112 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Heyes et al.
1
(MgSO4), and evaporated under reduced pressure to give a
colorless oil, which was purified by column chromatography
(CH2Cl2-1% MeOH/CH2Cl2). This gave 56 as a pale yellow oil
(750 mg, 74.0%); Rf 0.30 (3% MeOH/CH2Cl2). 1H NMR (δH):
7.74 (d, 2H, H(arom,Fmoc), J ) 7.6 Hz), 7.56 (d, 2H, H(arom,Fmoc), J
) 7.2 Hz), 7.37 (t, 2H, H(arom,Fmoc), J ) 7.5 Hz), 7.27 (d, 2H,
90.7%); Rf 0.6 (CH2Cl2:MeOH:NH3 92:7:1); mp 75-77 °C. H
NMR (δH): 7.72-7.60 (m, 1H, NH(amide)), 6.52 (s, 1H, H(arom,Mtr)),
6.42-6.19 (m, 3H, NH(guanidine)), 3.82 (s, 3H, OCH3(Mtr)), 3.62-
3.35 (m, 9H, CH2CH(OCH2)CH2OCH2), 3.35-3.11 (m, 3H,
CHNH2, NHCH2CH2), 2.69, 2.62 (2s, 6H, 2CH3(2,6-Mtr)), 2.17
(s, 3H, CH3(3-Mtr)), 2.09-1.89 (m, 2H, NH2), 1.73-1.41 (m, 8H,
NH2CHCH2CH2, OCH2CH2), 1.38-1.11 (m, 48H, CH2(lauryl/stearyl)),
0.88 (t, 6H, CH2CH3, J ) 6.4 Hz). HRMS: (M + H)x calcd for
49H94N5O6S, 880.6937; found, 880.6925. Anal. Calcd for
49H93N5O6S: C, H, N.
60. P r ep a r a t ion of L-Ar gin in e(2,3-d ila u r yloxy)p r op -
yla m id e (60). To a flask containing a mixture of TFA, phenol,
water, 1,2-ethane-dithiol, thioanisole, and TIS (81:5:5:5:2.5:
1) (20 mL) was added 58 (199 mg, 0.25 mmol), and the solution
was stirred for 8 h. The solvent was then removed under
reduced pressure, and the product was purified by column
chromatography (CH2Cl2-CH2Cl2:MeOH:NH3 92:7:1) to give
60 as a colorless oil (120 mg, 82.2%); Rf 0.1 (CH2Cl2:MeOH:
H
(arom,Fmoc), J ) 7.4 Hz), 6.94-6.82 (m, 1H, NH(amide)), 6.49 (s,
1H, H(arom,Mtr)), 6.23-5.90 (m, 3H, NH(guanidine)), 5.86 (d, 1H,
NH(carbamate)), 4.35 (t, 2H, CHCH2(Fmoc), J ) 7.0 Hz), 4.25-4.10
(m, 2H, CHNH, CHCH2(Fmoc)), 3.78 (s, 3H, OCH3(Mtr)), 3.54-
3.30 (m, 9H, CH2CH(OCH2)CH2OCH2), 3.30-3.10 (m, 2H,
NHCH2CH2), 2.69, 2.61 (2s, 6H, 2CH3(2,6-Mtr)), 2.10 (s, 3H,
CH3(3-Mtr)), 1.78-1.40 (m, 8H, NHCHCH2CH2, OCH2CH2),
1.30-1.10 (m, 36H, CH2(lauryl)), 0.87 (t, 6H, CH2CH3, J ) 6.6
Hz). 13C NMR (δC): 172.05 (CO(amide)), 158.99 (C(arom,Mtr)), 156.54
(NHC(dNH)NH), 144.16 (C(arom,Fmoc)), 141.69 (C(arom,Fmoc)), 139.04
C
C
(C(arom,Mtr)), 137.16 (C(arom,Mtr)), 133.73 (C(arom,Mtr)), 128.15 (C(ar
om,Fmoc)), 127.50 (C(arom,Fmoc)), 125.46 (C(arom,Fmoc)), 125.19 (C(arom
-
-
1
,Mtr)), 120.34 (C(arom,Fmoc)), 112.12 (C(arom,Mtr)), 72.23 (CHCH2O),
71.69 (CHCH2OCH2), 70.68 (CHOCH2), 67.54 (CHCH2(Fmoc)),
55.81 (OCH3(Mtr)), 54.64 (CHNH), 47.53 (CHCH2(Fmoc)), 41.00
(NHCH2CH2), 32.33 (CH2CH2CH3), 30.81-29.77 (CH2(lauryl)),
26.50 (OCH2CH2CH2), 25.51 (NH2CHCH2), 25.24 (NHCH2CH2),
24.52 (CH3(6-Mtr)), 23.10 (CH2CH3), 18.74 (CH3(2-Mtr)), 14.52
(CH2CH3), 12.35 (CH3(3-Mtr)). HRMS: (M + H)x calcd for
C59H94N5O7S, 1016.6835; found, 1016.6874.
