Synthesis and evaluation as MRI probe of the trifluoromethylated p-boronophenylalanine and its alcohol derivative

Article abstract of DOI:10.1016/j.bmc.2006.12.043

Boron-neutron capture therapy (BNCT) and magnetic resonance imaging (MRI) are quite attractive techniques for treatment and diagnosis of cancer, respectively. In order to develop practical tools for BNCT and MRI, novel compounds containing both the trifluoromethyl group and ¹⁰B atom in a single molecule were designed. In the present study, p-boronophenylalanine and p-boronophenylalaninol with the trifluoromethyl group were synthesized, and ¹⁹F NMR measurements of these compounds were carried out.

Full text of DOI:10.1016/j.bmc.2006.12.043

Bioorganic & Medicinal Chemistry 15 (2007) 2198–2205
Synthesis and evaluation as MRI probe of the trifluoromethylated
p-boronophenylalanine and its alcohol derivative
Yoshihide Hattori,a Hitoshi Yamamoto,b Hideya Ando,c,d Hirofumi Kondoh,c,d
Tomoyuki Asano,c,d Mitsunori Kirihata,c,d Yoshihiro Yamaguchia
and Tateaki Wakamiyaa,*
aFaculty of Science and Technology, Kinki University, Higashi-osaka, Osaka 577-8502, Japan
bGraduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
cBio-Research Inc., Kobe, Hyogo 650-0047, Japan
dGraduate School of Life & Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8231, Japan
Received 31 October 2006; revised 6 December 2006; accepted 7 December 2006
Available online 10 January 2007
Abstract—Boron-neutron capture therapy (BNCT) and magnetic resonance imaging (MRI) are quite attractive techniques for treat-
ment and diagnosis of cancer, respectively. In order to develop practical tools for BNCT and MRI, novel compounds containing
both the trifluoromethyl group and 10B atom in a single molecule were designed. In the present study, p-boronophenylalanine and
p-boronophenylalaninol with the trifluoromethyl group were synthesized, and 19F NMR measurements of these compounds were
carried out.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
useful technique for treatment of cancer.3 b-[4-
(10B)Boronophenyl]alanine (10Bpa)4 (1) and b-[4-
(10B)boronophenyl]alaninol (10Bpa-ol)5 (2), in which
each boron atom is enriched with 10B isotope, had been
created as novel 10B carriers for BNCT.
Magnetic resonance imaging (MRI) is commonly used
as a technique for diagnosis of cancer.1 The characteris-
tics of MRI are its relatively high resolution, the use of
probes (or contrast enhancers) that can be stored for a
long term, and the circumvention of danger resulting
from radiation exposure.
In order to create novel materials to use practically for
diagnosis and treatment of cancer by means of MRI
and BNCT, respectively, we had already synthesized
the compounds containing both fluorine6 and 10B
atoms in a single molecule such as DL-b-[4-(10B)
borono-2,6-difluorophenyl]alanine [DL-10Bpa(2,6F2)]
(3) or DL-3-[4-(10B)borono-2,6-difluorophenyl]alaninol
[DL-10Bpa(2,6F2)-ol] (4).7 As far as we examined, the
incorporated amounts of these compounds into C6
(rat glioma), KB (human melanoma), and HeLa
(human ephithelioma) cells are almost the same as that
of 10Bpa being clinically used for BNCT at present.
In particular, MRI based on the measurement of 19F
atom is becoming a remarkable method for diagnosis
of various diseases. For example, Higuchi et al. reported
that the intravenously administered 19F-containing sty-
rylbenzene derivative labels brain plaques and allows
them to be visualized in living amyloid b precursor
19
protein (APP) transgenic mice by F MRI.2
From the standpoint of treatment for brain cancer or
melanoma, the boron neutron capture therapy (BNCT)
based on the interaction between 10B isotope and
neutron has been highly noted in recent years as a quite
In the present study we newly designed and synthesized
DL-b-[4-(10B)borono-2-trifluoromethylphenyl]alanine
[DL-10Bpa(2CF3)] (5) and DL-3-[4-(10B)borono-2-trifluo-
romethylphenyl]alaninol [DL-10Bpa(2CF3)-ol] (6), in
which the fluorine atoms are increased in number com-
pared to difluoro derivatives 3 and 4 to raise the sensitiv-
ity of detection by 19F NMR (Fig. 1).
