for TBMPP against leukemia cell lines showed the en-
hanced expression of IER5, and then suppressed the expression of Cdc25B (Figure 5).14
Cdc25B is known to be a common factor of cell cycle progression for many kinds of
different tumor cell lines, therefore, phospha sugars TBMPP and DBMPP may be expected
Figure 2 Apoptosis (%) induced by TBMPP (20 μM) for 48 h at 37◦C.

218
R. MAKITA ET AL.
Figure 3 Western blot analysis by DBMPP against U937 cell lines.
to be quite efficient antitumor agents against many kinds of leukemia cell lines and to cure
effectively tumor patients suffered from different types of leukemia cell lines.
EXPERIMENTAL
Instruments
TLC (Silica gel: Wako Chromato sheet and/or Merk Kieselgel 60; El◦uent : CHCl3
: MeOH = 20 : 1, in Rf value); melting point apparatus (Gallenkamp, in C) and ther-
mal analysis instrument (Shimazu DTG-60A50AH, TGA and DSC, in oC); HPLC (GL
Science: GL-7410 HPLC Pump and GL-7450 UV Detector); MS (MALDI-TOF-MS: GL
Science, Voyager-DE Porimerix; Matrix: α-Cyano-4-hydroxycinnamic acid, in m/z); IR
Figure 4 Western blot analysis by DBMPP against K562 cell lines. (Color figure available online).

RESEARCH ON PHOSPHA SUGAR FOR ANTITUMOR DRUG
219
Figure 5 Western blot analysis; Acceleration of IER5 expression and suppression of Cdc25B expression by
TBMPP.
(JASCO FT/IR 410 (KBr), in cm−1); 1H-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 (1),15 was prepared and used as the start-
ing material.
Synthesis of 4-bromo-3-methyl-1-phenyl-2-phospholene 1-oxide (2)
3-Methyl-1-phenyl-2-phospholene 1-oxide (1: 0.96 g, 5.0 mmol; 1.0 eq.) was dis-
solved in CCl4 (5 mL), and to the solution NBS (1.33 g, 1.5 eq.) was added and stirred to
◦
make clear solution at 70 C. To the solution was then added AIBN (0.12 g, 0.75 mmol;
0.15 eq.) and the solution of the reaction mixture was refluxed for 1 day. The reaction
mixture was treated with saturated aqueous solution of sodium hydrogensulfite, and then
the solution was washed with saturated sodium hydrogencarbonate solution, brine, and
water followed by drying over anhydrous Na2SO4, which was followed by removal of the
solvent afforded crude product. Silica gel column chromatograpy of the product with the
mixed eluent of chloroform and methanol (20 : 1) afforded product 2 (0.88 g, 3.3 mmol) in
a yield of 65%; Molecular equation: C11H12BrOP; M.W.: 271.01; TLC: Rf = 0.42 (CHCl3
:MeOH = 20 : 1); MS (m/z), 271.5(MH+, 100), 273.5(MH+, isptope, 90); 1H-NMR (CDCl3,
300 MHz), δ = 2.23 (s, 3H, CH3), 2.70–2.89 (m, 1H, C5), 2.96–3.15 (m, 1H, C5), 5.14–5.20
(m, 1H, C4), 6.17 (t, J = 20.1 Hz, 1H, C2), 7.48–7.65(m, 3H, o,p-Ph), 7.80–7.87(m, 2H,
m-Ph).
Synthesis of 2,3-dibromo-3-methyl-1-phenylphospholane 1-oxide
(DBMPP (3))
3-Methyl-1-phenyl-2-phospholene 1-oxide (1: 0.21 g, 1.1 mmol; 1.0 eq.) was dis-
solved in CHCl3 (5 mL), and to the solution Cu(II)Br2 (0.12 g, 0.53 mmol; 0.49 eq.) was
added CHCl3 (10 mL) solution of bromine (0.5 mL; 2.7 g, 17 mmol; 16 eq.) dropwisely
and stirred for 1 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 hydrogencarbonate, 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 (20

220
R. MAKITA ET AL.
: 1) afforded 2,3-dibromo-3-methyl-1-phenylphospholane 1-oxide (3; 0.35 g, 0.99 mmol)
in 90% yield as a colorless crystal; Molecular equation: C11H13Br2OP; M.W.: 352; TLC:
Rf = 0.23 (CHCl3 :MeOH = 20 : 1); MS (m/z), 351.5 (MH+, 60), 353.5 (MH+, isptope
peak, 100), 355.5 (MH+, isptope peak, 40); 1H-NMR (CDCl3, 300 MHz), δ = 2.17 (s, 3H,
C3-CH3), 2.44–2.58 (m, 2H, C5), 2.60–2.83 (m, 2H, C4), 4.67 (dd, J = 1.5 Hz, J = 7.2 Hz,
1H, C2), 7.32–7.56 (m, 5H, Ph).