NH3 92:7:1). H NMR (δH): 8.01-7.88 (m, 2H, NH(amide), CH2-
CH2NH), 7.18-6.92 (m, 3H, C(NH2)NH), 3.86-3.63 (m, 3H,
CHNH2), 3.63-3.10 (m, 11H, NHCH2CH2, CH2CH(OCH2)CH2-
OCH2), 1.86-1.40 (m, 8H, NH2CHCH2CH2, OCH2CH2), 1.40-
1.17 (m, 36H, CH2(lauryl)), 0.86 (t, 6H, CH2CH3, J ) 6.6 Hz).
13C NMR (δC): 173.60 (CO(amide)), 157.83 (NH2C(dNH)NH),
72.23 (CHCH2O), 71.38 (CHCH2OCH2), 70.71 (CHOCH2), 54.04
(CHNH2), 41.09 (NCH2CH, NHCH2CH2), 32.33 (CH2CH2CH3),
31.07 (NH2CHCH2), 30.39-29.78 (CH2(lauryl)), 26.47 (OCH2-
CH2CH2), 24.97 (NHCH2CH2), 23.08 (CH2CH3), 14.49 (CH2CH3).
HRMS: (M + H)x calcd for C33H70N5O3, 584.5490; found,
584.5479.
57. P r ep a r a tion of N-r-F lu or en ylm eth oxyca r bon yl-
NG-4-m eth oxy-2,3,6-tr im eth ylben zen e Su lfon yl-L-a r gin -
in e(2-la u r yloxy-3-st ea r yl-oxy)p r op yla m id e (57). Com-
pound 57 was prepared analogously to 56 on a 1 mmol scale
to give, after chromatography (CH2Cl2-1% MeOH/CH2Cl2), 57
as a pale yellow oil (809 mg, 73.5%); Rf 0.3 (3% MeOH/CH2-
61. P r ep a r a t ion of L-Ar gin in e(2-la u r yloxy-3-st ea r yl-
oxy)p r op yla m id e (61). Compound 61 was prepared analo-
gously to 60 on a 0.25 mmol scale to give, after chromatogra-
phy (CH2Cl2-CH2Cl2:MeOH:NH3 92:7:1), 61 as a colorless oil
1
Cl2). H NMR (δH): 7.73 (d, 2H, H(arom,Fmoc), J ) 7.5 Hz), 7.56
(d, 2H, H(arom,Fmoc), J ) 7.2 Hz), 7.37 (t, 2H, H(arom,Fmoc), J ) 7.7
Hz), 7.26 (d, 2H, H(arom,Fmoc), J ) 6.8 Hz), 6.96-6.82 (m, 1H,
1
(140 mg, 83.8%); Rf 0.1 (CH2Cl2:MeOH:NH3 92:7:1). H NMR
NH(amide)), 6.49 (s, 1H, H(arom,Mtr)), 6.25-5.96 (m, 3H, NH(guani
-
(δH): 8.08-7.90 (m, 2H, NH(amide), CH2CH2NH), 7.16-6.99 (m,
3H, C(NH2)NH), 3.73-3.62 (m, 1H, CHNH2), 3.62-3.12 (m,
11H, NHCH2CH2, CH2CH(OCH2)CH2OCH2), 2.70-2.58 (m,
2H, NH2), 1.83-1.41 (m, 8H, NH2CHCH2CH2, OCH2CH2),
1.34-1.15 (m, 48H, CH2(lauryl/stearyl)), 0.88 (t, 6H, CH2CH3, J )
6.6 Hz). 13C NMR (δC): 173.63 (CO(amide)), 157.90 (NH2C(dNH)-
NH), 72.22 (CHCH2O), 71.42 (CHCH2OCH2), 70.71 (CHOCH2),
54.03 (CHNH2), 41.07 (NCH2CH, NHCH2CH2), 32.33 CH2CH2-
CH3), 31.07 (NH2CHCH2), 30.41-29.77 (CH2(lauryl/stearyl)), 26.48
(OCH2CH2CH2), 25.03 (NHCH2CH2), 23.09 (CH2CH3), 14.50
(CH2CH3). HRMS: (M + H)x calcd for C39H82N5O3, 668.6426;
found, 668.6418.
Liposom e For m u lation an d Lipoplex For m ation . DOPE
(48 µL of a 12.5 mg mL-1 CHCl3 solution, i.e., 0.6 mg, 0.8 µmol)
was added to a solution containing cationic lipid component
(0.6 mg, 1.2 µmol) in CHCl3 (0.5 mL) in a flask under argon.
One milliliter of sterilized HEPES buffer (20 mM, pH 7.8) was
added to the lipid solution, and the mixture was bath-sonicated
for 5 min until it became opalescent. The organic solvent was
removed in vacuo, and the liposome formulation was sonicated
for a further 5 min. Liposomes were then extruded through a
filter with a porosity of 200 nm using a LiposoFast Basic
manual extruder (Avestin Inc., Ottawa, Canada) at 37 °C.
