Polyamine-Based Cationic Lipids for DNA Delivery
463 473
Compound 12: Rf
=
0.30 (CH2Cl2/MeOH 9:1); 1H NMR (400 MHz,
196.9 (C=OÀDde) ppm; IR (CH2Cl2): n˜ = 1691, 1570 cmÀ1 (C=O); MS
(ES+): m/z (%): 600.3 (20) [M+H]+, 622.3 (5) [M+Na]+; HRMS (ES+):
m/z: calcd for C28H37F3N3O8 [M+H]+: 600.2527; found: 600.2539.
CDCl3): d = 1.05 (s, 12H; 2î(C(CH3)2), 1.87 (tt, 3J(H,H) = 7, 7 Hz,
4H; CH2CH2NHCH2CH2), 2.37 (s, 8H; COCH2), 2.57 (s, 6H; C=CCH3),
2.76 (t, 3J(H,H) = 7 Hz, 4H; CH2NHCH2), 3.51 (m, 4H; 2îCH2NHÀ
Dde), 13.44 (brs, 2H; NHÀDde) ppm; 13C NMR (CDCl3, 100 MHz): d =
18.3 (C=CCH3), 28.6 (C(CH3)2), 29.7 (CH2CH2NHCH2CH2), 30.5
(C(CH3)2), 41.7 (CH2NHÀDde), 47.2 (CH2NHCH2), 53.2 (COCH2), 108.3
Synthesis of polyamine scaffold resin 9: Compound
8 (3 equiv,
6.43 mmol) was dissolved in CH2Cl2 (15 mL), and then DIC (3.5 equiv,
7.63 mmol) and HOBt (3.5 equiv, 7.63 mmol) were added. After 10 min,
this solution was added to aminomethyl resin (1 equiv, 2.18 mmol) and
the suspension was shaken overnight. The resulting resin was washed
with CH2Cl2, MeOH, DMF, MeOH and CH2Cl2 (3î15 mL each) and
dried under high vacuum. The resin gave a negative ninhydrin test.
(C=CCH3), 174.0 (C=CCH3), 198.4 (C=O) ppm; IR (CH2Cl2): n˜
=
1628 cmÀ1 (C=O); MS (ES+): m/z (%): 460.4 (100) [M+H]+; HRMS
(ES+): m/z: calcd for C26H42N3O4 [M+H]+: 460.3170; found: 460.3172.
Compound 5: Rf
CDCl3):
=
0.26 (CH2Cl2/MeOH 9:1); 1H NMR (400 MHz,
1.05 (s, 6H; C(CH3)2, 1.75 1.92 (m, 4H;
Solid-phase synthesis of transfection agent library: synthesis of transfec-
tion library 1
d
=
CH2CH2NHCH2CH2), 2.39 (s, 4H; COCH2), 2.59 (s, 3H; C=CCH3), 2.77
(t, 3J(H,H) = 7 Hz, 2H; CH2CH2CH2NHDde), 2.84 (t, 3J(H,H) = 6 Hz,
2H; TfaNHCH2CH2CH2), 3.52 (m, 4H; NH(CH2CH2CH2)2), 5.03 (brs,
2H; NH), 13.45 (brs, 1H; NHÀDde) ppm; 13C NMR (CDCl3, 100 MHz):
Full experimental data for all library compounds 1,2, and 3 is given in
the Supporting Information.
General procedures
Dde deprotection: Resin (1 equiv) was pre-swollen in DMF for 30 min
and filtered. Hydrazine in DMF (5% v/v) was added to this resin and the
suspension was shaken for 2 h. The resin was filtered and washed succes-
sively with CH2Cl2, MeOH, DMF, MeOH and CH2Cl2 (positive ninhydrin
test).
d
= 18.2 (C=CCH3), 27.8 (TfaNHCH2CH2), 28.6 (C(CH3)2); 29.4
(CH2CH2NHDde);
30.5
(C(CH3)2);
39.9
(TfaNHCH2);
41.6
(CH2NHDde), 47.1 (CH2CH2CH2NHDde); 48.6 (TfaNHCH2CH2CH2);
53.2 (COCH2); 108.4 (C=CCH3); 116.5 (q, 1J = 288 Hz; CF3ÀCONH),
157.6 (q, 2J
=
36 Hz, CF3ÀCOÀNH), 174.0 (C=CCH3), 198.5 (COÀ
Dde) ppm; IR (CH2Cl2): n˜ = 1717 cmÀ1 (C=O); MS (ES+): m/z (%):
392.3 (16) [M+H]+; HRMS (ES+): m/z: calcd for C18H29F3N3O3 [M+H]+:
392.2156; found: 392.2145.
