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photodiode-array detector, 600S controller, and Millenium Chromatography Manager 2010 v. 2.15 (Waters
Corp., Milford, MA, USA); Rheodyne 77251 rotary valve fitted with a 5-ml loop (Rheodyne, Cotati, CA, USA);
1
Waters SymmetryTM-C8 column with integrated guard column (5 mm, 3.9 Â 150 mm); flow rate 0.5 ml min
;
mobile phase: A, H2O; B, MeCN; C 10% AcOH in H2O; linear gradient A/B/C 87:3 :10 ! 10 :80 :10 within
30 min. APCI-MS and APCI-MS/MS: Finnigan TSQ-700 triple-stage quadrupole instrument equipped with an
Â
atmospheric pressure chemical ionization (APCI) ion source (Finnigan, San Jose, CA, USA); APCI operating
conditions in the positive mode: vaporizer temp. 4508, corona voltage 5 kV, heated capillary temp. 2208, cheat
gas N2 with an inlet pressure of 40 psi, conversion dynode 15 kV; MS/MS experiments: collision gas Ar with a
relative pressure 2.5 ± 3.3 mTorr, collision-induced dissociation offset (Coff) 35 eV.
Ethyl (Æ)-3-{{3-{{4-[(3-Aminopropyl)amino]butyl}amino}propyl}amino}-3-(4-methoxyphenyl)propanoate
(19). To a soln. of spermine (17; 20.23 g, 99.98 mmol) in EtOH (100 ml) were added ethyl 3-(4-methoxyphenyl)-
3-oxopropanoate (18; 7.41 g, 33.34 mmol) in EtOH (35 ml) and AcOH (24.60 g, 409.66 mmol). The mixture was
refluxed for 2 h, cooled to r.t., and then MeOH (100 ml), AcOH (16.70 g, 278.10 mmol), and a soln. of
NaCNBH3 (2.40 g, 38.19 mmol) in MeOH (30 ml) were added. The mixture was stirred overnight at r.t. After
evaporation H2O (100 ml) was added, and the mixture was extracted with CHCl3 (3 Â 100 ml). The aq. phase
was alkalinized with K2CO3 and extracted with CHCl3 (1Â) and CHCl3/i-PrOH 4 :1 (5Â). After evaporation of
the extract, the residue was dissolved in EtOH and the soln. acidified with 32% aq. HCl soln. The precipitated
tetrahydrochloride of 19 was filtered off, washed with EtOH and Et2O, and recrystallized from AcOH (40 ml):
9.35 g (51% rel. to 18) of 19 ´ 2 HCl. TLC (silica gel, CHCl3/MeOH/25% aq. NH3 soln. 7:3 :1): Rf 0.40. IR
(KBr): 3435, 2986, 1735, 1613, 1515, 1463, 1300, 1255, 1188, 1180, 1112, 1030, 992, 845, 825, 750. 1H-NMR
(CD3OD): 7.48 (d, J 8.8, 2 arom. H); 7.02 (d, J 8.8, 2 arom. H); 4.66 (m, H C(3)); 4.06 (q, J 7.1, MeCH2);
3.82 (s, MeO); 3.18 ± 2.84 (m, 14 H); 2.24 ± 2.04 (m, 4 H); 1.83 (br. m, 4 H); 1.14 (t, J 7.1, MeCH2. 13C-NMR:
172.5 (s, CO); 162.5 (s, Cp); 131.2 (d, arom. C); 126.4 (s, Cipso); 115.8 (d, arom. C); 62.4 (t, C(2)); 60.2
(d, C(3)); 56.0 (q, MeO); 48.3, 48.2, 46.0, 44.1, 38.8, 38.0, 25.4, 24.2, 24.1 (9t, 9 CH2); 14.3 (q, Me). CI-MS: 409
(38, [M 1] ), 337 (12), 224 (26), 207 (86), 203 (100, [spermine H] ).