Keywords: Magnetic resonance imaging; Boron-neutron capture ther-
apy; b-[4-(10B)borono-2,6-trifluoromethylphenyl]alanine; 3-[4-(10B)-
borono-2,6-trifluoromethylphenyl]alaninol.
*
Corresponding author. E-mail: wakamiya@chem.kindai.ac.jp
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.12.043

Y. Hattori et al. / Bioorg. Med. Chem. 15 (2007) 2198–2205
2199
to give 4-bromo-2-trifluoromethylbenzoic acid (9). The
benzoic acid derivative 9 was converted into the benzyl-
alcohol derivative 10, and then the alcohol group was
protected with the MOM10 group to give the methoxy-
methyl ether derivative 11.
H N
2
R
HO
10
B
OH
In the next step, the phenylboric acid derivative 12 was
first tried to prepare from the compound 11 by the hal-
ogen-metal exchange reaction with i-PrMgCl and subse-
quent addition of 10B(OMe)3 in a similar manner as
reported previously for the preparation of
DL-10Bpa(2,6F2).7 Although, the bromo group was not
reacted with i-PrMgCl in the present case, the desired
boronation was successfully achieved via lithiation of
11 with n-BuLi.
1: R=CO H
2
2: R=CH OH
2
H N
F
H N
2
R
R
2
HO
HO
10
10
F
CF
3
B
B
OH
3: R=CO H
OH
5: R=CO H
2
2
4: R=CH OH
6: R=CH OH
2
2
Treatment of 12 with 3 M HCl to cleave the MOM
group gave the benzyl alcohol derivative 13. The
hydroxyl group in the compound 13 was changed to
bromide with PBr3, and the brominated product was
coupled with sodium diethyl acetamidomalonate.11
The diester moiety of the compound 14 was hydrolyzed
and decarboxylated to give Na-Ac-DL-10Bpa(2CF3)-OH
(15) that was hydrolyzed with 3 M HCl to give
DL-10Bpa(2CF2) (5).
Figure 1. 10Bpa (1) and the related compounds 2–6.
2. Chemistry
The synthesis of DL-10Bpa(2CF3) was carried out by the
conventional method based on the reaction of alkyl ha-
lide with sodium diethyl acetamidomalonate8 as shown
in Scheme 1.
The a-amino group of 5 was first protected with the Boc
group, and the carboxyl group of the product 16 was
then reduced via methyl ester12 to provide the alcohol
derivative 17. The Boc group of 17 was finally cleaved
with 4 M HCl/AcOEt to give DL-10Bpa(2CF3)-ol (6) as
HCl salt.
2-Aminobenzotrifluoride (7) was brominated according
to the method reported by Roche et al.,9 and the amino
group was converted into 5-bromo-2-iodobenzotrifluo-
ride (8) with t-BuNO2 and I2. The benzotrifluoride
derivative 8 was reacted with i-PrMgCl and then CO2
NH2
CF3
I
CO2H
CF3
OMOM
CF3
a
b
d
c
e
OH
CF3
Br
Br
CF3
Br
Br
7
8
9
10
CO2Et
11
AcHN
CO2H
AcHN
CO2Et
OMOM
OH
CF3
h
f
i
g
HO
HO
10B
CF3
10B
OH
HO
HO
10B
CF3
15
10B
OH
CF3
OH
OH
12
13
14
H2N
CO2H
BocHN
CO2H
BocHN
CH2OH
H2N
CH2OH
HCl
l
k
j
HO
HO
HO
HO
10B
OH
CF3
10B
OH
CF3
10B
OH
CF3
10B
OH
CF3
6
5
16
17
Scheme 1. Reagents and conditions: (a) 1—KBr, (NH4)6Mo7O24Æ4H2O, NaBO3Æ4H2O, AcOH, rt, 6 h; 2—t-BuNO2, I2, MeCN, rt, 16 h, 91.4%; (b)
1—i-PrMgCl, THF, À20 ꢁC, 2 h; 2—CO2, À20 ꢁC, 1 h, 67.8%; (c) 1—ClCO2CH(CH3)2, Et3N, THF, 1.5 h, À10 ꢁC; 2—NaBH4, H2O, 0 ꢁC, 8 h,