Guanidation: The resulting resin was pre-swollen in THF for 30 min and
filtered. A solution of N,N’-bis(tert-butoxycarbonyl)-S-methylisothiourea
(4 equiv, 1m) in THF and a few drops of pyridine were added and the
suspension was shaken overnight. The solution was collected and the
resin was washed as above (negative ninhydrin test).
N1-1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)ethyl-N5-(4-(allyloxycarbo-
nylmethoxy)phenyl-methoxycarbonyl)-(N9-trifluoroacetyl)-norspermidine
(7): Compound 5 (1 equiv, 5.42 g, 13.85 mmol) was dissolved in DMF
(10 mL), linker 6 (1.2 equiv, 6.43 g, 16.62 mmol) was added, and the reac-
tion mixture was stirred overnight. It was then poured into KHSO4 (1m,
100 mL) and extracted with EtOAc (2î100 mL). The combined organic
layers were washed with water, dried over Na2SO4 and evaporated to
dryness. The crude product was purified on silica gel, with elution with
CH2Cl2 to CH2Cl2/MeOH (97:3), to afford the desired product 7 (7.77 g,
88%). Rf = 0.37 (CH2Cl2/MeOH 20:1); 1H NMR (400 MHz, CDCl3): d
= 0.96 (s, 6H; C(CH3)2), 1.69 (brs, 2H; TfaHNCH2CH2), 1.83 (brs, 2H;
DdeHNCH2CH2), 2.29 (s, 4H; 2îCOCH2ÀDde), 2.42 (s, 3H; C=CCH3),
3.18 3.35 (m, 8H; N(CH2CH2CH2)2), 4.59 (s, 2H; OCH2C6H4), 4.63 (m,
2H; OCH2CH=CH2), 5.02 (s, 2H; C6H4OCH2CO), 5.18 5.30 (m, 2H;
Tfa deprotection: A mixture of 1m KOH/THF/MeOH (4:3:1) was added
to the resin (pre-swollen in THF and filtered). This suspension was
shaken for 2 h. The resin was washed successively with CH2Cl2, MeOH,
DMF, MeOH and CH2Cl2 (positive ninhydrin test).
Couplingof carboxylic acid : The resin was pre-swollen for 30 min in
CH2Cl2 and filtered. A solution of carboxylic acid (4 equiv, 0.5m), DIC
(4 equiv, 0.5m) and HOBt (4 equiv, 0.5m) in CH2Cl2 was added to this
resin. The suspension was shaken for 2 h and the resin was washed as de-
scribed above (negative ninhydrin test).
Cleavage of the product from the resin: The resin was pre-swollen in
CH2Cl2 for 30 min and filtered. A cocktail of TFA/CH2Cl2/H2O/thioani-
sole (8:0.5:0.5:1) was added and the suspension was shaken for 2 h. The
resin was filtered and the solution was collected. The solvent was re-
moved under vacuum, and the crude product was re-dissolved in H2O
and extracted with CH2Cl2. The aqueous layer was evaporated under re-
duced pressure to afford the pure products in 76 100% yield (% yield
based on the loading of the aminomethyl resin and product as 4¥TFA
salts).
Synthesis of N1,N9-bis-1,1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl-
N5-(3-carboxypropanoyl)-norspermidine (13): Succinic anhydride (0.48 g,
4.74 mmol) was added to a solution of 12 (1.98 g, 4.31 mmol) in CH2Cl2
(5 mL) and the mixture was stirred for 1 h. The reaction mixture was
poured into water (200 mL) and extracted with EtOAc (2î100 mL). The
combined organic layers were dried over anhydrous Na2SO4 and evapo-
rated under reduced pressure to afford the title compound as an amor-
phous solid (2.22 g, 95%).
3
OCH2CH=CH2), 5.85 (m, 1H; OCH2CH=CH2), 6.81 (d, J(H,H) = 9 Hz,
3
2H; ArH), 7.22 (d, J(H,H) = 9 Hz, 2H; ArH), 7.93 (brs, 1H; NHÀTfa),
13.44 (brs, 1H; NHÀDde) ppm; 13C NMR (CDCl3, 100 MHz): d = 18.3
(C=CCH3), 27.3 (TfaHNCH2CH2), 28.3 (DdeHNCH2CH2), 28.6
(C(CH3)2), 30.5 (C(CH3)2), 36.4 (TfaHNCH2), 41.1 (TfaHNCH2CH2CH2),
44.3 (DdeHNCH2), 44.5 (DdeHNCH2CH2CH2), 53.2 (COCH2ÀDde), 65.7
(OCH2C6H4), 66.3 (OCH2CH=CH2), 67.8 (C6H4OCH2CO), 108.4 (C=
CCH3), 115.2 (CHarom), 115.8 (OCH2CH=CH2), 119.6 (OCH2CH=CH2),
129.8 (Carom), 130.6 (CHarom), 131.8 (Carom), 158.3 (C=O amide), 168.8 (C=
O ester), 174.1 (C=CCH3), 198.4 (C=OÀDde) ppm; IR (CH2Cl2): n˜
=
1757, 1718 cmÀ1 (C=O); MS (ES+): m/z (%): 639.9 (100) [M+H]+, 661.9
(70) [M+Na]+; HRMS (ES+): m/z: calcd for C31H40F3N3O8Na [M+Na]+:
662.2659; found: 662.2641.
