2308
ZORKO ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
was extracted with CH2Cl2, dried (Na2SO4), and concentrated to obtain an oil
(0.79 g, 75%). For 1H NMR, ␦ values were 0.88 (t, 3H, CH3; J ϭ 6.6 Hz), 1.28
[m, 20H, (CH2)6, (CH2)4], 1.63 (m, 2H, COCH2CH2), 2.01 (m, 4H, CH2CH ϭ
CHCH2), 2.37 (m, 2H, COCH2), 2.86 and 2.87 (2t, 4H, 2ϫ CH2NH2; J1 ϭ 6.8
Hz, J2 ϭ 6.9 Hz), 3.39 (m, 4H, CH2NCH2), and 5.35 (m, 2H, CH ϭ CH)
(underlining denotes protons appearing in the 1H NMR signal). For MS (FAB
[fast atom bombardment]), m/z was 368.3 (Mϩ ϩ 1).
(ii) Oleic acid (4-aminobutyl)-(3-aminopropyl)amide (compound 2). The
compound was prepared in the same manner as compound 1, starting from
N-[4-(trifluoroacetamido)butyl]-N-[3-(trifluoroacetamido)propyl]amine. For 1H
NMR, ␦ values were 0.88 (t, 3H, CH3; J ϭ 6.6 Hz), 1.28 [m, 20H, (CH2)6,
(CH2)4], 1.38 to 1.77 [m, 8H, COCH2CH2, NCH2CH2, NCH2(CH2)2CH2NH2],
2.01 (m, 4H, CH2CH ϭ CHCH2), 2.30 (m, 2H, COCH2), 2.62 to 2.78 (m, 4H, 2ϫ
CH2NH2), 3.20 to 3.46 (m, 4H, CH2NCH2), and 5.34 (m, 2H, CH ϭ CH). For
MS (FAB), m/z was 410.4 (Mϩ ϩ 1).
(iii) Oleic acid bis(2-DEAE)amide (compound 3). Oleoyl chloride (1.5 ml, 4.5
mmol) was added to a cold (0°C) solution of tetraethylenediethylenetriamine
(1.0 ml, 3.9 mmol), Et3N (0.7 ml, 5.0 mmol), and DMAP (cat.) in dry CH2Cl2 (25
ml) and stirred at RT overnight. The concentrated residue was partitioned
between 0.5 M H2SO4 (20 ml) and ether (20 ml). The aqueous layer was washed
again with ether (20 ml) and basified to pH 10 with 2 M NaOH. The product was
extracted with CH2Cl2 (2 ϫ 20 ml), dried (Na2SO4), and concentrated to obtain
a slightly yellowish oil (1.51 g, 81%). For 1H NMR, ␦ values were 0.88 (t, 3H,
CH3; J ϭ 7.3 Hz), 1.03 (t, 12H, 4ϫ NCH2CH3; J ϭ 7.2 Hz), 1.29 [m, 20H,
(CH2)6, (CH2)4], 1.64 (m, 2H, COCH2CH2), 2.00 (m, 4H, CH2CH ϭ CHCH2),
2.31 (t, 2H, COCH2; J ϭ 7.6 Hz), 2.56 [m, 12H, 2ϫ (CH3CH2)2NCH2], 3.38 (m,
4H, 2ϫ NHCH2), and 5.34 (m, 2H, CH ϭ CH). For MS (FAB), m/z was 480.5
(Mϩ ϩ 1).
mixture was washed with 1 M HCl, 5% aqueous NaHCO3, and H2O. The organic
layer was concentrated, and the crude extract was purified by chromatography on
silica gel, eluting with CH2Cl2/EtOAc/Et3N (40:10:0.25), to obtain a light yel-
lowish oil (1.43 g). This N-TFA protected product was dissolved in MeOH (30
ml) and cooled to 0°C, and NaBH4 (0.50 g, 13.2 mmol) was added. The mixture
was stirred at RT overnight and concentrated. The residue was partitioned be-
tween H2O (30 ml) and CH2Cl2 (30 ml), and the organic layer was dried
(Na2SO4). After concentration, the residue was dissolved in MeOH (25 ml), and
36% HCl was added to precipitate the product as its HCl salt. This was sus-
pended in H2O and basified to pH 10 with 1 M NaOH, and the product was
extracted with CH2Cl2, dried (Na2SO4), and concentrated to obtain an oil which
solidified partially upon standing (0.75 g, 64%). For 1H NMR, ␦ values were
0.86 (t, 3H, CH3; J ϭ 6.6 Hz), 1.25 [m, 20H, (CH2)6, (CH2)4], 1.48 (m, 2H,
COCH2CH2), 1.98 (m, 4H, CH2CH ϭ CHCH2), 2.31 (m, 2H, COCH2), 2.85 to
3.64 [m, 12H, N(CH2CH2)2, 2ϫ COCH2NH2], and 5.33 (m, 2H, CH ϭ CH). For
MS (FAB), m/z was 482.4 (Mϩ ϩ 1).
(viii) (N-Oleoyl-L-prolyl)-[N,N-bis(2-aminoethyl)]amide (compound 8). Oleoyl
chloride (1.5 ml, 4.5 mmol) was added to a cold (0°C) suspension of L-proline
(1.00 g, 8.7 mmol), Et3N (1.4 ml, 10.5 mmol), and DMAP (cat.) in CH2Cl2 (40
ml) and stirred at RT overnight. The reaction mixture was washed with 1 M HCl
and saturated aqueous NaCl solution and dried (Na2SO4). The concentrated
residue was purified by chromatography on silica gel, eluting with ether/hexane/
AcOH (10:10:0.2) to obtain N-oleoyl-L-proline as a colorless oil (2.21 g). Oxalyl
chloride was added to a cold (0°C) solution of N-oleoyl-L-proline and DMAP
(cat.) in CH2Cl2 (25 ml) (0.56 ml, 6.4 mmol) and stirred at RT for 3 h. This
solution was added to a cold (0°C) solution of N,N-bis[2-(trifluoroacetamido)
ethyl]amine (1.80 g, 6.1 mmol) and Et3N (1.7 ml, 12 mmol) in CH2Cl2 (25 ml)
and stirred at RT overnight. The mixture was washed with 5% aqueous NaHCO3
and saturated aqueous NaCl solution, dried (Na2SO4), and concentrated to
obtain a light yellowish oil (1.80 g). This N-TFA protected compound was
suspended in MeOH (20 ml) and cooled to 0°C, and NaBH4 (1.32 g, 35 mmol)
was added. The mixture was stirred at RT overnight and concentrated. The
residue was partitioned between H2O (50 ml) and CH2Cl2 (50 ml), and the
organic layer was dried (Na2SO4). The concentrated residue was dissolved in
MeOH (30 ml), and HCl gas was bubbled in to precipitate the product as its HCl
salt. It was then suspended in H2O and basified to pH 10 with 1 M NaOH, and
the product was extracted with CH2Cl2, dried (Na2SO4), and concentrated to
obtain an oil (0.73 g, 35%). For 1H NMR (CDCl3), ␦ values were 0.88 (t, 3H,
CH3; J ϭ 6.6 Hz), 1.28 [m, 20H, (CH2)6, (CH2)4], 1.62 (m, 2H, COCH2CH2),
1.86 to 2.35 [m, 10H, CH2CH ϭ CHCH2, COCH2, CH(CO)(CH2)2], 2.79 to 3.15
(m, 4H, 2ϫ CH2NH2), 3.39 to 3.75 (m, 6H, CH2NHCH2, NCH2), 4.88 (m, 1H,
CH), and 5.34 (m, 2H, CH ϭ CH). For MS (FAB), m/z was 465.4 (Mϩ ϩ 1).
(ix) (N-Oleoyl-L-prolyl)-N,N-bis[2-(diethylamino)ethyl]amide (compound 9).
Acid chloride of N-oleoyl-L-proline (0.77 g, 1.94 mmol) in CH2Cl2 (5 ml) was
added to a cold (0°C) solution of tetraethylenediethylenetriamine (0.5 ml, 1.95
mmol), Et3N (0.8 ml, 5.8 mmol), and DMAP (cat.) in CH2Cl2 (25 ml). The
mixture was stirred at RT overnight and washed with 5% aqueous NaHCO3 and
saturated aqueous NaCl solution and dried (Na2SO4). After concentration, a
light yellowish oil (0.86 g, 77%) was obtained. For 1H NMR (CDCl3), ␦ values
were 0.88 (t, 3H, CH3; J ϭ 6.6 Hz), 1.05 [m, 12H, 2ϫ N(CH2CH3)2], 1.28 [m,
20H, (CH2)6, (CH2)4], 1.63 (m, 2H, COCH2CH2), 1.84 to 2.35 and 2.51 to 2.92
[2m, 22H, CH2CH ϭ CHCH2, COCH2, NCH(CH2)2, 2ϫ CH2N(CH2CH3)2],
3.28 to 3.76 (m, 6H, CH2NCH2, NCH2), 4.74 (m, 1H, CH), and 5.34 (m, 2H,
CH ϭ CH). For MS (FAB), m/z was 577.5 (Mϩ ϩ 1).
(iv) N1-Oleoyldiethylenetriamine (compound 4). Diethylenetriamine (300 mg,
2.9 mmol) and benzophenoneimine (1.2 ml, 7.2 mmol) in CH2Cl2 (20 ml) were
stirred at RT overnight. The mixture was concentrated and washed with petro-
leum ether (2 ϫ 15 ml). The residue was concentrated and suspended in dioxane
(10 ml), and 2 M HCl (10 ml) was added. The mixture was stirred at RT for 3 h,
partially concentrated, diluted with H2O (10 ml), and washed with ether. The
aqueous phase was basified to pH 10 with 2 M NaOH and extracted with CH2Cl2.
The concentrated residue was dissolved in MeOH (10 ml), and 36% HCl was
added to obtain a precipitate. This precipitate was filtered and then dissolved in
H2O and basified to pH 10 with 1 M NaOH, and the product was extracted with
CH2Cl2 (2 ϫ 15 ml). The combined extracts were dried (Na2SO4) and concen-
trated to obtain an oil which solidified partially upon standing (0.10 g, 36%). For
1H NMR, ␦ values were 0.88 (t, 3H, CH3; J ϭ 6.6 Hz), 1.28 [m, 20H, (CH2)6,
(CH2)4], 1.69 (m, 2H, COCH2CH2), 2.00 (m, 4H, CH2CH ϭ CHCH2), 2.16 (t,
2H, COCH2; J ϭ 7.8 Hz), 2.68 (m, 2H, NH2CH2), 2.79 (m, 4H, CH2NHCH2),
3.65 (m, 2H, CH2NHCO), 5.34 (m, 2H, CH ϭ CH), and 6.19 (br s, 1H, NHCO).
For MS (FAB), m/z was 368.4 (Mϩ ϩ 1).
(v) Oleic acid bis[2-(2-aminoethylamino)ethyl]amide (compound 5). (2,2,2-
Trifluoroacetylamino)ethylbromide (0.93 mg, 4.3 mmol) was added to a solution
of compound 1 (75 mg, 0.21 mmol) and N, N-diisopropylethylamine (110 l, 0.63
mmol) in toluene, and the mixture was refluxed for 5 h. The cold mixture was
filtered and concentrated and redissolved in MeOH (5 ml), and NaBH4 was
added (47 mg, 1.25 mmol). After being stirred at RT overnight, the concentrated
residue was partitioned between H2O (10 ml) and CH2Cl2 (10 ml). The organic
layer was dried (Na2SO4) and concentrated. The residue was dissolved in MeOH
(5 ml), and HCl gas was bubbled in followed by ether to precipitate the product
as its HCl salt (75 mg, 60%). For analyses, the product was suspended in H2O,
basified to pH 10 with 1 M NaOH, extracted with CH2Cl2, dried (Na2SO4), and
concentrated to obtain an oil. For 1H NMR, ␦ values were 0.88 (t, 3H, CH3; J ϭ
6.6 Hz), 1.28 [m, 20H, (CH2)6, (CH2)4], 1.62 (m, 2H, COCH2CH2), 2.01 (m, 4H,
CH2CH ϭ CHCH2), 2.18 (m, 2H, COCH2), 2.43 to 2.93 [m, 12H, 2ϫ CH2NH
(CH2)2NH2], 3.33 (m, 4H, CH2NCH2), and 5.34 (m, 2H, CH ϭ CH). For MS
(FAB), m/z was 454.4 (Mϩ ϩ 1).
