Y. Wang et al. / Bioorg. Med. Chem. 13 (2005) 5592–5599
5597
if the enhanced therapeutic efficacy of DHA–HCPT is
due to a prolonged half-life in vivo and/or a selective
accumulation by tumors. We will perform experiments
to answer these and other questions related to the mech-
anism of action of DHA–HCPT and will report the
results in due course.
CH@CH), 3.40 (s, 2H, CH2), 3.29 (br s, 4H, 2· CH2),
2.83–2.75 (m, 10H, 5· CH2), 2.35 (br s, 4H, 2· CH2),
2.27 (t, 4H, J = 4.80 Hz, 2· CH2), 2.04–2.00 (m, 2H,
CH2), 1.38 (s, 9H, 3· CH3), 0.91 (t, 3H, J = 7.20 Hz,
CH3). Anal. Calcd for (C38H55N3O3Æ4.1H2O) C, 67.55;
H, 9.42; N, 6.21. Found: C, 67.91; H, 9.42; N, 5.86.
4.1.3. 10-[4-[[4-(cis-4,7,10,13,16,19-Docosahexenoyl)ami-
no]benzyl]-1-piperazino]carbonyloxy-camptothecin. Com-
pound 5 (0.85 g, 1.4 mmol) was dissolved in ethyl
acetate (10 mL), and saturated anhydrous HCl in ethyl
acetate (7 mL) was added. The reaction mixture was
stirred at room temperature for 30 min. The precipitate
was filtered, and the solid was washed with ethyl ether to
afford 6 (0.68 g, 90% yield). The product was used for
the next reaction without further purification.
4. Experimental
4.1. Chemistry
Melting points were measured using a Mel-Temp II
instrument and are uncorrected. H NMR spectra were
1
recorded at ambient temperature on an NT-400 spec-
trometer. Mass spectra (electrospray ionization, EI)
were recorded using a Micromass Q-Tof 1 mass spec-
trometer. Atlantic Microlab, Inc., Norcross, GA, per-
formed the elemental analyses, and the results were
within 0.4% of the theoretical values unless otherwise
noted. Analytical thin-layer chromatography was
performed on silica-coated plastic plates (silica gel 60
F-254, Merck) and was visualized under UV light.
Preparative separations were performed by flash
chromatography on silica gel (Merck, 70–230 mesh).
HCPT (1.0 g, 2.76 mmol) in tetrahydrofuran (THF,
200 mL) was treated with 4-nitrophenyl chloroformate,
7 (2.16 g, 10.8 mmol), and triethylamine (4.0 mL), and
the reaction was allowed to proceed at room tempera-
ture for 1 h. The product was extracted with ethyl ace-
tate (1 L) and the organic phase was washed with
water (500 mL · 3). The solution was dried with sodium
sulfate and the solvent was removed in vacuo. The prod-
uct was purified by column chromatography, eluting
with ethyl acetate to afford 8 as a yellow solid (1.32 g,
91% yield). The product was used for the next reaction
without further purification. Carbonate 8 (0.66 g,
1.25 mmol) in THF (60 mL) was treated with amine 6
(0.85 g, 1.58 mmol) in the presence of triethylamine
(2.4 mL), and the reaction mixture was stirred at room
temperature for 2 h. The solvent was removed in vacuo,
and the product was purified by column chromatogra-
phy, eluting with ethyl acetate and methanol (15:1,
v/v) to afford DHA–HCPT as a yellow solid (0.8 g,
72% yield), mp 196–199 ꢁC. MS m/z (M++H): 893.0.