N1-1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)ethyl-N5-(4-(carboxyme-
thyl)phenyl-methoxy-carbonyl))-N9-(trifluoroacetyl)-norspermidine (8):
Compound 7 (1 equiv, 6.34 g, 9.91 mmol) in CH2Cl2/THF (1:1, 30 mL
each) was purged with N2 (g) for 1 h. [Pd(PPh3)4] (0.1 equiv, 1.14 g,
0.99 mmol) and thiosalicylic acid (2 equiv, 3.06 g, 19.82 mmol) were
added in one portion, and the reaction mixture was left to stir for 2 h.
The reaction mixture was evaporated to dryness and the crude product
was purified on silica gel, with elution with EtOAc to 50% MeOH/
Rf = 0.48 (CH2Cl2/MeOH 9:1); 1H NMR (400 MHz, [D6]DMSO): d =
1.08 (s, 12H; 2îC(CH3)2ÀDde); 1.87 (m, 2H; NCH2CH2CH2NH), 1.99
(m, 2H; NHCH2CH2CH2N), 2.39 (s, 8H; COCH2), 2.50 (m, 2H;
NCOCH2), 2.56 (s, 3H; C=CÀCH3ÀDde), 2.62 (m, 5H; m, C=CÀ
CH3+CH2CO2H), 3.47 (m, 6H; CH2NCH2CH2CH2NH), 3.58 (m, 2H;
NHCH2CH2CH2N), 13.34 (brs, 1H; NH), 13.42 (brs, 1H; NH) ppm; 13C
=
0.23 (CH2Cl2/MeOH 2:1); 1H
NMR (100 MHz, [D6]DMSO):
(NCH2CH2CH2NH), 30.2
(C(CH3)2+NHCH2CH2CH2N), 32.4 (NCH2), 43.0 (NHCH2CH2CH2N),
43.4 (NCH2CH2CH2NH), 45.1 (NCH2CH2CH2NH), 46.9
d
=
20.0 (C=CÀCH3), 29.8
EtOAc, to give 8 (4.29 g, 72%). Rf
(CH2CO2H), 33.6
NMR (400 MHz, [D6]DMSO): d = 0.96 (s, 6H; C(CH3)2), 1.75 1.90 (m,
4H; CH2CH2NHCH2CH2), 2.36 (s, 4H; COCH2ÀDde), 2.55 (s, 3H; C=
CCH3); 3.20 3.40 (m, 8H; HN(CH2CH2CH2)2), 4.33 (s, 2H; OCH2C6H4),
5.07 (s, 2H; OCH2CO2H), 6.90 (d, 3J(H,H) = 9 Hz, 2H; ArH), 7.32 (d,
3J(H,H) = 9 Hz, 2H; ArH), 9.57 (brs, 1H; NHÀTfa), 13.38 (brs, 1H;
NHÀDde) ppm; 13C NMR (100 MHz, [D6]DMSO): d = 17.7 (C=CCH3),
27.3 (TfaHNCH2CH2), 28.3 (DdeHNCH2CH2+C(CH3)2), 30.2 (C(CH3)2),
37.5 (TfaHNCH2), 41.5 (TfaHNCH2CH2CH2), 45.0 (DdeHNCH2), 49.0
(DdeHNCH2CH2CH2), 52.8 (COCH2ÀDde), 66.6 (OCH2C6H4), 67.7
(OCH2CO2H), 107.4 (C=CCH3), 114.8 (CHarom), 128.5 (Carom), 129.6
(CHarom), 131.8 (Carom), 159.1 (C=O amide), 173.3 (C=CCH3+CO2H),
(NHCH2CH2CH2N), 55.1 (2îCH2ÀDde), 109.7 (C=CÀCH3), 174.0 (C=
CÀCH3), 175.5, 175.7 (NCOCH2CH2CO2H), 199.1, 199.2 (C=O) ppm; IR
(film): n˜ = 1725, 1635 cmÀ1 (C=O); MS (ES+): m/z (%): 528.4 (100)
[M+H]+; HRMS (ES+): m/z: calcd for C30H46N3O7 [M+H]+: 560.3330;
found: 560.3341.
Synthesis of compound library 2: Hydrazine in DMF (5%, v/v) (10 mL)
was added to the resin 9 (1.18 g, 0.76 mmol, pre-swollen in DMF and fil-
tered) and the suspension was shaken for 2 h. The resin was washed with
471
Chem. Eur. J. 2004, 10, 463 473
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim