A Liposaccharide Drug Delivery Agent
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1255
a Perkin-Elmer Sciex API 3000 mass spectrometer operating
in positive ion electrospray mode (ESI-MS) or an Applied
Biosystems Qstar Pulsar electrospray qtof mass spectrometer
operating in positive ion electrospray mode (HRMS), using 70%
B at 0.3 mL/min. NMR spectra were recorded on either a
Varian Gemini 300 instrument (1H, 300 MHz; 13C, 75 MHz)
or a Bruker AM 500 instrument (1H, 500 MHz; 13C, 125 MHz).
The following abbreviations were used to indicate the peak
multiplicity: s ) singlet; d ) doublet, t ) triplet; m )
multiplet, br ) broad. Proton signals were assigned using
gradient COSY45 spectra recorded on the Bruker instrument.
IR spectra were recorded on a Shimadzu FTIR-8201PC by KBr
disk (2 mg of compound in 200 mg of KBr). The purity of 11
was determined by HPLC-MS using the Perkin-Elmer Sciex
API 3000 mass spectrometer operating in positive ion electro-
spray mode linked to a HPLC system (Shimadzu SCL-10A VP
Controller, two LC-10AT VP Pumps, DGU-12A Degasser and
an Agilent 1100 Series Auto Injector) operating at a flow rate
of 0.3 mL/min. The chromatography conditions were as fol-
lows: CN, Agilent Zorbax SB-CN column (5 µm), 2.1 × 50 mm,
linear gradient of 10% B to 100% B over 10 min; ODS, Agilent
Zorbax SB-C18 column (5 µm) 2.1 × 50 mm, linear gradient
of 30% B to 100% B over 10 min. An aliquot (10 µL) of a
solution of 11 (0.1 mg/mL in 1:1 A/B) was injected and the
purity calculated as the percent area of integrated peaks in
the total ion count chromatogram (TIC) (mass range 120-1000
by 0.1 amu). The reported peaks displayed mass spectra
consistent with compound 11, a mixture of diastereomers.
72.3, 71.8, 70.1, 68.6, 20.6, 20.4, 20.4, 20.4; FT-IR 2954, 1728,
1427, 1373, 1211, 1042 cm-1
.
2-(ter t-Bu toxyca r bon yla m in o)-D,L-tetr a d eca n oic Acid
(5). Compound 5 was prepared by a slightly modified literature
procedure.23 Sodium (3.81 g, 166 mmol) was dissolved in
ethanol (100 mL) under nitrogen and diethyl acetamido
malonate (30.0 g, 138 mmol) was added, followed by 1-bromo-
dodecane (48.2 g, 193 mmol). The solution was refluxed
overnight under an atmosphere of nitrogen. Upon cooling the
mixture was poured onto crushed ice (600 mL) and the
precipitated product collected, air-dried, and then refluxed
overnight in a solution of concentrated HCl/DMF (9:1, 200 mL).
Upon cooling, the product precipitated and was collected,
washed with ice cold water, and then air-dried to afford
2-amino-D,L-tetradecanoic acid hydrochloride (37.6 g, 97%):
ESI-MS, m/z 244 [M + H]+. 2-Amino-D,L-tetradecanoic acid
hydrochloride (35.2 g, 126 mmol) was suspended in a solution
of tert-butyl alcohol/water (2:3, 750 mL) and the pH was
adjusted to 13 with sodium hydroxide (5 M). Di-tert-butyl
dicarbonate (41.2 g, 189 mmol) in tert-butyl alcohol (75 mL)
was added and the pH of the reaction maintained at 13 during
the first 3 h by addition of sodium hydroxide (5 M). The
solution was then left to stir overnight. The mixture was
diluted with water (300 mL), and solid citric acid was added
to acidify the mixture to pH 3. The mixture was extracted with
ethyl acetate (5 × 200 mL), and the combined extracts were
dried (MgSO4) and evaporated to yield a crude product which
was recrystallized from warm acetonitrile to afford the title
compound (5) (24.2 g, 56%): mp 55-56 °C, literature mp 62-
64 °C;23 ESI-MS, m/z 366 [M + Na]+, 361 [M + NH4]+, 344 [M
Acetic 1,2,3,4-Tetr a -O-a cetyl-â-D-glu cop yr a n u r on ic An -
h yd r id e (3).27 D-Glucuronic acid (6.0 g, 31 mmol) was sus-
pended in acetic anhydride (85 mL) and stirred at 0 °C in a
250 mL round-bottom flask fitted with a condenser and a
calcium chloride drying tube. Iodine (510 mg, 2.01 mmol) was
added slowly and the mixture allowed to warm to room
temperature overnight (ca. 18 h). The solvent was coevapo-
rated with toluene to give a pale, brown solid which was
washed with ether and collected in a Buchner funnel. Repeated
ether trituration afforded the title compound (3) as a white
solid (7.9 g, 71%): TLC Rf 0.65 (ethyl acetate, ceric ammonium
sulfate dip); mp 132 °C; ESI-MS, m/z 427 [M + Na]+, 422 [M
1
+ H]+, 288 [M - t-Bu]+, 244 [M - Boc]+; H NMR (300 MHz,
CDCl3) δ 7.55 (1H, br s, COOH), 5.03 (1H, d, J 8.1, CONH),
4.28 (1H, m, R-CH), 1.9-1.5 (2H, m, â-CH2), 1.43 (9H, s,
C(CH3)3), 1.24 (20H, m, 10CH2), 0.87 (3H, t, J 6.2, CH3); 13C
NMR (75 MHz, CDCl3) δ 177.6, 155.6, 80.0, 53.4, 32.4, 31.9,
29.6, 29.5, 29.4, 29.3, 29.2, 28.3, 25.3, 22.7, 14.1.
2-(ter t-Bu toxyca r bon yla m in o)-D,L-tetr a d eca n oic Acid
Ben zyl Ester (6). Carboxylic acid 5 (4.10 g, 11.9 mmol) was
dissolved in methanol (300 mL). The solution was neutralized
with a 20% solution of Cs2CO3 (ca. 30 mL, ca. 18 mmol Cs2-
CO3) and the solvent removed in vacuo. DMF (50 mL) was
added and the solvent was removed in vacuo. Addition and
evaporation of DMF (50 mL) was repeated to afford the solid
cesium salt, which was dried under reduced pressure. The
cesium salt was dissolved in DMF (150 mL) and benzyl
chloride (1.66 g, 1.51 mL, 13.1 mmol) was added. The mixture
was stirred at 70 °C for 4 h. The solvent was removed in vacuo
and the crude product taken up in ethyl acetate (200 mL) and
washed with 5% HCl (2 × 100 mL), saturated bicarbonate (2
× 100 mL), and brine (100 mL). The organic layer was dried
(MgSO4), filtered, and evaporated to afford the title compound
(6) (3.88 g, 75%) as an oil: ESI-MS, m/z 566 [M + Cs]+, 451
[M + NH4]+, 434 [M + H]+; 1H NMR (300 MHz, CDCl3) δ 7.34
(5H, m, Ar-H), 5.16 (3H, m, Ar-CH2 overlapping with
CONH), 4.34 (1H, m, R-CH), 1.9-1.5 (2H, m, â-CH2), 1.44 (9H,
s, C(CH3)3), 1.26 (20H, m, 10CH2), 0.89 (3H, t, J 10.9, CH3);
13C NMR (75 MHz, CDCl3) δ 172.7, 155.3, 135.4, 128.4, 128.2,
128.1, 79.6, 66.7, 53.4, 32.5, 31.8, 29.5, 29.4, 29.3, 29.1, 28.2,
25.1, 22.6, 14.0; HRMS calcd for [M + H]+ 434.3264, found
434.3270.