97.5%; (d) MOM–Cl, DIEA, CH2Cl2, reflux, 24 h, 86.2%; (e) 1—n-BuLi, THF, À78 ꢁC, 1 h; 2—10B(OMe)3, À78 ꢁC then rt, 5 h, 71.3%; (f) 3 M HCl,
THF, 60 ꢁC, 8 h; 90.5%; (g) 1—PBr3, Et2O, 0 ꢁC, 4 h; 2—sodium diethyl acetamidomalonate, THF, rt, 16 h, 79.9%; (h) 1—1 M NaOH, 80 ꢁC, 8 h;
2—3 M HCl, 80 ꢁC 5 h, 86.8%; (i) 1—3 M HCl, 80 ꢁC, 24 h; 2—propylene oxide, i-PrOH, rt, 6 h, 83.8%; (j) (Boc)2O, Na2CO3, acetone, H2O, rt, 7 h,
90.8%; (k) 1—MeI, KHCO3, rt, 24 h; 2—NaBH4, LiCl, THF, MeOH, 0 ꢁC then rt, 2 h, 91.1%; (l) 4 M HCl/AcOEt, rt, 30 min, 92.2%.

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Y. Hattori et al. / Bioorg. Med. Chem. 15 (2007) 2198–2205
3. Results and discussion
integrated counts (Fig. 4b). However, detailed assign-
ments of these two peaks are not achieved yet.
In order to apply the compounds 5 and 6 to 19F MRI, we
first examined detection-sensitivity in the measurement of
19F NMR of these fluorinated compounds in deuterium
saline13 (2.4, 0.24, 0.024, and 0.0024 mM). As shown in
Figures 2 and 3, 19F signals of the trifluoromethylated
compounds 5 and 6 are detectable much more easily than
those of difluorinated derivatives 3 and 4.
From the elucidations mentioned above, the following
results were given, that is, (i) all fluorinated DL-10Bpa
derivatives may be usable as 19F MRI probe, (ii) the
carboxyl type compound is more sensitive than the
corresponding alcohol derivative in 19F NMR
measurement, and (iii) the trifluoromethylated com-
pound 5 is better than the compound 3 as a 19F MRI
probe.
From the standpoint of future MRI study, we next
examined whether these fluorinated p-boronophenyl-
alanine derivatives incorporated into cancer cells are
detectable by 19F NMR measurement. For this purpose,
Ihara cells (human melanoma)14 incorporating the com-
pounds 3 and 5 were loaded into micro NMR tube and
then subjected to the measurement of 19F NMR (Fig. 4).
Biological evaluation of the trifluoromethylated com-
pounds 5 and 6 as boron carrier is currently being
undertaken, and the results will be reported soon else-
where. Furthermore, we are planning to apply these
compounds to 19F MRI for diagnosis of melanoma.
In the measurement of compound 3 incorporated in Iha-
ra cells, 19F signal is detectable even by four integrated
counts (ca. 1 min). When integrated counts were in-
creased to 64 (ca. 10 min) (Fig. 4a), an extra small signal
was observed beside a major one, though the reason is
not clarified. In the case of the measurement of com-
pound 5, two major signals were detected even by four
4. Experimental
4.1. General
All of the melting points are uncorrected and were mea-
sured by Yanaco MP-J3 (Yanaco CO., Ltd, Kyoto,
Figure 2. 19F NMR spectra of compounds 3 and 4.

Y. Hattori et al. / Bioorg. Med. Chem. 15 (2007) 2198–2205
2201
Figure 3. 19F NMR spectra of compounds 5 and 6.
