Preparation of phospha sugar analogues and their evaluation as novel molecular targeting anticancer agents

Article abstract of DOI:10.1080/10426507.2010.530326

Phospha sugars were prepared by a novel synthetic route starting from phosphorus heterocyclic compounds, 2-phospholenes. The anhydro- and deoxy-phospha sugar derivatives have been revealed to have potential anticancer activities against human leukemia of

Full text of DOI:10.1080/10426507.2010.530326

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Preparation of Phospha Sugar Analogues
and Their Evaluation as Novel Molecular
Targeting Anticancer Agents
Kenji Tsunekawa a , Mitsuji Yamashita a , Michio Fujie a b , Taishi
Niimi a , Takuya Suyama a , Kazuhide Asai a , Satoru Ito a , Junko
Yamashita a , Manabu Yamada a , Nobuhisa Ozaki a & Satoki
Nakamura c
a Department of Nano Materials, Graduate School of Science and
Technology , Shizuoka University , Hamamatsu , Japan
b Research Equipment Center , Hamamatsu University School of
Medicine , Hamamatsu , Japan
c Department of Internal Medicine III , Hamamatsu University School
of Medicine , Hamamatsu , Japan
Published online: 25 Apr 2011.
To cite this article: Kenji Tsunekawa , Mitsuji Yamashita , Michio Fujie , Taishi Niimi , Takuya
Suyama , Kazuhide Asai , Satoru Ito , Junko Yamashita , Manabu Yamada , Nobuhisa Ozaki & Satoki
Nakamura (2011) Preparation of Phospha Sugar Analogues and Their Evaluation as Novel Molecular
Targeting Anticancer Agents, Phosphorus, Sulfur, and Silicon and the Related Elements, 186:4,
936-944, DOI: 10.1080/10426507.2010.530326
To link to this article: http://dx.doi.org/10.1080/10426507.2010.530326
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Phosphorus, Sulfur, and Silicon, 186:936–944, 2011
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2010.530326
PREPARATION OF PHOSPHA SUGAR ANALOGUES
AND THEIR EVALUATION AS NOVEL MOLECULAR
TARGETING ANTICANCER AGENTS
Kenji Tsunekawa,1 Mitsuji Yamashita,1 Michio Fujie,1,2
Taishi Niimi,1 Takuya Suyama,1 Kazuhide Asai,1 Satoru Ito,1
Junko Yamashita,1 Manabu Yamada,1 Nobuhisa Ozaki,1
and Satoki Nakamura3
1Department of Nano Materials, Graduate School of Science and Technology,
Shizuoka University, Hamamatsu, Japan
2Research Equipment Center, Hamamatsu University School of Medicine,
Hamamatsu, Japan
3Department of Internal Medicine III, Hamamatsu University School of Medicine,
Hamamatsu, Japan
Abstract Phospha sugars were prepared by a novel synthetic route starting from phosphorus
heterocyclic compounds, 2-phospholenes. The anhydro- and deoxy-phospha sugar derivatives
have been revealed to have potential anticancer activities against human leukemia of K562
and U937 cell lines. In this article, deoxybromophospha sugars with different numbers of
bromo substituents were prepared, and their anticancer activities were evaluated by MTT
method. The order of the activities depending on the number of bromo substituent was first
revealed, and trideoxytribromotetrofuranose type phospha sugar [2,3,4-tribromo-3-methyl-1-
phenylphospholane 1-oxide (4: TBMPPAO)] was the most active among these phospha sugars
prepared. Diasteromers of dideoxydibromotetrofuranose-type phospha sugar [2,3-dibromo-3-
methyl-1-phenylphospholane 1-oxide (2: DBMPPAO)] were separated into four components,
and the structure as well as the character of each component was assigned by spectral and
chromatographic data as well as by MTT method.
The deoxybromophospha sugars have higher antileukemia activity than Gleevec against U937
cells.
Keywords Antileukemia; deoxybromophospha sugars; MTT in vitro evaluation; phosphorus
heterocycles
INTRODUCTION
Well known typical pseudo sugars are carba-, aza-, and thia-sugars,1–3 with a carbon,
nitrogen, and sulfur atom, respectively, instead of the oxygen atom in the hemiacetal ring of
Received 30 July 2010; accepted 4 October 2010.
The authors greatly acknowledge financial support by the Satellite Shizuoka and the Headquarters of Japan
Science and Technology Agency (JST); Grants-in-Aid for Scientific Research by Japan Society for the Promotion
of Science (JSPS), the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT); and the
Ministry of Health, Labour and Welfare, Japan (MHLW).
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 432-8561, Japan. E-mail:
tcmyama@ipc.shizuoka.ac.jp
936

PREPARATION OF PHOSPHA SUGAR ANALOGUES
937
the normal sugars. These pseudo sugars are known to exist in nature and are also prepared by
synthetic sugar chemistry, where generally pentoses or hexoses of normal carbohydrates are
used as the starting materials for the syntheses.4 Pseudo sugars exert important biological
activities; therefore, many studies on not only the isolation and synthesis of pseudo sugars
but also on their characterization and evaluation for biologically active materials are actively
performed. On the other hand, phospha sugars, one new category of the pseudo sugars that
have a phosphorus atom in the hemiacetal ring of sugars, are not yet found in nature, and
the syntheses of them are rather difficult compared with typical pseudo sugars.5–8
We have been searching for biologically active phospha sugars and we have first found
several new phospha sugar derivatives with antitumor activities against leukemia cell lines
by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) in vitro
evaluation methods for phospha sugar analogues.9 In this article, we will first deal with the
catalytic addition reaction of bromine to prepare branched deoxybromophospha sugars, e.g.,
2,3-dibromo-3-methyl-1-phenylphospholane 1-oxide (2: DBMPPAO) and 2,3,4-tribromo-
3-methyl-1-phenylphospholane 1-oxide (4: TBMPPAO), and the evaluation of the branched
deoxybromophospha sugar analogues 2–4 by MTT in vitro method against human leukemia
cell lines. The activity of these branched deoxybromophospha sugar analogues synthesized
were compared with that of Gleevec (imatinib mesylate), clinically being used as an
anticancer agent at present. The present results showed that DBMPPAO (2) and TBMPPAO
(4) are superior to Gleevec against U937 leukemic cell lines.
RESULTS AND DISCUSSION
Dideoxydibromotetrofuranose-type phospha sugar analogues [Scheme 1; 2,3-
dibromo-3-methyl-1-phenylphospholane 1-oxide (DBMPPAO (2): R = Ph) and
2,3-dibromo-3-methyl-1-methoxyphospholane 1-oxide (R = OMe)] were prepared by
an addition reaction of bromine with 3-methyl-1-phenyl- and 3-methyl-1-methoxy-2-
phospholene 1-oxide (1; R = Ph and OMe). The addition reaction of bromine to the
electron deficient C C double bond of 2-phospholene 1 was achieved by catalysts, i.e.,
manganese(IV) oxide and manganese(II) bromide, under the mild reaction conditions in
dichloromethane (Scheme 1).
Scheme 1 Synthesis of 2,3-dibromo-3-methyl-1-phenylphospholane 1-oxide [2: DBMPPAO (R = Ph)] and 2,3-
dibromo-3-methyl-1-methoxyphospholane 1-oxide (R = OMe). (The numbering for 2-phospholene and phospho-
lane rings is shown in the structures.)
The yield of DBMPPAO (2), by using manganese(II) catalyst, was increased from
33% (without catalyst) to 78% (with MnO2 catalyst) and 96% (with MnBr2 catalyst)
(Table 1). Entries 4–7 in the table imply that the reactions were more efficiently catalyzed
by MnBr2 than MnO2, probably because the former catalyst acts homogeneously in the

