design and synthesis of tumor seeking closo-dodecaborate- containing amino acids as boron carrier...

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


FluorescenceMicrograph (ICC)


Merge imageMerge image

Micro-distribution of Boron-Amino Acids (in C6 cell)

NH2

O

H2N +

BHO

OH

1-

3b
















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






NH2

O

H2N +

BHO

OH

1-

3b
















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.

design and synthesis of tumor seeking closo-dodecaborate- containing amino acids as boron carrier...

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