Japan). Silica-gel column chromatography was carried
out with silica gel PSQ100B (Fuji Silysia Chemical
Ltd, Aichi, Japan). 1H NMR spectra were measured
on a Varian Mercury 300 [300 MHz, Varian CO.,
Ltd, USA] spectrometer. The chemical shifts in 1H
NMR are given in d values from TMS used as the
internal standard. 19F NMR spectra were measured
on a Varian Inova 600 [564 MHz, Varian Co., Ltd,
USA] spectrometer. Matrix-assisted laser desorption
ionization time of flight mass spectra (MALDI-TOF
MS) were obtained on a KRATOS KOMPACT MAL-
DI IV mass spectrometer (Shimadzu Co. Ltd, Kyoto,
Japan). Measurement of high resolution mass was car-
ried out by fast-atom bombardment mass spectrometry
(FAB-MS) using a JEOL JMS-700 TKM mass spec-
trometer (JEOL Co. Ltd, Tokyo, Japan). Elemental
analyses were performed at the MICRO CORDER
JM10 (J-Science Lab Co. Ltd, Kyoto, Japan).
10B(OMe)3 was purchased from Stella Chemifa Corpo-
ration (Osaka, Japan).

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Y. Hattori et al. / Bioorg. Med. Chem. 15 (2007) 2198–2205
Figure 4. 19F measurements of compounds 3 and 5 incorporated into Ihara cells.
4.2. Synthesis
(50 mL) was added i-PrMgCl (2.0 M solution in THF,
26.5 mL, 53.0 mmol) dropwise at À20 ꢁC under Ar
atmosphere. After stirring for 2 h, CO2 gas was bubbled
into the reaction mixture for 1 h under cooling at
À20 ꢁC. To the reaction mixture was added saturated
NH4Cl aq (100 mL) and extracted with AcOEt (3·
60 mL). The combined extracts were dried over anhy-
drous MgSO4 and concentrated in vacuo. The residual
solid was recrystallized from AcOEt and hexane to give
9 as dark yellowish brown crystals (9.50 g, 66.4%): mp
4.2.1. 4-Bromo-2-iodobenzotrifluoride (8). To a solution
of 2-aminobenzotrifluoride (1.37 g, 8.50 mmol) in AcOH
(10 mL) were added KBr (1.20 g, 10.1 mmol), NaBO3Æ
4H2O (1.43 g, 9.30 mmol), and (NH4)6Mo7O24Æ4H2O
(18.7 mg, 0.08 mmol), and the mixture was stirred for
3 h at room temperature. The reaction mixture was basi-
fied with saturated Na2CO3 aq and extracted with AcOEt
(3· 50 mL). The combined extracts were washed with sat-
urated NaHCO3 aq (3· 40 mL) and brine (3· 40 mL), and
dried over anhydrous MgSO4. The dried extract was con-
centrated in vacuo, and the residue was dissolved in
MeCN (5 mL). The solution was added to a solution of
I2 (6.47 g, 25.5 mmol) and 90% tert-butyl nitrite (1.47 g,
12.8 mmol) in MeCN (5 mL). After stirring for 16 h at
room temperature, the reaction mixture was poured into
10% Na2SO3 aq (50 mL), and extracted with CH2Cl2
(3· 30 mL). The combined extracts were washed with
10% Na2SO3 aq (3· 30 mL), H2O (3· 30 mL) and brine
(3· 30 mL), and dried over anhydrous MgSO4. The dried
extract was concentrated in vacuo, and the residual solid
was recrystallized from hexane to give 8 as yellow crystals
(2.73 g, 91.4%): mp 125–129 ꢁC. 1H NMR (CDCl3) d 7.34
(dd, 1H, J = 2.4 Hz, 8.1 Hz), 7.77 (d, 1H, J = 2.4 Hz),
7.87 (d, 1H, J = 8.1 Hz); Anal. Calcd for C7H3BrF3I:
C, 23.96; H, 0.86. Found: C, 23.42; H, 1.04.
113–117 ꢁC. 1H NMR (CDCl3)
d 7.81 (d, 1H,
J = 8.1 Hz), 7.89 (d, 1H, J = 8.1 Hz), 7.96 (s, 1H); Anal.
Calcd for C7H3BrF3I: C, 23.96; H, 0.86. Found: C,
23.42; H, 1.04.