938
K. TSUNEKAWA ET AL.
Table 1 Preparation of 2,3-dibromo-3-methyl-1-phenylphospholane 1-oxide [2: DBMPPAO; R = Ph)] and 2,3-
dibromo-3-methyl-1-methoxyphospholane 1-oxide (R = OMe)
Yield [%]
Column
Enrty
R
Catalyst
E
Time [h]
chromatography
Recrystallization
1
2
3
4
5
6
7
Ph
Ph
Ph
Ph
Ph
Ph
OMe
None
—
2
2
48
8
12
12
12
1
33
78
—
92
94
96
64
—
—
(24)
(16)
—
MnO2
MnBr2
MnBr2
MnBr2
MnBr2
MnO2
1
0.5
0.5
2
—
—
16
medium. And then the catalytic reaction with 0.5 equivalents of MnBr2 for 1 h gave up
to 96% yield of DBMPPAO (2). The addition reaction of bromine to the double bond of
2-phospholenes in anhydrous media hardly proceeded in the absence of catalyst; how-
ever, the reaction in aqueous media is reported to afford 2-bromo-3-hydroxyphospholane
derivatives.10,11 Therefore, the manganese compounds catalyzed the addition reaction of
bromine to the electron deficient C C double bond of compound 1, probably because of
either higher concentration of bromonium intermediate produced or higher nucleophilicity
of bromide ion complex with Mn catalyst than that of the simple bromide ion.
4-Bromo-3-methyl-1-phenyl-2-phospholene 1-oxide (3: MBMPPEO) was subjected
to the bromine addition reaction. The addition reaction of bromine to MBMPPEO (3)
proceeded without catalyst at refluxing temperature of carbon tetrachloride to afford
tribromotrideoxytetrofuranose-type phospha sugar analogue [or 2,3,4-tribromo-3-methyl-
1-phenylphospholane 1-oxide (4: TBMPPAO)] in moderate yield (Scheme 2).
Scheme 2 Preparation of 2,3,4-tribromo-3-methyl-1-phenylphospholane 1-oxide (4: TBMPPAO).
The plausible intermediate for the addition reaction of bromine with 2-phospholene
1 in the presence of MnBr2 gave the mixture of 2 (DBMPPAO), and the structures of the
isolated four diastereomers 2a–d are shown in Scheme 3. The addition of bromine to a
C C double bond is generally known,12 and the reaction proceeds via a nucleophilic attack
of bromide ion to the bromonium intermediate. According to the reaction mechanism, the
addition of bromine under manganese(II) bromide catalyst may proceeds by nucleophilic
attack of MnBr3− at either position of C(3) or C(2) of the formed bromonium intermediate
via SN2, and then the two vicinal bromo substituents should be added in trans-fashion to
give trans-dibromo adducts 2a and 2b of DBMPPAO. The cis-dibromo adducts 2c and 2d of
DBMPPAO may be formed, either by the reaction of intermedially formed carbocation12 on

PREPARATION OF PHOSPHA SUGAR ANALOGUES
939
Scheme 3 Addition reaction mechanism of bromine to 2-phospholene 1 under MgBr2 catalyst and the structures
of diastereomers 2a to 2d of DBMPPAO.
the third position (C(3)+) with bromo anion via SN1 or the isomerization of the trans-fused
dibromo adducts via a more stable tertiary carbonium intermediate at C(3) position by SN1
mechanism, which was observed for 3-methylphospholane 1-oxides.11
The antileukemia activities for these deoxybromophospha sugars—MBMPPEO,
DBMPPAO, and TBMPPAO—having different number of bromo substituents were eval-
uated by MTT in vitro method against K562 and U937 cell lines, and the results against
U937 cell lines are shown in Figure 1.
The axes of the ordinate and the abscissa of Figure 1 show cell viability (%) or
survival ratio (%), which represent the effect of drugs on the cell proliferation of U937
cells based on the absorbance of visible light of 560 nm by MTT in vitro evaluation and the
concentration (µM) of the agents evaluated, respectively (see the Experimental section).
Figure 1 shows that the antiproliferative activity of phospha sugars against leukemic cells
is proportionally affected by the number of bromo substituents on the phospha sugars,
and that TBMPPAO (4) has the highest activity against U937 cells among the branched
Figure 1 MTT in vitro evaluation for deoxybromophospha sugars—MBMPPEO, DBMPPAO, and
TBMPPAO—against U937 leukemia cell lines. [MBMPPEO (◦: IC50 = 83.3 µM), DBMPPAO (ꢁ: IC50
=
27.5 µM), TBMPPAO (ꢀ: IC50 = 2.3 µM), and Gleevec (•).]

