1. Design and Synthesis of Tumor Seeking closo-Dodecaborate- Containing Amino Acids as Boron Carrier for BNCT Yoshihide Hattori 1), Miki Ishimura 1), Mari Mukumoto 1), Yoichiro Ohta 1), Hiroshi Takenaka 2), Kouki Uehara 2), Tomoyuki Asano 2), Minoru Suzuki 3), Shin-ichiro Masunaga 3), Koji Ono 3), ○Mitsunori Kirihata 1) 1 Research Center of Boron Neutron Capture Therapy, Osaka Prefecture University, 2 Stella Pharma Co. 3 Kyoto University Research Reactor Institute

2. In the course of our developing studies on new boron carriers for BNCT, we have designed and synthesized dodecaborate ([B 12 H 11 ] 2- -) unit-containing L-amino acids (DBAA), a new class of tumor seeking and water soluble amino acids. [B 1 ２ H 1 ２ ] 2- Dodecaborate Unit BSH [B 1 ２ H 1 ２ S] 2- BOH [B 1 ２ H 1 ２ O] 2- BNH 2 [B 1 ２ H 13 N] 1-

3. 10 B-Boron moiety 10 B-Boron moiety Tumor seeking moiety Tumor seeking moiety Hydrophilic part Hydrophobic Linker part Hydrophilic part Design Concept of DBAA [closo-B 12 H 11 ] 2- - Linker ◇   -amino acid： ◇  -amino acid： ◇   -amino acid amide： BS-

4. BSH-AA and BNH 2 -AM, two classes of dodecaborate containig amino acids were newly developed for BNCT. Type A: BS-AA Thiododecaborate AA Type B: BN-AM Anmoniodecaborate AM Hydrophilic moiety Hydrophobic moiety

5. Type A: Thiododecaborate-containing AAs (BS-AA) BS-CB-AA BS-CH-AA n=1,2,3,5 BS-AA BSH unit

6. Synthetic Method: S-Alkylation of BSH with alkylhalides Mixture of mono- and di-alkylated BSH was generated. R-X + ◆ Direct alkylation D. Gabel et al., Inorg. Chem. 1993, 32, 2276-2278. ◆ Stepwise alkylation S-cyanoethyl-BS NMe 4 OH R-X

7. Syntheses of BS-AAs (Type A) : stepwise S-alkylation by Combinatorial Chemistry BSH n: 1, 2, 3, 5 Hydantoins R-X (Halides) X: Cl, Br, I + BS-AA Library Etc. Combi- Chem

8. NMe4OH / MeOH 2NM 4 + MeCN A typical synthetic route for cis-BS-CB-AA 2NM 4 + 81% 1) 0.5N NaOH 2) IR-124(H + ) 3) IR-124(Na + ) 98% NMe 4 OH / MeCN 81% 2NM 4 + 2Na + I-I- cis-BS-CB-AA

9. First screening: Boron uptake by tumor cells 2-2- 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

10. trans ACBC-BS 2-2- trans ACBC-BSH can deliver large amount of boron to glioma cells. First screening: Boron uptake by tumor cells

11. Boron concentrations ± SD （ µg 10 B/g ） Ratios b Agent Time a (h)nBloodBrainTumorT/BloodT/Brain ACBC-BS CED c 30min 140.8±0.30.1±0.010.7±3.013.3147.2 630.9±0.60.2±0.10.5± 2.8 2430.8±0.1 ±0.00.4±0.30.46.2 CED d 24h 1 33.3±2.21.5±1.321.1±3.36.414.2 6 31.3±0.00.4±0.317.8±10.113.742.4 2430.5±0.10.0± ±0.1 1.4 iv e 146.3±1.70.2±0.02.7±0.50.414.9 637.0±3.40.3±0.16.0±1.70.918.6 BPA iv f 138.5±0.33.0±0.619.7±1.42.36.7 634.1±1.33.1±1.314.4±3.43.64.7 Result in vivo biodistribution study Boron concentrations (Blood, Brain, Tumor) CED: Convection Enhanced Delivery

12. （ µg 10 B/g ） Boron concentration (Tumor) Tumor Boron concentrations Result in vivo biodistribution study trans-ACBC

13. Type B: Ammoniododecaborate-containing Amide (BNH 2 -AM) BNH 2 Ammonioundecahydro- closo-dodecaborate L-Val-BNH 2 L-Phe-BNH 2 L-BPA-BNH 2 etc.

14. New synthetic route of BNH 2 -AM by amidation (Typical synthesis of BNH 2 -L-Val ) Overall yield 66% H 2 /10% Pd/C IR 120 (H + ), NaOH L-Val-BNH 2 MeOH NMe 4 + NaH Na+ NMe 4 + DMF Intermediate DMF

15. Boron uptake by tumor cells in vitro 1-1- R=H; Phe-BNH 2 (3a) R=B(OH) 2 ; BPA-NH 2 (3b) Phe-BNH 2 (3a) BPA-BNH 2 (3b) Incorporated amounts of AA-BNH 2 are lower than those of BS-AA.

16. Second Screening: Cell-Killing Effect Despite of the incorporated amounts of AA-BNH 2 are lower than that of another boron compounds, the cell-killing effects of these are very high.

17. BPA 2-2- 2Na + 2-2- 1 2b2b Phase-contrast micrograph Fluorescence Micrograph (ICC) Fluorescence Micrograph (ICC) Merge image Micro-distribution of Boron-Amino Acids (in C6 cell) 1-1- 3b Phase-contrast micrograph Fluorescence Micrograph (ICC) Fluorescence Micrograph (ICC) Merge image Phase-contrast micrograph Fluorescence Micrograph (ICC) Fluorescence Micrograph (ICC) Merge image Phase-contrast micrograph Fluorescence Micrograph (ICC) Fluorescence Micrograph (ICC) Merge image

18. Distributed over the whole cell including the cell nuclei. Aggregated on the fringe of the cell nuclei. BPA 2-2- 2Na + 2-2- 1 2b2b Phase-contrast micrograph Fluorescence Micrograph (ICC) Fluorescence Micrograph (ICC) Merge image 1-1- 3b Phase-contrast micrograph Fluorescence Micrograph (ICC) Fluorescence Micrograph (ICC) Merge image Phase-contrast micrograph Fluorescence Micrograph (ICC) Fluorescence Micrograph (ICC) Merge image Phase-contrast micrograph Fluorescence Micrograph (ICC) Fluorescence Micrograph (ICC) Merge image

19. Summury cis ACBC-BS (2a) trans ACBC-BS (2b) 2-2- 2-2- BSH-amino acid (1) 1-1- R=H; Phe-BNH 2 (3a) R=B(OH) 2 ; BPA-BNH 2 (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. BNH 2 containing amino acid derivatives 3a and 3b were distributed over the whole cancer cell including cell nuclei, and the cell-killing effects of AA-BNH 2 is higher than that 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 – BNH 2 was distributed over the whole cell including the cell nuclei similary to L-BPA.