To form the lipoplexes for transfection, plasmid DNA
containing the lacZ gene under the control of the Elongation
Factor promoter (pEFlacZ) was diluted to a concentration of
0.1 mg mL-1 in phosphate buffered saline (PBS). DNA solution
(4 µL) was then dispensed into a 500 µL Eppendorf tube for
each well of cells to be transfected. The corresponding amount
of liposome formulation to be used in the experiment (typically
3-5 µL, at a total lipid concentration of 1.2 mg mL-1) was then
added to the DNA, and the total volume was made up to 32
µL with PBS. Each tube was then vortexed briefly and left to
stand for 15-20 min.
dine)), 5.90 (d, 1H, NH(carbamate), J ) 6.8 Hz), 4.35 (t, 2H,
CHCH2(Fmoc) 7.0 Hz), 4.25-4.10 (m, 2H, CHNH,
,
J
)
CHCH2(Fmoc)), 3.79 (s, 3H, OCH3(Mtr)), 3.54-3.30 (m, 9H,
CH2CH(OCH2)CH2OCH2), 3.30-3.11 (m, 2H, NHCH2CH2),
2.69, 2.62 (2s, 6H, 2CH3(2,6-Mtr)), 2.10 (s, 3H, CH3(3-Mtr)), 1.79-
1.40 (m, 8H, NHCHCH2CH2, OCH2CH2), 1.30-1.10 (m, 48H,
CH2(lauryl/stearyl)), 0.87 (t, 6H, CH2CH3, J ) 6.6 Hz). 13C NMR
(δC): 172.33 (CO(amide)), 158.96 (C(arom,Mtr)), 156.63 (NHC(dNH)-
NH), 144.17 (C(arom,Fmoc)), 141.65 (C(arom,Fmoc)), 139.01 (C(arom,Mtr)),
137.10 (C(arom,Mtr)), 133.63 (C(arom,Mtr)), 128.13 (C(arom,Fmoc)), 127.50
(C(arom,Fmoc)), 125.50 (C(arom,Fmoc)), 125.24 (C(arom,Mtr)), 120.36
(C(arom,Fmoc)), 112.13 (C(arom,Mtr)), 72.18 (CHCH2O), 71.66 (CHCH2-
OCH2), 70.75 (CHOCH2), 67.56 (CHCH2(Fmoc)), 55.80 (OCH3(Mtr)),
54.79 (CHNH), 47.51 (CHCH2(Fmoc)), 40.98 (NHCH2CH2), 32.33
(CH2CH2CH3), 30.56-29.77 (CH2(lauryl/stearyl)), 26.50 (OCH2-
CH2CH2), 25.70 (NH2CHCH2), 25.44 (NHCH2CH2), 24.54
(CH3(6-Mtr)), 23.10 (CH2CH3), 18.75 (CH3(2-Mtr)), 14.52 (CH2CH3),
12.35 (CH3(3-Mtr)). HRMS: (M + Na)x calcd for C65H105N5O7-
SNa, 1122.7653; found, 1122.7632.
58. P r ep a r a tion of NG-4-Meth oxy-2,3,6-tr im eth ylben -
zen esu lfon yl-L-ar gin in e(2,3-dilau r yloxy)pr opylam ide (58).
Compound 58 was prepared analogously to 49 on a 0.7 mmol
scale to give, after chromatography (CH2Cl2-CH2Cl2:MeOH:
NH3 92:7:1), 58 as a colorless oil (501 mg, 89.9%); Rf 0.6 (CH2-
Cl2:MeOH:NH3 92:7:1). 1H NMR (δH): 7.71-7.61 (m, 1H,
NH(amide)), 6.52 (s, 1H, H(arom,Mtr)), 6.42-6.22 (m, 3H, NH(guani
-
dine)), 3.82 (s, 3H, OCH3(Mtr)), 3.63-3.33 (m, 9H, CH2CH(OCH2)-
CH2OCH2), 3.33-3.10 (m, 3H, CHNH2, NHCH2CH2), 2.69, 2.61
(2s, 6H, 2CH3(2,6-Mtr)), 2.17 (s, 3H, CH3(3-Mtr)), 1.94-1.81 (m,
2H, NH2), 1.69-1.40 (m, 8H, NH2CHCH2CH2, OCH2CH2),
1.40-1.15 (m, 36H, CH2(lauryl)), 0.87 (t, 6H, CH2CH3, J ) 6.6
Hz). HRMS: (M + H)x calcd for C43H82N5O6S, 796.5986; found,
796.6028. Anal. Calcd for C43H81N5O6S: C, H, N.
59. P r ep a r a tion of NG-4-Meth oxy-2,3,6-tr im eth ylben -
zen esu lfon yl-L-a r gin in e(2-la u r yloxy-3-stea r yloxy)p r op -
yla m id e (59). Compound 59 was prepared analogously to 49
on a 0.7 mmol scale to give, after chromatography (CH2Cl2-
CH2Cl2:MeOH:NH3 92:7:1), 59 as a white solid (559 mg,
Cell Cu ltu r e a n d Tr a n sfection . All tissue culture media
and reagents were obtained from Life Technologies, Inc.
(Paisley, U.K.) unless otherwise stated. V79 (chinese hamster
lung) and HT29 (human colon adenocarcinoma) cell lines were
obtained from the European Collection of Cell Cultures (Sal-