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T. Nishimaru et al. / Bioorg. Med. Chem. 17 (2009) 57–63
uct was purified by silica-gel column chromatography (PSQ 100B,
80 g, hexane/AcOEt = 1:3). The fractions containing the desired
product were combined and concentrated in vacuo to give 19 as
yellow oil (2.87 g, 85.3%). 1H NMR (CDCl3): d 1.42 (9H, s), 1.51–
1.71 (12H, m), 3.03–3.27 (16H, m), 3.95 (2H, s), 4.64 (1H, br s),
5.02 (1H, br s), 5.09 (2H, s), 6.74 (1H, br s), 7.32–7.36 (5H, m),
7.58–7.76 (9H, m), 7.95–8.07 (3H, m); MS Found m/z 1142.6. Calcd
for C47H61 N9O17S3Na: [M+Na]+, m/z 1142.3.
alyst was filtered off, and the filtrate was concentrated in vacuo to
obtain colorless oily residue. To a solution of the residue in DMF
(3 mL) were added Et3N (44.3
lL, 318 lmol) and Hcc-OSu
(88.4 mg, 292 mol), and the solution was stirred for 3 h at rt.
l
The reaction mixture was diluted with H2O (5 mL), and the result-
ing suspension was extracted with CHCl3 (3Â 3 mL). The extract
was washed with 10% citric acid (3Â 3 mL) and brine (3Â 3 mL),
and dried over anhydrous MgSO4, followed by concentration in va-
cuo. The crude product was purified by silica-gel column chroma-
tography (PSQ 100B, 20 g, CHCl3/MeOH = 97:3). The fractions
containing the desired product were combined and concentrated
in vacuo to give 22 as yellow oil (226 mg, 82.5%). 1H NMR (CDCl3)
4.2.15. N12-Benzylcarbonyl-4,8-di(tert-butoxycarbonyl)-N1-[N-
(N4-tert-butoxycarbonyl-4-aminobutyl)-N-(tert-butoxycarbon-
yl)glycyl]-4,8-diaza-1,12-dodecanediamine (20)
To a suspension of 19 (1.33 g, 1.19 mmol) in CH3CN (25 mL)
d 1.42 (18H, s), 1.43 (18H, s), 1.51 (8H, m4), 1.71 (6H, m), 2.78 (2H,
were added Cs2CO3 (2.32 g, 7.12 mmol) and PhSH (550
l
L,
d), 3.02–3.24 (14H, m), 4.99 (1H, m), 6.17 (1H, br s), 6.76 (1H, s),
6.78 (1H, d), 7.37 (1H, m), 8.53 (1H, s); MS Found m/z 1055.9. Calcd
for C50H80N8O15Na: [M+Na]+, m/z 1055.6.
5.36 mmol). The mixture was stirred for 15.5 h at rt, and then
the solvent was removed in vacuo to obtain colorless oily residue.
The residue was dissolved in AcOEt (30 mL), and the solution was
once dried over anhydrous MgSO4, followed by concentration in
vacuo. To the solution of the thus-obtained crude product in
CH2Cl2 (2.5 mL) were added Et3N (0.765 mL, 5.49 mmol) and Boc2O
(817 mg, 4.28 mmol), and the solution was stirred for 21 h at rt.
The solvent was removed in vacuo, and the residue was dissolved
in AcOEt (100 mL). The solution was washed with 10% citric acid
(3Â 30 mL), saturated aqueous NaHCO3 (3Â 30 mL) and brine
(3Â 30 mL). The organic layer was dried over anhydrous MgSO4,
and then concentrated in vacuo. The crude product was purified
by silica-gel column chromatography (PSQ 100B, 20 g, benzene/
AcOEt = 1:1). The fractions containing the desired product were
combined and concentrated in vacuo to give 20 as colorless oil
(913 mg, 76.7%). 1H NMR (CDCl3) d 1.44 (37H, s), 1.61–1.74 (4H,
m), 2.52–2.59 (2H, m), 2.94–3.26 (18H, m),3.81 (2H, s), 4.51 (1H,
br s), 4.68 (1H, br s), 5.12 (2H, s), 6.44 (1H, br s), 6.92 (1H, br s),
7.33–7.36 (5H, m); MS Found m/z 887.5. Calcd for C44H76N6O11Na:
[M+Na]+, m/z 887.5.
4.2.18. 4,8-Di(tert-butoxycarbonyl)-N1-[N-(N4-tert-butoxy-
carbonyl-4-aminobutyl)-N-(tert-butoxycarbonyl)glycyl]-N12
[(7-hydroxycoumarin-4-acetyl)asparaginyl]-4,8-diaza-1,12-
dodecanediamine (23)
-
To a solution of 21 (300 mg, 306 lmol) in MeOH (5 mL) were
added 10% Pd(OH)2–C (150 mg) and AcOH (5 mL). The mixture
was stirred for 2 h at rt under an atmosphere of hydrogen. The cat-
alyst was filtered off, and the filtrate was concentrated in vacuo to
obtain colorless oily residue. To a solution of the residue in DMF
(5 mL) were added Et3N (51.1
arin-4-acetic acid succinimidyl ester (Hca-OSu) (107 mg,
337 mol), and the solution was stirred for 3 h at rt. The reaction
lL, 367 lmol) and 7-hydroxycoum-
l
mixture was diluted with H2O (5 mL), and the resulting suspension
was extracted with CHCl3 (3Â 3 mL). The extract was washed with
10% citric acid (3Â 3 mL) and brine (3Â 3 mL), and dried over anhy-
drous MgSO4, followed by concentration in vacuo. The crude prod-
uct was purified by silica-gel column chromatography (PSQ 100B,
20 g, CHCl3/MeOH = 97:3). The fractions containing the desired
product were combined and concentrated in vacuo to give 23 as
yellow oil (254 mg, 79.3%). 1H NMR (CDCl3) d 1.42 (18H, s), 1.43
(18H, s), 1.51 (8H, m), 1.71 (6H, m), 2.58 (2H, s), 2.78 (2H, d),
3.02–3.24 (14H, m), 4.99 (1H, m), 6.17 (1H, br s), 6.76 (1H, s),
6.78 (1H, d), 7.37 (1H, m), 8.53 (1H, s); MS Found m/z 1069.4. Calcd
for C51H82N8O15Na: [M+Na]+, m/z 1069.6.