940
K. TSUNEKAWA ET AL.
Table 2 Diastereomers 2a–d of 2,3-dibromo-3-methyl-1-phenylphospholane 1-oxide (2: DBMPPAO) prepared
by MnO2 and MnBr2 catalyst
Ratio of
diastereomerc
[%]
Catalyst
1H NMRb chemical shift δ [ppm]
(coupling constant JHP [Hz])
Retention
time of
Compound
HPLCa [min]
C(2)
H
C(3) Me
C(4,5)
H
P(1) Ph
H
MnO2
MnBr2
2a
2b
2c
2d
8.1
9.1
9.9
4.19 (7.8)
4.51 (7.3)
4.51 (5.1)
4.20 (7.2)
1.56
1.55
1.56
1.52
2.20–3.10
2.24–2.95
2.24–3.13
2.22–3.05
7.53–7.86
7.51–7.84
7.51–7.88
7.48–7.84
27
32
23
18
3
36
6
11.5
55
aThe retention time was observed by HPLC analysis (column: Wakopak, Wakosil ꢀ 4.6 mm × 250 mm;
eluent:CHCl3:MeOH = 30:1; flow rate: 0.5 mL/min).
b1H NMR data of proton chemical shift (in ppm) and coupling constant (in Hz) were obtained by a 300 MHz
(CD3OD) instrument.
cDiastereomer ratio is based on that of HPLC peak area.
deoxybromophospha sugars evaluated and Gleevec (imatinib mesylate), which is known
as a molecule-targeting chemotherapeutical agent against leukemia cells.13 TBMPPAO (4)
was much more active against U937 cells by ten times (IC50 was 2.3 µM) than that of
DBMPPAO (2).
The diastereomeric components of mixture 2 (DBMPPAO) in the product (entries
2 and 5 of Table 1) were separated by HPLC (column: Wakopak Wakosil ꢀ 20.0 mm ×
250 mm; eluent: CHCl3:MeOH = 30:1; flow rate: 5.0 mL/min) into the four diastereomers
2a–d. The structures of diastereomers 2a–d of the separated fractions with the retention
times of 8.1, 9.1, 9.9, and 11.5 min should be assigned to 2a, 2b, 2c, and 2d of DBMPPAO,
respectively, based on the spectral and chromatographical data (Scheme 3 and Table 2).
The structural assignment is based on the chemical shift values of C(2) H and
C(3) Me on the 2- and 3-positions, respectively, of the phospholane ring whose proton
chemical shifts in 1H NMR spectra are affected by the magnetic anisotropic effect caused by
the phenyl group on the phosphorus heterocycles. By this anisotropic effect, the hydrogens
on the second position (C(2)H) of the phospholane rings of 2a to 2d of DBMPPAO being
located on the same side of the phenyl group were shifted to relatively higher magnetic
field; on the other hand, the hydrogens on the opposite side of the phenyl group shifted to
relatively lower field. The methyl group on the third position (C(3)CH3) was also shifted by
the anisotropy effect to the higher or lower magnetic field depending on the same or opposite
side of the phenyl group, respectively.14 The largest retention time of HPLC analysis for 2d
may be supported by the largest dipole moment (7.05 D) calculated by MOPAC among the
diastereomers of DBMPPAO (2) (the calculated dipole moments for 2a, 2b, and 2c were
5.43, 5.65, and 4.05 D, respectively).
Evaluations of 2,3-dibromo-3-methyl-1-phenylphospholane 1-oxide (2: DBMPPAO;
mixture of diastereomers) and the separated four diastereomers 2a–2d as inhibitors on
proliferation of leukemia cells were carried out by in vitro MTT assay method.15 K562
and U937 cells of leukemia cell lines were incubated with the diastereomer mixture of
DBMPPAO (2) or isolated diastereomers 2a–d of DBMPPAO at the indicated concen-
trations (0–500 µM) at 37◦C for 48 h. Each component of DBMPPAO (mixture 2 and
diastereomers 2a–d) strongly suppressed the cell proliferation of K562 and U937 cells