1
+ NH4]+, 345 [M - OAc]+; H NMR (500 MHz, CDCl3) δ 5.83
(1H, d, J 1,2 6.8, H-1), 5.38 (1H, t, J 9.0 H-4), 5.30 (1H, t, J 8.7,
H-3), 5.12 (1H, t, J 7.4, H-2), 4.35 (1H, d, J 4,5 9.0, H-5), 2.28
(3H, s, Ac), 2.13 (3H, s, Ac), 2.06, 2.04 (9H, 2s, 3Ac overlap-
ping); 13C NMR (125 MHz, CDCl3) δ 169.7, 169.2, 169.1, 168.5,
164.7, 162.5, 91.3, 72.9, 71.2, 70.0, 68.0, 21.9, 20.6, 20.4, 20.3;
FT-IR 2955, 1828, 1766, 1427, 1373, 1211, 1088 cm-1
.
1,2,3,4-Tetr a -O-a cetyl-â-D-glu cop yr a n u r on ic Acid (4).
Meth od A.26 D-Glucuronic acid (6.00 g, 31 mmol) was sus-
pended in acetic anhydride (85 mL) and stirred at 0 °C. Iodine
(427 mg, 1.68 mmol) was added slowly over 0.5 h and stirring
was continued at 0 °C for 2 h then a further 1 h at 25 °C. The
solution was cooled to 0 °C, dry methanol (30 mL, 740 mmol)
was added dropwise, and then the solution was allowed to stir
for 18 h at 25 °C. The solution was concentrated and taken
up in DCM (100 mL), washed with sodium thiosulfate (1 M
solution, 2 × 100 mL), dried (MgSO4), filtered, and evaporated
in vacuo to afford a pale, brown gum (8.3 g). The crude product
was taken up in a mixture of ether, hexane, and chloroform
(1:1:1, 120 mL) and crystallized upon concentration of the
mixture. The crystalline product was triturated with ether to
afford the title compound (4) (6.17 g, 55%) as a white powder.
Meth od B.27,28 Compound 3 (2.04 g, 5.05 mmol) was
dissolved in a mixture of THF (40 mL) and water (10 mL) and
stirred overnight (ca. 18 h). The solvent was removed in vacuo
to afford peracetylated glucuronic acid as a white powder in
quantitative yield: TLC Rf 0.41 (MeOH/DCM 1:3, ceric am-
monium sulfate dip); mp 149 °C; ESI-MS, m/z 385 [M + Na]+,
380 [M + NH4]+, 303 [M - OAc]+; 1H NMR (500 MHz, CDCl3)
δ 8.57 (1H, br s, COOH), 5.77 (1H, d, J 1,2 7.6, H-1), 5.28 (2H,
m, H-3 and H-4 overlapping), 5.10 (1H, t, J 8.0, H-2), 4.23 (1H,
d, J 4,5 9.0, H-5), 2.08, 2.01, 2.01, 2.00 (12H, 4s, 4Ac); 13C NMR
(125 MHz, CDCl3) δ 170.1, 169.8, 169.7, 169.3, 169.0, 91.2,
1-D,L-(Ben zyloxyca r bon yl)tr id ecyla m m on iu m Tr iflu o-
r oa ceta te (7). Benzyl ester 6 (471 mg, 1.09 mmol) was
dissolved in DCM/TFA (20 mL, 1:1) and stirred for 1 h. The
solvent was removed in vacuo and the residue was dissolved
in acetonitrile/water (1:1) and lyophilized to afford the title
1
compound (7) (463 mg, 95%): ESI-MS, m/z 334 [M + H]+; H
NMR (300 MHz, CDCl3) δ 7.34 (5H, m, Ar-H), 5.24 (1H, d, J
12.1, Ar-CH), 5.13 (1H, d, J 12.1, Ar-CH), 4.01 (1H, m, R-CH),
1.91 (2H, m, â-CH2), 1.23 (20H, m, 10CH2), 0.90 (3H, t, J 6.4,
CH3); 13C NMR (75 MHz, CDCl3) δ 169.7, 134.5, 128.7, 128.6,
128.5, 68.1, 53.1, 31.9, 30.5, 29.6, 29.6, 29.5, 29.3, 29.1, 29.0,
24.6, 22.7, 14.1; HRMS calcd for [M + H]+ 334.2740, found
334.2731.