Synthesis of 2,3,4-tribromo-3-methyl-1-phenylphospholane 1-oxide
(TBMPP (4))
Similarly, the reaction of 4-bromo-3-methyl-1-phenyl-2-phospholene 1-oxide (2:
0.16 g, 6.0 mmol; 1.0 eq.) with bromine (1.2 mL; 6.6 g, 41 mmol; 6.8 eq.) in CCl4 under
reflux temperature for 24 h and work-up procedure afforded TBMPP (1.7 g, 4.0 mmol)
in 69% yield; Molecular equation: C11H12Br3OP; M.W.: 431; TLC: Rf = 0.62 (CHCl3 :
◦
MeOH = 20 : 1); Mp: 142–144 C; MS (m/z): 429.0 (MH+, 45), 431.1 (MH+, isptope,
100), 433.1 (MH+, isptope, 95), 435.1 (MH+, isptope, 30); 1H-NMR (CDCl3, 300 MHz),
δ = 2.14 (s, 3H, C3-CH3), 2.73–2.90 (m, 1H, C5), 3.04–3.14 (m, 1H C5), 4.26 (d, J =
1.8 Hz,1H, 4C), 4.68–4.86 (m, 1H, C2), 7.52–7.79 (m, 5H, Ph).
Synthesis of 2-bromo-3-hydroxy-3-methyl-1-phenylphospholane
1-oxide (5)
The reaction of 3-methyl-1-phenyl-2-phospholene 1-oxide (1: 0.24 g, 1.3 mmol; 1.0
eq.) with bromine (0.5 mL; 2.7 g, 17 mmol; 13 eq.) in the mixed solvent of CHCl3 (2 mL) and
water (8 mL (340 eq.) of diluted sodium hydroxide aqueous solution) under stirring for 3 d at
room temperature was progressed. The reaction mixture was treated with saturated sodium
hydrogensulfite solution and the product was extracted with CHCl3. The extract was washed
with saturated sodium hydrogencarbonate solution, brine, and water, and then the solution
of the product was dried over anhydrous Na2SO4. Evaporation of the solvent followed by
silica gel column chromatography with the mixed eluent of chloroform and methanol
(20 : 1) afforded product 5 (0.20 g, 0.69 mmol) in 55% yield; Molecular equation
C11H14BrO2P; M.W.: 289; TLC: Rf = 0.45 (CHCl3 : MeOH = 20 : 1); MS (m/z): 289.5
1
(MH+), 291.5 (MH+, isptope); H-NMR (CDCl3, 300 MHz): δ = 1.69 (s, 3H, C3-CH3),
2.01–2.51 (m, 2H, C4, C5), 4.32 (m, 1H, C2), 7.48–7.71 (m, 5H, Ph).