PREPARATION OF PHOSPHA SUGAR ANALOGUES
941
Figure 2 Inhibition from cell proliferation of leukemia cells by treatment with the mixture 2 and the separated
diastereomers 2a–d of DBMPPAO against K562 cells (top) and U937 cells (bottom), respectively, at 37 ◦C for 48
h. [Mixture (ꢁ), 2a(ꢂ), 2b(ꢃ), 2c(ꢁ), and 2d(•) of DMPPAO (2).]
in a dose-dependent manner, and as the result, the intensity of absorbance at the wave
length of 560 nm in MTT method decreased (Figure 2). The half values of the absorbance
intensity against K562 and U937 cells afforded IC50 of 32 µM and 27 µM, respectively,
for DBMPPAO (2) (top and bottom graphs, respectively, of Figure 2).
In conclusion, we have prepared branched dideoxydibromotetrofuranose-type phos-
pha sugar or 2,3-dibromo-3-methyl-1-phenylphospholane 1-oxide (2: DBMPPAO) in 96%
yields from 2-phospholene 1. DBMPPAO (2) was separated into four diastereomers 2a–d.
Branched trideoxytribromotetrofuranose-type phospha sugar analogue, 2,3,4-tribromo-3-
methyl-1-phenylphospholane 1-oxide (4: TBMPPAO), was also prepared by the addition of
bromine to the C C double bond of 2-phospholene 3. The MTT in vitro bio-assay method
for these branched deoxybromophospha sugar analogues revealed that DBMPPAO (2) and
TBMPPAO (4) have quite efficient anticancer activities against leukemia cells in manners of
(i) wider spectra than Gleevec, (ii) high activities, and (iii) high specificities and selectivities
against cancer cells (no damage was observed to normal leukocyte). Deoxybromophospha
sugar analogues 2 and 4 were also active against solid cancer cells. Optimization of the
structure and activity relationships for phospha sugars against solid and solution cancer
cells is now in progress.
To innovate in tumor therapy, research on developing both advanced medicinal ma-
terials for curing and diagnosing cancer cells is incleasingly demanded.16