The product was dissolved in a mixture of THF and eth-
yl acetate, and anhydrous HCl in ethyl acetate was then
added. The precipitate was filtered and the solid was
dried to afford DHA–HCPT as a HCl salt, mp 230 ꢁC
4.1.1. tert-Butyl 4-(4-nitrobenzyl)-1-piperazinecarboxyl-
ate (3). To a solution of tert-butyl 1-piperazinecarboxyl-
ate, 1 (0.74 g, 4 mmol), and 4-nitrobenzyl bromide, 2
(0.86 g, 4 mmol), in N,N-dimethylformamide (DMF,
5 mL) was added potassium carbonate (3 g), and the
reaction mixture was stirred at room temperature for
4 h. The product was extracted with ethyl acetate
(50 mL), and the organic phase was washed with water
(20 mL · 3). The solution was dried with sodium sulfate
and the solvent was removed in vacuo. The product was
purified by column chromatography, eluting with ethyl
acetate to afford 3 as a solid (1.22 g, 95% yield), mp
99–100 ꢁC. 1H NMR (DMSO-d6): 8.21–8.19 (d, 2H,
J = 8.8 Hz, ArH), 7.61–7.59 (d, 2H, J = 8.0 Hz, ArH),
3.63 (s, 2H, CH2), 3.34–3.30 (m, 4H, partially obscured,
2· CH2), 2.35–2.32 (m, 4H, 2· CH2), 1.39 (s, 9H, 3·
CH3). Anal. (C16H23N3O4) C, H, N.
1
(dec). H NMR (DMSO-d6): 9.89 (s, 1H, NH), 8.66–
7.26 (m, 9H, ArH), 6.53 (s, 1H, OH), 5.43–5.30 (m,
16H), 3.68 (s, 1H, CH2), 3.50 (br s, 4H, 2· CH2),
3.21–3.18 (m, 2H, CH3CH2), 2.84–2.76 (m, 10H), 2.48
(br s, 4H, 2· CH2), 2.35 (br s, 4H, 2· CH2), 2.05–2.01
(m, 2H, CH2), 1.91–1.84 (m, 2H, CH2), 1.29 (t, 1H,
J = 8.0 Hz, CH3), 0.93–0.87 (m, 6H, 2· CH3). Anal.
Calcd for (C54H61N5O7ÆHClÆ5H2O): C, 63.67; H, 7.13;
N, 6.87. Found: C, 63.88; H, 6.03; N, 6.71.
4.1.2. tert-Butyl 4-[4-(cis-4,7,10,13,16,19-docosahexe-
noyl)amino]benzyl-1-piperazinecarboxylate (5). To com-
pound 3 (0.68 g, 2.1 mmol) in ethyl acetate (100 mL)
was added 10% Pd/C (0.1 g), and the reaction mixture
was hydrogenated for 2 h under a hydrogen pressure
of 60 lb/in.2. The product was filtered, and the solvent
was removed in vacuo to afford amine 4 (0.6 g, 97%
yield). Without further purification, to amine 4 (0.6 g,
2.06 mmol) dissolved in acetonitrile (90 mL) was added
cis-4,7,10,13,16,19-docosahexenoic acid (DHA, 0.67 g,
2 mmol), 2-(1-benzotriazol-1-yl)-1,1,3,3-tetramethyluro-
nium hexafluorophosphate (HBTU, 0.94 g, 2.5 mmol),
and N,N-diisopropylethylamine (2.1 mL). The reaction
mixture was stirred overnight. The solvent was removed
in vacuo, and the product was purified by column chro-
matography, eluting with ethyl acetate to afford 5 as an
4.1.4. 10-[4-(1-Piperidino)-1-piperidino]carbonyloxycam-
ptothecin. Carbonate 8 (0.53 g, 1 mmol) in THF (55 mL)
was treated with 4-piperidinopiperidine (0.17 g, 1 mmol)
and triethylamine (1 mL), and the reaction mixture was
stirred at room temperature for 2 h. The solvent was re-
moved in vacuo, and the product was purified by col-
umn chromatography, eluting with dichloromethane
and methanol (5:1, v/v) to afford PP–HCPT as a yellow
solid (0.41 g, 73% yield). The product was dissolved in a
mixture of THF and ethyl acetate, and anhydrous HCl
in ethyl acetate was added. The precipitate was filtered,
and the solid was dried to afford PP–HCPT as a HCl
1
oil (0.91 g, 73% yield). H NMR (DMSO-d6): 9.84 (s,
1H, NH), 7.53–7.51 (d, 2H, J = 8.80 Hz, ArH), 7.19–
7.17 (d, 2H, J = 8.40 Hz, ArH), 5.35–5.31 (m, 12H,