Synthesis of 2-bromo-3-(2-hydroxyethoxy)-3-methyl-1-
phenylphospholane 1-oxide (6)
The CHCl3 (2 mL) solution of the mixture of 3-methyl-1-phenyl-2-phospholene 1-
oxide (1: 0.050 g, 0.26 mmol; 1.0 eq.), ethylene glycol (3 mL, 3.3 g, 53 mmol; 200 eq.), and
bromine (0.3 mL; 1.6 g, 10 mmol; 39 eq.) was stirred for 2 d at room temperature. The work-
up procedure of the reaction mixture by treating with saturated sodium hydrogensulfite
and washing with sodium hydrogencarbonate, brine, and water followed by drying over
anhydrous Na2SO4 and removal of the solvent, and chromatography on silica gel (eluent:
CHCl3 : MeOH = 20; 1) afforded product 6 (0.077 g, 0.23 mmol) in a yield of 88%;
Molecular equation: C13H18BrO3P; M.W.: 333; TLC: Rf = 0.15 (CHCl3 : MeOH = 20 : 1);

RESEARCH ON PHOSPHA SUGAR FOR ANTITUMOR DRUG
221
MS (m/z): 333.1 (MH+, 100), 335.1 (MH+, isptope, 85); 1H-NMR (CDCl3, 300 MHz), δ =
1.57 (s, 3H, C3-CH3), 2.10–2.31 (m, 2H, C5), 2.40–2.44 (m, 2H, C4), 3.63–3.65 (m, 2H,
-CH2OH), 3.82–3.85 (m, 2H, -OCH2-), 4.19 (s, 1H, -OH), 7.50–7.82 (m, 5H, Ph)
Synthesis of 2-bromo-3-(2-(2-hydroxyethoxy)-ethoxy)-3-methyl-1-
phenylphospholane 1-oxide (7)
Similarly, the reaction of 3-methyl-1-phenyl-2-phospholene 1-oxide (1: 0.050 g,
0.26 mmol; 1.0 eq.) with bromine (0.5 mL; 1.6 g, 10 mmol; 38 eq.) in diethylene glycole
(3 mL; 3.3 g, 31 mmol; 120 eq.) for 2 d at room temperature under stirring and the work-up
procedure followed by silica gel column chromatography (eluent: CHCl3 : MeOH = 20 : 1)
afforded product 7 (0.055 g, 0.15 mmol) in 58% yield; Molecular equation C15H22BrO4P;
M.W.: 377; TLC: Rf = 0.15 (CHCl3 : MeOH = 20 : 1); MS (m/z): 377.3 (MH+, 100), 379.3
1
(MH+, isptope, 90); H-NMR (CDCl3, 300 MHz), δ = 1.53 (s, 3H, C3-CH3), 2.07–2.13
(m, 2H, C5), 2.34–2.53 (m, 2H, C4), 3.84 (s, 1H, OH), 3.59–3.84 (m, 8H, -CH2CH2-),
4.15–4.17 (m, 1H, C2), 7.46–7.77 (m, 5H, Ph)
Synthesis of 2-Imidazolyl-3-methyl-1-phenyl-2-phospholane 1-oxide (8)
To an acetonitril (3 mL) solution of 4-bromo-3-methyl-1-phenyl-2-phospholene 1-
oxide (2: 0.050 g, 0.18 mmol; 1.0 eq.) was added imidazole (0.062 g, 0.91 mmol; 5.1
eq.) and the mixture was stirred for 24 h at 60◦C. The reaction mixture was worked-up by
addition of CHCl3 followed by washing with saturated sodium hydrogencarbonate solution,
brine, and water, and then dried over anhydrous Na2SO4 followed by removal of the solvent
to afford crude product 8, whose chromatography on silica gel with an eluent of mixed
solvent (CHCl3 : MeOH = 20 : 1) afforded product 8 (0.042 g, 0.16 mmol) in a yield of
89%; Molecular equation: C14H15N2OP; M.W.: 258; TLC: Rf = 0.2 (CHCl3 : MeOH = 20
: 1); MS (m/z): 259.5 (MH+, 100); 1H-NMR (CDCl3, 300 MHz), δ = 1.88 (s, 3H, C3-CH3),
2.36–2.49 (m, 1H, C5), (2.85–2.95 (m, 1H, C5), 5.16(s, 1H, C2), 6.31 (d, J = 21.6 Hz,
im-C4-H), 7.13 (d, J = 20.9 Hz, im-C5-H), 7.52–7.72 (m, 5H, Ph), 7.63 (s, 1H, im-C2-H)
Evaluations and Analyses
MTT In Vitro Evaluation for Phospha Sugars Against Leukemic Cells13
Antitumor activities of phospha sugars 2–8 prepared were evaluated by MTT method
against K562 (human chronic myelogenous leukemia) and U937 (human acute mye-
logenous leukemia) cell lines as well as HL60, NB4, YRK2, NOMO-1, CEM, MOLT4,
SUP-B15, MEG-01, and SHG3 cell lines. Cells were seeded in 96-well flat-bottomed mi-
croplates 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 h at 37◦C, and then 10 μL solution of 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) was added to each well at the final
concentration of 1.0 μg/mL/well. After incubation for 24 h or 48 h at 37◦C, absorbance
was measured at a wavelength of 560 nm by using a microplate reader for the in vitro
evaluation.