4.2.3. 4-Bromo-2-trifluoromethylbenzyl alcohol (10). To a
solution of 9 (3.47 g, 12.9 mmol) and Et3N (1.96 g,
19.4 mmol) in anhydrous THF (50 mL) was added
ClCOOCH(CH3)2 (2.38 g, 19.4 mmol) dropwise over a
10-min period at À20 ꢁC. After stirring for 1.5 h at
À10 ꢁC, to the reaction mixture was added ice-cooled
water (50 mL), followed by an addition of NaBH4
(2.44 g, 64.5 mmol) in limited amounts at 0 ꢁC. After
stirring for 8 h at room temperature, the reaction mix-
ture was acidified with citric acid and diluted with
H2O (50 mL) to dissolve precipitated inorganic materi-
als. After evaporation of THF, the aqueous solution
was extracted with AcOEt (3· 40 mL). The combined
extracts were washed with saturated NaHCO3 aq (3·
30 mL) and brine (3· 30 mL), and dried over anhydrous
4.2.2. 4-Bromo-2-trifluoromethylbenzoic acid (9). To a
solution of 8 (18.6 g, 53.0 mmol) in anhydrous THF

Y. Hattori et al. / Bioorg. Med. Chem. 15 (2007) 2198–2205
2203
MgSO4. The dried extract was concentrated in vacuo,
and the residue was purified by silica-gel column chro-
matography (silica gel: 90 g, hexane/AcOEt = 3:1) to
give 10 as colorless oil (3.21 g, 97.5%): 1H NMR
(CDCl3) d 1.97 (t, 1H, J = 5.7 Hz), 4.84 (d, 2H,
J = 5.7 Hz), 7.62 (d, 1H, J = 8.4 Hz), 7.71 (d, 1H,
J = 8.4 Hz), 7.77 (s, 1H).
ture was added ice-cooled water (50 mL), and Et2O layer
was once taken out. The aqueous layer was extracted
with Et2O (3· 40 mL). The combined Et2O layer and ex-
tracts were washed with water (3· 50 mL) and brine (3·
50 mL). The organic layer was dried over anhydrous
MgSO4 and concentrated in vacuo. The residual solid
was dissolved in anhydrous DMSO (5 mL), and the
solution was added to a suspension of sodium diethyl
acetamidomalonate8 (1.31 g, 5.47 mmol) in anhydrous
DMSO (5 mL) under Ar atmosphere. After stirring for
6 h, the reaction mixture was acidified with 1 M HCl
and extracted with AcOEt (3· 30 mL). The combined
extracts were washed with saturated NaHCO3 (3·
20 mL) and brine (3· 20 mL). The organic layer was
dried over anhydrous MgSO4 and concentrated in vac-
uo. The crystalline residue was recrystallized from
AcOEt and hexane to give 14 as colorless crystals
(1.52 g, 79.9%): mp 131–134 ꢁC. 1H NMR
(CD3COCD3) d 1.17 (t, 6H, J = 7.4 Hz), 2.00 (s, 3H),
3.85 (s, 2H), 4.00–4.22 (m, 4H), 7.37 (d, 1H, J =
7.8 Hz), 7.51 (s, 2H), 7.99 (d, 1H, J = 7.8 Hz), 8.14 (s,
1H). Anal. Calcd for C17H2110BF3NO7: C, 48.81; H,
5.06; N, 3.35. Found: C, 48.43; H, 5.15; N, 3.14.
4.2.4. 4-Bromo-2-trifluoromethylbenzyl methoxymethyl
ether (11). To a solution of 10 (2.77 g, 10.9 mmol) in
CH2Cl2 (50 mL) were added DIEA (3.53 g, 27.3 mmol)
and MOM–Cl (1.76 g, 21.8 mmol), and the reaction
mixture was refluxed for 24 h. After evaporation of the
solvent, the residue was dissolved in AcOEt (50 mL).
The solution was washed with 10% aqueous citric acid
(3· 20 mL), saturated NaHCO3 (3· 20 mL) and brine
(3· 20 mL), and dried over anhydrous MgSO4. The
dried organic layer was concentrated in vacuo, and the
residue was purified by silica-gel column chromatogra-
phy (silica gel: 60 g, hexane/AcOEt = 9:1) to give 11 as
1
colorless oil (2.81 g, 86.2%): H NMR (CDCl3) d 3.42
(s, 3H), 4.72 (s, 2H), 4.75 (s, 2H), 7.58 (d, 2H,
J = 8.4 Hz), 7.68 (d, 2H, J = 8.4 Hz), 7.78 (s, 1H).