a
4.2.16. N12-(N -Benzyloxycarbonylasparaginyl)-4,8-di(tert-
butoxycarbonyl)-N1-[N-(N4-tert-butoxycarbonyl-4-amino-
butyl)-N-(tert-butoxycarbonyl)glycyl]-4,8-diaza-1,12-dodec-
anediamine (21)
To a solution of 20 (901 mg, 1.02 mmol) in MeOH (5 mL) were
added 10% Pd(OH)2–C (500 mg) and AcOH (5 mL). The mixture
was stirred for 6 h at rt under an atmosphere of hydrogen. The cat-
alyst was filtered off, and the filtrate was concentrated in vacuo to
obtain colorless oily residue. To the solution of the thus-obtained
residue in DMF (5 mL) were added Et3N (0.154 mL, 1.11 mmol)
and Z-Asn-ONp (434 mg, 1.12 mmol), and the solution was stirred
for 24 h at rt. The reaction mixture was concentrated in vacuo, and
the residue was dissolved in CHCl3 (20 mL). The solution was
washed with 10% citric acid (3Â 5 mL), saturated aqueous NaHCO3
(3Â 10 mL) and brine (3Â 10 mL). The organic layer was dried over
anhydrous MgSO4, and concentrated in vacuo. The crude product
was purified by silica-gel column chromatography (PSQ 100B,
20 g, CHCl3/MeOH = 97:3). The fractions containing the desired
product were combined and concentrated in vacuo to give 21 as
colorless oil (800 mg, 80.1%). 1H NMR (CDCl3) d 1.24–1.30 (5H,
m), 1.33–1.45 (36H, s), 1.65–1.89 (6H, m), 2.98–3.34 (16H, m),
4.45–4.58 (1H, m), 5.11 (2H, s), 7.34–7.39 (5H, m); MS Found m/z
1001.3. Calcd for C48H82N8O13Na: [M+Na]+, m/z 1001.6.
4.2.19. Hcc,Abg-[des-Dhpa,Lys-(NPTX-594)] (4)
To a solution of 22 (50.3 mg, 48.7 lmol) in CH2Cl2 (1.5 mL) was
added TFA (3.5 mL), and the mixture was stirred for 3 h at rt. The
solvent was removed in vacuo, and the residue was dissolved in
H2O (3 mL), followed by reconcentration in vacuo. The thus-ob-
tained crude product was purified by RP-HPLC. The fractions con-
taining the desired product were combined and lyophilized to
give 4 (20.6 mg, 38.8%) as yellow powdery 4 TFA salt. 1H NMR
(D2O) d 1.34 (2 H, q), 1.50 (2H, m), 1.59 (2H, m), 1.81 (6H, m),
1.99 (2H, m), 2.71–2.91 (4H, m), 3.00 (8H, m), 3.17 (2H, q,
J = 6.6 Hz), 3.27 (2H, s), 3.84 (1H, t, J = 6.6 Hz), 6.72 (1H, s), 6.81
(1H, d), 7.56 (1H, m), 8.58 (1H, s); MS Found m/z 633.1. Calcd for
C30H49N8O7: [M+H]+, m/z 633.4.
4.2.20. Hca,Abg-[des-Dhpa,Lys-(NPTX-594)] (5)
To a solution of 23 (50.9 mg, 48.6 lmol) in CH2Cl2 (1.5 mL) was
added TFA (3.5 mL). The mixture was stirred for 4 h at rt. The sol-
vent was removed in vacuo, and the residue was dissolved in H2O
(3 mL), followed by reconcentration in vacuo. The thus-obtained
crude product was purified by RP-HPLC. The fractions containing
the desired product were combined and lyophilized to give 5
(32.5 mg, 60.6%) as yellow powdery 4 TFA salt. 1H NMR (D2O) d
1.34 (2H, q), 1.50 (2H, m), 1.59 (2H, m), 1.81 (6H, m), 1.99 (2H,
m), 2.56 (2H, s), 2.71–2.91 (4H, m), 3.00 (8H, m), 3.17 (2H, m),
4.2.17. 4,8-Di(tert-butoxycarbonyl)-N1-[N-(N4-tert-butoxy-
carbonyl-4-aminobutyl)-N-(tert-butoxycarbonyl)glycyl]-N12
-
[(7-hydroxycoumarin-3-carbonyl)asparaginyl]-4,8-diaza-1,12-
dodecanediamine (22)
To a solution of 21 (259 mg, 265 lmol) in MeOH (5 mL) were
added 10% Pd(OH)2–C (150 mg) and AcOH (5 mL). The mixture
was stirred for 2 h at rt under an atmosphere of hydrogen. The cat-