222
R. MAKITA ET AL.
Cell Cycle Analyses13
Propidium iodide (PI) (Sigma) staining was used to analyze cellular DNA content.
Cells treated with Imatinib mesylate (Glivec, Gleevec) (1 μM) or phospha sugars DBMPP
or TBMPP were cultured at 37◦C in 2 mL of complete medium containing 1 × 106 cells.
After incubation for 24 h or 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. For apoptosis analysis, 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).
Western Blot Analyses13,14
For Phospha Sugar Treated Leukemia Cells. Leukemia cells treated with
phospha sugars (DBMPP or TBMPP) were harvested, and then washed with cold PBS,
and resuspended in lysis buffer containing 0.5% Nonidet P-40, 50 mM Tris-HCl (pH 8.0),
0.1 mM EDTA, 150 mM NaCl, 1 mM sodium orthovanadate, and 1 mM dithiothreitol
supplemented with one Complete Mini protease inhibitor tablet (Boehringer Mannheim
GmBH) per 20 mL lysis buffer immediately before use. The proteins were separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred
to polyvinylidene difluoride membranes (Millipore). The membranes were then blocked
with 0.5% milk in PBS for 1 h at room temperature. After being washed with Tris-buffered
saline Tween (TBS-T), the membranes were incubated for 1 h at room temperature with
horseradish peroxidase-conjugated goat anti-mouse IgG or anti-rabbit IgG (Amersham
Biosciences Inc.) for 1 h and exposed to X-ray film at room temperature. The signal was
detected by chemiluminescence using an ECL detection kit (Amersham Bioscience Inc.).
The following commercially available antibodies and dilutions were used for Western blot
analysis: rabbit polyclonal anti-FoxM1 antibody (MPP2 K-19, 1:500) (Santa Cruz Biotech-
nology, Inc.), rabbit polyclonal anti-p27Kip1 antibody (1:1000) (Santa Cruz Biotechnology,
Inc.), mouse monoclonal anti-p21Cip1 antibody (1:1000) (Santa Cruz Biotechnology, Inc.),
rabbit polyclonal anti-Cdc25B2 antibody (1:500) (Santa Cruz Biotechnology, Inc.), mouse
monoclonal anti-Cyclin D1 antibody (1:500) (Santa Cruz Biotechnology, Inc.), mouse
monoclonal anti-Cyclin A antibody (1:500) (Santa Cruz Biotechnology, Inc.), rabbit poly-
clonal anti-KIS antibody (1:500) (ABGENT, Inc.), rabbit polyclonal anti-Aurora-B anti-
body (1:500) (ABGENT, Inc.), mouse monoclonal anti-bcl-2 antibody (BD Biosciences
Pharmingen), mouse monoclonal anti-caspase-9 antibody (BD Biosciences Pharmingen),
mouse monoclonal anticaspase-3 (CPP32) antibody (BD Biosciences Pharmingen), and
mouse monoclonal anti-PARP antibody (BD Biosciences Pharmingen). To ensure equal
protein volume loading, similar experiments were performed by using a mouse monoclonal
antiactin antibody (C-4; ICN Biomedicals, Inc., Aurora, OH) as an internal control.
For Transfected Cells. Cells transfected with scrambled shRNA or with IER5
shRNA-#1 or -#2 were harvested. After 3 d, Western blot analysis was performed using the
following antibodies: goat polyclonal anti-IER5 (Abcam), rabbit polyclonal anti-Cdc25B
(Santa Cruz), anti-CHK1 (Santa Cruz), anti-WEE1 (Santa Cruz), anti-Aurora-B (Santa
Cruz), mouse monoclonal anti-Cyclin B1 (Santa Cruz), and anti-Survivin (Santa Cruz). To
ensure equal protein volume loading, similar experiments were performed by using a mouse
monoclonal antiactin antibody (C-4; ICN Biomedicals, Inc., Aurora, OH) as an internal
control.

RESEARCH ON PHOSPHA SUGAR FOR ANTITUMOR DRUG
223
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