942
K. TSUNEKAWA ET AL.
EXPERIMENTAL
Instrumentation Details
TLC (silica gel: Wako Chromato Sheet and/or Merk Kieselgel 60; ◦Eluent:
CHCl3:MeOH = 20:1, in Rf value); melting point apparus (Gallenkamp, in C) and
thermal analysis instrument (Shimazu: DTG-60A50AH, TGA & DSC, in ◦C); 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
[JASCO FT/IR 410 (KBr), in cm−1]; 1H NMR [JEOL JNM-AL300 (300 MHz) and Hitach
R90H (90 MHz); Solvent: CDCl3, in δ (ppm) from TMS] were used for analyzing the
products.
Synthesis of 2,3-Dibromo-3-methyl-1-phenylphospholane
1-Oxide (2: DBMPPAO)
To CH2Cl2 (10 mL), a solution of 3-methyl-1-phenyl-2-phospholene 1-oxide (1;
0.28 g, 1.5 mmol) and manganese(II) bromide (0.16 g, 0.74 mmol; 0.5 eq.) was added
dropwise CH2Cl2 (10 mL) to a solution of bromine (0.40 mL, 9.8 mmol; 7.0 eq.), and
the reaction mixture was stirred for 1 h under Ar atmosphere at room temperature. The
reaction was quenched by the addition of saturated sodium sulfite aqueous solution (10 mL).
The aqueous mixture was extracted with chloroform (3 × 10 mL). The organic layer was
neutralized with saturated NaHCO3 aqueous solution, washed with saturated NaCl solution,
and dried with anhydrous sodium sulfate. The solvent of the filtrate was evaporated under
reduced pressure to give an oily mixture of product 2. The mixture was purified by column
chromatography on silica gel by using chloroform and methanol (30:1) as the eluent to
give 2,3-dibromo-3-methyl-1-phenylphospholane 1-oxide (2: DBMPPAO; 0.49 g) in 96%
yield.
Rf on TLC: 0.52; M.P.: 189.20◦C; B.P.: 280.24◦C; MS (m/z): 353.24 (M—H+
1
(Molecular peak −1); IR: 1126 cm−1 (P O), 748 cm−1, 1396 cm−1 (C-Br); H NMR
(CDCl3, 300 MHz): δ: 1.67 (s, 3H, CH3), 2.36–2.46 (m, 2H, C(4)H2), 2.97–3.02 (m, 2H,
C(5)H2), 4.28–4.31 (m, 1H, C(2)H), 7.51–7.70 (m, 5H, Ph).
The diastereoisomers of product 2 were separated by preparative HPLC [column:
Wakopac, Wakosil ꢀ 20.0 mm × 250 mm (Wako Gel); eluent: chloroform:methanol =
40:1; flow rate: 5.0 mL/min] to give the four diastereomeric components 2a–d (1H NMR
and HPLC, data are shown in Table 2). The MOPAC calculation for dipole moments of 2d
was 7.05 D, which was a larger value than those of 5.43, 5.65, and 4.05 D for 2a, 2b, and
2c, respectively.
In Vitro MTT Assay
2,3-Dibromo-3-methyl-1-phenylphospholane 1-oxide (2: DBMPPAO), being evalu-
ated by the in vitro MTT method, was dissolved in dimethylsulfoxide (DMSO) (Sigma
Chemical Company, St. Louis, MO, USA) as the solvent, and was diluted into appropriate
concentration with DMSO in culture medium immediately before use. The final concen-
trations of 2 in DMSO in all experiments were less than 0.010%, and all the treatment
conditions were compared with vehicle controls. The control experiments for the evalua-
tion were carried out by using DMSO, and the absorption change by DMSO in the MTT
method was not observed for K562 and U937 cells at 37◦C for 48 h.

PREPARATION OF PHOSPHA SUGAR ANALOGUES
943
Cells grown in the presence of the medium alone were used as the controls. Af-
ter incubation of the control and the sample solutions being added by MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] at 37◦ for 4 h, absorbance
was measured at a wavelength of 560 nm by using a microplate reader for in vitro evalua-
tion. The concentration-dependent absorbance changes for sample solutions in wells seeded
by cells and added by different concentrations of phospha sugars were carried out for 48 h
incubation and additional incubation for 4 h at 37◦C after addition of MTT. The observed
absorbance (ordinate) at 560 nm visible light was plotted vs. the concentration (abscissa)
of the sample for the MTT in vitro evaluation (top and bottom graphs of Figure 2), and the
sample concentration corresponding to the half intensity of the absorption affords IC50.
Human Tumor Cell Lines and Culture
Chronic myeloid leukemia (K562) and promyeloid leukemia (U937) cells were pur-
chased from American Type Culture Collection (ATCC; Rockville, MD, USA). The cells
were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf
serum (FCS) 292 mg/L (or 2.0 mmol) L-glutamine, 100 µg/mL streptomycin, and 200 U/mL
penicillin (GIBCO-BRL, Gaithersburg, MD, USA). All cells were maintained in a humid-
ified 5% CO2 atmosphere at 37◦C.
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