4.2.5. 4-(10B)Borono-2-trifluoromethylbenzyl methoxy-
methyl ether (12). To a solution of 11 (2.54 g, 8.49 mmol)
in anhydrous THF (30 mL) was added a solution of 1.6 M
n-BuLi in hexane (5.57 mL, 8.91 mmol) at À78 ꢁC under
Ar atmosphere. After stirring for 1 h at À78 ꢁC, to the
reaction mixture was added 10B(OMe)3 (1.31 g,
12.7 mmol), and the solution was stirred for 5 h at
À78 ꢁC. To the reaction mixture was added saturated
NH4Cl aq (30 mL) at room temperature, and the mixture
was extracted with Et2O (3· 60 mL). The combined
extracts were washed with 10% aqueous citric acid (3·
60 mL) and brine (3· 60 mL). The organic layer was dried
over anhydrous MgSO4 and concentrated in vacuo. The
residue was purified by silica-gel column chromatography
(silica gel: 60 g, hexane/AcOEt = 1:1) to give 12 as color-
less oil (1.59 g, 71.3%): 1H NMR (CD3COCD3) d 3.36 (s,
3H), 4.74 (s, 2H), 4.77 (s, 2H), 7.49 (s, 2H), 7.75 (d, 1H,
J = 7.8 Hz), 8.12 (d, 1H, J = 7.8 Hz), 8,18 (s, 1H).
4.2.8. Na-Acetyl-DL-b-[4-(10B)borono-2-trifluoromethyl-
phenyl]alanine (15). A mixture of 14 (1.00 g, 2.39 mmol)
in 1 M NaOH (40 mL) was stirred for 8 h at 80 ꢁC. To
the cooled reaction mixture was added 3 M HCl
(20 mL), and the solution was stirred for 4 h at 80 ꢁC.
The reaction mixture was extracted with AcOEt (3·
40 mL), and the combined extracts were washed with
brine (3· 30 mL). The organic layer was dried over
anhydrous MgSO4 and concentrated in vacuo. The crys-
talline residue was recrystallized from AcOEt and hex-
ane to give 15 as colorless crystals (660 mg, 86.8%):
mp 158–162 ꢁC. 1H NMR (CD3COCD3) d 1.87 (s,
3H), 3.08–3.48 (m, 2H), 4.78–4.86 (m, 1H), 7.51–7.58
(m, 4H), 8.01 (d, 1H, J = 6.0 Hz), 8.18 (s, 1H); MAL-
DI-TOF MS: found m/z 319.2 [M+H]+ (calcd for
C12H1310BF3NO5+H: 319.1); Anal. Calcd for
C12H1310BF3NO5: C, 45.29; H, 4.12. N, 4.40; Found:
C, 45.64; H, 4.38; N. 4.09.
4.2.6. 4-(10B)Borono-2-trifluoromethylbenzyl alcohol (13).
The solution of 12 (1.57 g, 5.96 mmol) in THF (30 mL)
and 3 M HCl (30 mL) was stirred for 8 h at 60 ꢁC. The
reaction mixture was extracted with Et2O (3· 40 mL),
and the combined extracts were washed with water (3·
50 mL) and brine (3· 50 mL). The organic layer was
dried over MgSO4, and the solution was concentrated
in vacuo. The residue was purified by silica-gel column
chromatography (silica gel: 50 g, CH2Cl2/MeOH = 9:1)
to give 13 as colorless crystals (1.18 g, 90.5%): mp
289–294 ꢁC. 1H NMR (CD3COCD3) d 4.77 (s, 2H),
7.58 (s, 2H), 7.71 (d, 1H, J = 7.8 Hz), 8.11 (d, 1H,
J = 7.8 Hz), 8.18 (s, 1H). Anal. Calcd for C8H810BF3O3:
C, 43.84; H, 3.68. Found: C, 43.45; H, 3.93.