(x) (N␣-Oleoyl–L-lysyl–L-prolyl)-N,N-bis[2-(diethylamino)ethyl]amide (com-
pound 10). Oleoyl chloride (2.3 ml, 5.95 mmol) was added to a cold (0°C)
solution of Nε-(trifluoroacetyl)–L-lysyl–L-proline (2.0 g, 5.89 mmol), Et3N (1.67
ml, 12.1 mmol), and DMAP (cat.) in dry CH2Cl2 (50 ml). The mixture was stirred
at RT overnight and washed with 1 M HCl, 5% aqueous NaHCO3, and H2O and
dried (Na2SO4). The solvent was evaporated, and the crude extract was purified
by chromatography on silica gel, eluting with CH2Cl2/EtOAc/Et3N (30:10:0.25)
to obtain a light yellowish oil (1.99 g). Oxalyl chloride (0.30 ml, 3.46 mmol) was
added to a cold (0°C) solution of [N␣-oleoyl-Nε-(trifluoroacetyl)–L-lysyl]–L-pro-
line and DMAP (cat.) in CH2Cl2 (30 ml). The solution was stirred at RT for 3 h.
This solution was added to a cold (0°C) solution of tetraethylenediethylenetri-
amine (0.85 ml, 3.33 mmol) and Et3N (1.3 ml, 9.89 mmol) in CH2Cl2 (25 ml) and
stirred at RT overnight. The mixture was washed with 5% aqueous NaHCO3 and
saturated aqueous NaCl solution, dried (Na2SO4), and concentrated to obtain a
light yellowish oil (1.53 g). This N-TFA protected product was suspended in
MeOH (20 ml) and cooled to 0°C, and NaBH4 (0.43 g, 11.5 mmol) was added.
The mixture was stirred at RT overnight and concentrated. The residue was
partitioned between H2O (40 ml) and CH2Cl2 (40 ml), and the organic layer
was dried (Na2SO4). The concentrated residue was dissolved in MeOH (30
(vi) Oleic acid [4-(2-aminoethylamino)butyl]-[3-(2-aminoethylamino)propyl]
amide (compound 6). The compound was prepared in the same manner as
that for compound 4, starting from compound 2. For 1H NMR, ␦ values
were 0.88 (t, 3H, CH3; J ϭ 6.6 Hz), 1.02 to 1.85 [m, 28H, (CH2)6, (CH2)4,
NCH2(CH2)2CH2NH2, NCH2CH2, COCH2CH2], 1.92 (m, 4H, CH2CH ϭ
CHCH2), 2.22 (m, 2H, COCH2), 2.28 to 2.83 [m, 12H, 2ϫ CH2NH(CH2)2NH2],
3.09 to 3.32 (m, 4H, CH2NCH2), and 5.33 (m, 2H, CH ϭ CH). For MS (FAB),
m/z was 368.4 (Mϩ ϩ 1).
(vii) N,N-Bis[2-(glycinamido)ethyl]oleoylamide (compound 7). (2,2,2-Trifluo-
roacetylamino)acetyl chloride (1.13 g, 5.85 mmol) in CH2Cl2 was added to a cold
(0°C) solution of compound 1 (0.90 g, 2.45 mmol), Et3N (1.0 ml, 7.53 mmol), and
DMAP (cat.) in CH2Cl2 (25 ml) and stirred at RT overnight. The reaction