(Æ)-8-(4-Methoxyphenyl)-1,5,9,13-tetraazacycloheptadecan-6-one ((Æ)-Buchnerine; 14). For the prepa-
ration of the free base, 19 ´ 4 HCl (4.18 g, 7.54 mmol) was dissolved in H2O (85 ml), the soln. alkalinized with
solid K2CO3 and extracted with CHCl3/i-PrOH 4 :1 (5 Â 50 ml), the extract evaporated, the residue dissolved in
CHCl3, and the soln. filtered and evaporated. The free base 19 was refluxed with benzene (200 ml) for 2 h in a
flask equipped with a H2O remover. The soln. was cooled to 08, 1m Sb(OEt)3 in toluene (9.65 mmol) was added,
and the mixture was refluxed for 14 h. After cooling to 08, the mixture was quenched with MeOH (50 ml) and
chromatographed (silica gel, CHCl3/MeOH/25% aq. NH3 soln. 78 :19 :3 ! 7:3 :1): 1.46 g (53%) of 14. Colorless
oil. TLC (silica gel, CHCl3/MeOH/25% aq. NH3 soln. 7 :3 :1): Rf 0.45. IR (CHCl3): 2995, 2930, 2840, 2675, 1650,
1612, 1510, 1465, 1305, 1250, 1178, 1120, 1035, 909, 833, 658. 1H-NMR (CDCl3): 8.48 (t, J 4.8, CONH); 7.26 ±
7.12 (m, 2 arom. H); 6.90 ± 6.79 (m, 2 arom. H); 4.03 ± 2.27 (m, 21 H); 1.89 ± 1.36 (m, 8 H). 13C-NMR (CDCl3):
171.7 (s, CO); 158.6 (s, Cp); 135.4 (s, Cipso); 127.8, 113.9 (2d, arom. C); 59.7 (d, C(8)); 55.2 (q, MeO); 48.8, 48.6,
48.2, 47.7, 46.7, 45.7, 38.6, 28.2, 27.9, 27.3, 27.0 (11t, 11 CH2). ESI-MS: 363 ([M H] ).
8-(4-Methoxyphenyl)-1,13-(4-tolylsulfonyl)-1,5,9,13-tetraazacycloheptadecan-6-one (24) and 9-(4-Bromo-
butyl)-8-(4-methoxyphenyl)-6-oxo-1,13-(4-tolylsulfonyl)-1,5,9,13-tetraazacycloheptadecane-5-carboxylic Acid
(25). To a soln. of 23 (2.74 g, 4.44 mmol) in dry DMF (445 ml) Cs2CO3 (3.25 g, 9.97 mmol) was added. The
mixture was heated at 608 for 40 min and stirred for a further 40 min at r.t. Then 1,4-dibromobutane (1.07 g,
4.96 mmol) in DMF (94 ml) was added dropwise. The mixture was stirred for 30 h at r.t. and evaporated. The
residue was chromatographed (AcOH, then AcOH/MeOH 12 :1, 10 :1, and 9 :1): 2.59 g (87%) of 24 and 0.22 g
(6%) of 25.
Data of 24: TLC (silica gel, AcOH/MeOH 6 :1): Rf 0.38. ESI-MS: 671 ([M H] ). Spectroscopic data: see
[16][19].
Data of 25: Colorless glass-like solid. TLC (silica gel, AcOH/MeOH 6 :1): Rf 0.57. 1H-NMR: br. signals, no
interpretation possible. 13C-NMR (CDCl3): 180.00 ± 131.18 (br. s, several C); 129.65, 128.36, 126.98, 113.93
(d, arom. CH); 62.82 (br. d, C(8)); 55.19 (q, MeO); 50.00 ± 24.15 (br. t, several CH2); 28.38 (q, Me). ESI-MS:
849 ([M H] ), 871 ([M Na] ), 887 ([M K] ). Anal. calc. for C39H53BrN4O8S2 (849.90): C 55.11, H 6.29,
Br 9.40, N 6.59, S 7.55; found: C 56.43, H 6.29, Br 8.84, N 6.87, S 7.56.
Hemin/H2O2 Oxidation of Dihydroxyverbacine (10). Dihydroxyverbacine (10) (3 mg, 0.0060 mmol) and
hemin (1.3 mg, 0.0019 mmol) were dissolved in MeCN (0.15 ml) and 0.1m eq. K2CO3 (0.15 ml). Then 10% H2O2
soln. (20 ml, ca. 0.0588 mmol) was added, and the mixture was shaken for 10 min. For analysis, the mixture was
filtered through a 0.45-mm centrifugal filter tube (Eppendorf-Netheler-Hinz GmbH, Hamburg). The mixture
was analyzed by HPLC/MS and HPLC/MS/MS (aliquots of 5 ml were injected; see Fig.).