4.2.9. DL-b-[4-(10B)Borono-2-trifluoromethylphenyl]ala-
nine (5). A mixture of 15 (588 mg, 1.85 mmol) in 3 M
HCl was stirred for 12 h at 80 ꢁC. The reaction mixture
was washed with Et2O (3· 30 mL) and concentrated in
vacuo. The residue was dissolved in i-PrOH (10 mL),
and to the solution was added propylene oxide (215 mg,
3.70 mmol). After stirring for 24 h, the reaction mixture
was concentrated in vacuo. The crystalline residue was
recrystallized from water to give 5 as colorless crystals
(428 mg, 83.8%): mp 295–300 ꢁC (dec). 1H NMR (D2O)
d 2.63–2.88 (m, 2H), 3.66 (t, 1H, J = 7.8 Hz), 6.81 (d,
1H, J = 7.4 Hz), 7.22 (d, 1H, J = 7.4 Hz), 7.35 (s, 1H);
MALDI-TOF MS: found m/z 277.3 [M+H]+ (calcd for
C10H1110BF3NO4+H: 277.1); Anal. Calcd for C10H1110B
F3NO4+2H2O: C, 38.47; H, 4.84; N, 4.49; Found: C,
38.23; H, 5.04; N. 4.40.
4.2.7. 2-Acetylamino-2-[4-(10B)borono-2-trifluoromethyl-
phenyl]methylmalonic acid diethyl ester (14). To a solu-
tion of 13 (1.00 g, 4.56 mmol) in Et2O (50 mL) was
added slowly PBr3 (1.48 g, 5.47 mmol) at 0 ꢁC, and the
mixture was stirred for 4 h at 0 ꢁC. To the reaction mix-
4.2.10. Na-tert-Butoxycarbonyl-DL-b-[4-(10B)borono-2-
trifluoromethylphenyl]alanine (16). To a solution of 5
(200 mg, 0.724 mmol) in water (10 mL) and acetone

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Y. Hattori et al. / Bioorg. Med. Chem. 15 (2007) 2198–2205
(10 mL) were added Na2CO3(84.3 mg, 0.796 mmol) and
Boc2O (174 mg, 0.796 mmol), and the mixture was stir-
red for 12 h. The reaction mixture was acidified with
10% aqueous citric acid, and acetone was removed by
evaporation in vacuo. The aqueous solution was extract-
ed with AcOEt (3· 40 mL), and the combined extracts
were washed with 10% aqueous citric acid (3· 30 mL)
and brine (3· 30 mL). The organic layer was dried over
anhydrous MgSO4 and concentrated in vacuo. The crys-
talline residue was recrystallized from AcOEt and hex-
ane to give 16 as colorless crystals (247 mg, 90.8%):
in Dulbecco’s Minimum Essential Medium (DMEM)
supplemented with 10% fetal bovine serum, 2 mM gluta-
mine, 24 mM sodium bicarbonate at 37 ꢁC in a 5% CO2
atmosphere.
Cells in mono-layer were harvested with 0.25% trypsin /
0.02% EDTA in Ca2+-free phosphate-buffered saline
(PBS).
4.3.2. Boron incorporation into Ihara cells7. Cultures
were inoculated with 4.0 · 107 cells/dish, and cells were
grown for 24 h in DMEM. The medium was replaced
with that containing DL-10Bpa(2,6F2) (3) or
DL-10Bpa(2CF3) (5) (final concentration was 2.0 mM in
each case), and then the cells were cultured for 24 h.
1
mp 291–292 ꢁC (dec). H NMR (CD3COCD3) d 1.32
(s, 9H), 3.08–3.51 (m, 2H), 4.44–4.53 (m, 1H), 6.49–
6.51 (br, 1H), 7.44 (br, 2H), 7.55 (d, 1H, J = 7.8 Hz),
8.03 (d, 1H, J = 7.8 Hz) 8.17 (s, 1H); Anal. Calcd for
C15H1910BF3NO6: C, 47.87; H, 5.09; N, 3.72. Found:
C, 48.48; H, 5.37; N, 3.66.
4.3.3. 19F NMR measurement of fluorinated DL-10Bpa
incorporated into Ihara cell. The medium was removed
by aspiration, and the cells were washed twice with
PBS and deuterium saline. After centrifugation, the
clear supernatant was removed by aspiration, and the
residual cells (250 lL) are transferred into micro NMR
tube. The 19F signals of fluorinated DL-10Bpa incorpo-
rated into the cells were measured by 19F NMR.
4.2.11. Na-tert-Butoxycarbonyl-DL-3-[4-(10B)borono-2-
trifluoromethylphenyl]alaninol (17). To a solution of 16
(2.92 g, 7.76 mmol) in DMF (25 mL) were added KHCO3
(1.55 g, 15.5 mmol) and MeI (2.20 g, 15.5 mmol), and the
mixture was stirred for 24 h. The reaction mixture was
concentrated in vacuo, and the residue was suspended in
AcOEt (100 mL). The suspension was washed with 10 %
citric acid (3· 30 mL), saturated NaHCO3 aq (3·
30 mL) and brine. The organic layer was dried over
MgSO4 and concentrated in vacuo. To the solution of
the residue in THF (50 mL) were added LiCl (1.64 g,
38.8 mmol), NaBH4 (2.94 g, 77.6 mmol), and MeOH
(50 mL) at 0 ꢁC. After stirring for 6 h, to the reaction mix-
ture was added 10% citric acid (100 mL), and the aqueous
solution was extracted with AcOEt (3· 60 mL). The com-
bined extracts were dried over MgSO4 and concentrated
in vacuo. The oily residue thus obtained was purified by
silica-gel column chromatography (silica gel: 150 g,
CH2Cl2/MeOH = 19:1) to give 17 as colorless amorphous
Acknowledgment
This work was supported in part by Grants-in-Aid for
Scientific Research Nos. 11680953 and 11680678 from
the Ministry of Education, Science, Sports, and Culture
of Japan.
References and notes
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1
solid (2.56 g, 91.1%): H NMR (CD3COCD3) d 1.30 (s,
9H), 2.88–2.99 (m, 2H), 3.15–3.35 (m, 2H), 3.55–3.66
(m, 1H), 7.40 (br, 2H), 7.41 (d, 1H, J = 7.8 Hz), 8.01 (d,
1H, J = 7.8 Hz) 8.16 (s, 1H).
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4.2.12.
DL-3-(4-(10B)Borono-2-trifluoromethylphenyl)-
alaninol (6) hydrochloride. The compound 17 (900 mg,
2.72 mmol) was dissolved in 4 M HCl/AcOEt (10 mL),
and the solution was stirred for 10 min. The reaction
mixture was concentrated in vacuo, and the residue
was dissolved in H2O (20 mL). The aqueous solution
was washed with Et2O (3· 10 mL) and concentrated in
vacuo. The crystalline residue was recrystallized from
MeOH and Et2O to give 6 as colorless crystals
(671 mg, 92.6 %): mp 211–218 ꢁC (dec). 1H NMR
(D2O) d 2.87–2.91 (m, 2H), 3.37–3.42 (m, 2H), 3.49–
3.54 (m, 1H), 7.21 (d, 1H, J = 7.8 Hz), 7.64 (d, 1H,
J = 7.8 Hz), 7.79 (s, 1H); MALDI-TOF MS: found
m/z 263.2 [M+H]+ (calcd for C10H1310BF3NO3 +H:
263.1); Anal. Calcd for C10H1410BClF3NO3: C, 40.21;
H, 4.72; N, 4.69. Found: C, 40.07; H, 4.96; N 4.55.
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10. Abbreviations according to IUPAC-IUB commission Eur.
J. Biochem. 1984, 9, 138 are used. Ac, acetyl; AcOH, acetic
acid; AcOEt, ethyl acetate; Boc, tert-butoxycarbonyl;
n-BuLi, n-butyllithium; DIEA, diisopropylethylamine;
DMF, N,N-dimethylformamide, DMSO, N,N-dimethyl-
sufoxide; Et2O, diethylether; MeCN, acetonitrile; MOM,
methoxy methyl; THF, tetrahydrofuran; i-PrMgCl, i-pro-
pylmagnesium chloride; PBS, phosphate-buffered saline.
13. Deuterium saline means PBS prepared with D2O.
14. The best incorporation of 3 and 5 into several tumor cells
was observed in Ihara cells.15
15. Hattori, Y.; Kurihara, K.; Niki, Y.; Kondoh, H.; Asano,
T.; Kirihata, M.; Takagaki, M.; Yamamoto, H.;
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