design and synthesis of tumor seeking closo-dodecaborate- containing amino acids as boron carrier...
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Design and Synthesis of Tumor Seeking closo-Dodecaborate-Containing Amino Acids as
Boron Carrier for BNCT
Yoshihide Hattori1), Miki Ishimura1), Mari Mukumoto1), Yoichiro Ohta1), Hiroshi Takenaka2), Kouki Uehara2), Tomoyuki Asano2), Minoru
Suzuki3), Shin-ichiro Masunaga3), Koji Ono3), ○Mitsunori Kirihata1)
1 Research Center of Boron Neutron Capture Therapy, Osaka Prefecture University,
2 Stella Pharma Co.3 Kyoto University Research Reactor Institute
In the course of our developing studies on new boron carriers for BNCT, we have designed and synthesized dodecaborate ([B12H11]2--) unit-containing L-amino acids (DBAA), a new class of tumor seeking and water soluble amino acids.
2-
: B
[B1 2 H1 2 ]2-
Dodecaborate Unit
2-
SH
BSH
[B1 2 H1 2 S]2-
2-
OH
BOH
[B1 2 H1 2 O]2-
1-
NH3+
BNH2
[B1 2 H13N]1-
: BH
10B-Boronmoiety
Tumor seekin
gmoietyn
Hydrophilic part HydrophobicLinker part
Hydrophilic part
Design Concept of DBAA
[closo-B12H11]2--
Linker
◇ -amino acid :R CO2H
NH2R'
◇ -amino acid :R CO2H
NH2H
◇ -amino acid amide :
R CONH-
NH2H
2-
S
BS-
BSH-AA and BNH2-AM, two classes of dodecaborate containig amino acids were newly developed for BNCT.
Type A: BS-AAThiododecaborate AA
1-
NH2
H2N
O
+
Type B: BN-AMAnmoniodecaborate AM
Hydrophilicmoiety
Hydrophobicmoiety
CO2H
NH2
2-
S
2-
SNH2
CO2H
2-
SNH2
CO2H
Type A: Thiododecaborate-containing AAs (BS-AA)
BS-CB-AABS-CH-AA
2-
SCO2H
NH2
n
n=1,2,3,5
BS-AA
2-
S H
BSH unit
: BH: B
Synthetic Method: S-Alkylation of BSH with alkylhalides
Mixture of mono- and di-alkylated BSHwas generated.
2-
SH R-X2-
SR
+
2-
SR
R+
◆ Direct alkylation
D. Gabel et al., Inorg. Chem. 1993, 32, 2276-2278.
◆ Stepwise alkylation
2-
SCN
S-cyanoethyl-BS
2-
SCN
R
+
NMe4OH
2-
SR
R-X
: BH : B
Syntheses of BS-AAs (Type A) : stepwise S-alkylation by Combinatorial Chemistry
2-
SH
BSH
XNH
HNO
O
X
NH
HNO
O
X CO2H
NH2
X NH2
CO2H
CO2H
NH2
X
HBr
n
n: 1, 2, 3, 5
XCO2H
NH2
Hydantoins
R-X (Halides)
X: Cl, Br, I
+BS-AA
Library
: BH
Etc.
Combi-Chem
: B
NMe4OH / MeOH
INH
HNO
O2-
S NH
HNO
O
CN
+2-
SCN
2NM4+
MeCN
2-
S
CO2H
NH2
A typical synthetic route for cis-BS-CB-AA
2NM4+
2-
S NH
HNO
O
81%
1) 0.5N NaOH
2) IR-124(H+)
3) IR-124(Na+)
98%
NMe4OH / MeCN
81%
2NM4+
2Na+
I-
cis-BS-CB-AA
0 0.5 1 1.5 2
SAShuman
carcinoma
B16mouse
melanoma
C6rat glioma
Boron concentration (mgB/107cell)
1a (2mM)
1b (2mM)
1c (2mM)
L-BPA (2mM)
First screening: Boron uptake by tumor cells
SCOOH
NH2
n
2-
BS-AA (1a-c)a: n=2, b: n=3, c: n=6
・ BS-AAs (1a-c) were uptaken by tumor cells as L-BPA.・ The length of side carbon chain is not correlated with incorporation amount.
Type A
trans ACBC-BS
2-
S
CO2H
NH2
2Na+
trans ACBC-BSH can deliver large amount of boron
to glioma cells.
First screening: Boron uptake by tumor cells
Boron concentrations ± SD ( µg10B/g ) Ratiosb
Agent Timea(h) n Blood Brain Tumor T/Blood T/Brain
ACBC-BS
CEDc
30min
1 4 0.8 ± 0.3 0.1 ± 0.0 10.7 ± 3.0 13.3 147.26 3 0.9 ± 0.6 0.2 ± 0.1 0.5 ± 0.5 0.5 2.8
24 3 0.8 ± 0.1 0.1 ± 0.0 0.4 ± 0.3 0.4 6.2
CEDd
24h
1 3 3.3 ± 2.2 1.5 ± 1.3 21.1 ± 3.3 6.4 14.26 3 1.3 ± 0.0 0.4 ± 0.3 17.8 ± 10.1 13.7 42.424 3 0.5 ± 0.1 0.0 ± 0.0 0.0 ± 0.1 0.1 1.4
ive 1 4 6.3 ± 1.7 0.2 ± 0.0 2.7 ± 0.5 0.4 14.96 3 7.0 ± 3.4 0.3 ± 0.1 6.0 ± 1.7 0.9 18.6
BPA ivf 1 3 8.5 ± 0.3 3.0 ± 0.6 19.7 ± 1.4 2.3 6.76 3 4.1 ± 1.3 3.1 ± 1.3 14.4 ± 3.4 3.6 4.7
Resultin vivo biodistribution study
Boron concentrations (Blood, Brain, Tumor)
CED: Convection Enhanced Delivery
( µg10B/g )
Bo
ron
co
nce
ntr
atio
n (
Tu
mo
r)
Tumor Boron concentrationsResult
in vivo biodistribution study
S
CO2H
NH2
2Na+
trans-ACBC
Type B: Ammoniododecaborate-containing Amide (BNH2-AM)
: BH: B
1-
H3N+
BNH2
Ammonioundecahydro-closo-dodecaborate
1-
NH2
H2N
O
+
L-Val-BNH2
1-
NH2
O
NH2
+
L-Phe-BNH2
1-
NH2
O
NH2BHO
HO
+
L-BPA-BNH2
etc.
New synthetic route of BNH2-AM by amidation (Typical synthesis of BNH2-L-Val )
Overall yield 66%
1- H2
N+
O
NH2
H2/10% Pd/CIR 120 (H+), NaOH
L-Val-BNH2
MeOH
NHZ
O
O NO2
1-
NH2+
NMe4+
NaH
Na+
2-
NH2
NMe4+ DMF
Intermediate
DMF
Boron uptake by tumor cells in vitro
NH2
O
H2N +
R
1-
R=H; Phe-BNH2 (3a)R=B(OH)2; BPA-NH2 (3b)
Phe-BNH2(3a)
BPA-BNH2 (3b)
Incorporated amounts of AA-BNH2 are lower than those of BS-AA.
Second Screening: Cell-Killing Effect
Despite of the incorporated amounts of AA-BNH2 are lower than that of another boron compounds, the cell-killing effects of these are very high.
BPA
BHO
OH
CO2HH2N
S
COOH
NH2
2-
2Na+
2-
S
NH2
COOH
1
2b
Phase-contrastmicrograph
Phase-contrastmicrograph
FluorescenceMicrograph (ICC)
FluorescenceMicrograph (ICC)
Merge imageMerge image
Micro-distribution of Boron-Amino Acids (in C6 cell)
NH2
O
H2N +
BHO
OH
1-
3b
Phase-contrastmicrograph
Phase-contrastmicrograph
FluorescenceMicrograph (ICC)
FluorescenceMicrograph (ICC)
Merge imageMerge image
Phase-contrastmicrograph
Phase-contrastmicrograph
FluorescenceMicrograph (ICC)
FluorescenceMicrograph (ICC)
Merge imageMerge image
Phase-contrastmicrograph
Phase-contrastmicrograph
FluorescenceMicrograph (ICC)
FluorescenceMicrograph (ICC)
Merge imageMerge image
Distributed over the whole cell including
the cell nuclei.
Aggregated on the fringe
of the cell nuclei.
BPA
BHO
OH
CO2HH2N
S
COOH
NH2
2-
2Na+
2-
S
NH2
COOH
1
2b
Phase-contrastmicrograph
Phase-contrastmicrograph
FluorescenceMicrograph (ICC)
FluorescenceMicrograph (ICC)
Merge imageMerge image
NH2
O
H2N +
BHO
OH
1-
3b
Phase-contrastmicrograph
Phase-contrastmicrograph
FluorescenceMicrograph (ICC)
FluorescenceMicrograph (ICC)
Merge imageMerge image
Phase-contrastmicrograph
Phase-contrastmicrograph
FluorescenceMicrograph (ICC)
FluorescenceMicrograph (ICC)
Merge imageMerge image
Phase-contrastmicrograph
Phase-contrastmicrograph
FluorescenceMicrograph (ICC)
FluorescenceMicrograph (ICC)
Merge imageMerge image
Summury
cis ACBC-BS (2a)trans ACBC-BS (2b)
2-
S
2Na+
CO2H
NH2
2-
S
COOH
NH2
2Na+
BSH-amino acid (1)
NH2
O
H2N +
R Na+
1-
R=H; Phe-BNH2 (3a)R=B(OH)2; BPA-BNH2 (3b)
• We have accomplished the synthesis of dodecaborate unit containing amino acid.
• BSH containing amino acids 1 and 2 were uptaken into several tumor cells, especially trans-ACBC-BS (2b), can deliver large amount of boron to glioma cells.
• BNH2 containing amino acid derivatives 3a and 3b were distributed over the whole
cancer cell including cell nuclei, and the cell-killing effects of AA-BNH2 is higher thanthat of L-BPA and BSH-containing amino acids.
• BS-AAs were aggregated on the fringe of the cell nuclei, on the other hand L-BPA – BNH2 was distributed over the whole cell including the cell nuclei similary to L-BPA.