Chlorambucil Anticancer Ether Lipid Prodrugs
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3413
7.5 Hz, 2H), 2.36 (t, J ) 7.5 Hz, 2H), 1.90 (p, J ) 7.5 Hz, 2H),
1.54 (m, 2H), 1.32-1.26 (m, 30H), 0.88 (t, J ) 6.9 Hz, 3H). 13C
NMR (75 MHz, 4:1 CDCl3/CD3OD) δ 174.1, 144.9, 130.8, 130.1
(2C), 112.6 (2C), 72.3 (d, J ) 8.6 Hz), 72.1, 69.6, 66.9, 64.5 (d, J
) 5.2 Hz), 59.4 (d, J ) 5.2 Hz), 54.5, 54.4, 54.4, 53.9 (2C), 40.9
(2C), 34.3, 34.0, 32.3, 30.1, 30.0, 29.9, 29.9, 29.8, 27.3, 26.4, 23.1,
14.3. IR (neat) 2923, 2852, 2366, 1734, 1518, 1247, 1088, 750
cm-1. m/z (M + Na+) 817.44.
(S)-(2,3-Di-O-tert-butyldimethylsilyl)glyceryl 2-Cyanoethyl-N,N-
diisopropylphosphoramidite (11). Alcohol 10 (904 mg, 2.82 mmol)
and diisopropylethylamine (1.0 mL, 5.92 mmol) were dissolved in
anhydrous CH2Cl2 (10 mL) under an atmosphere of N2. 2-Cyano-
ethyl-N,N-diisopropylchlorophosphoramidite (1.0 g, 4.22 mmol) was
added dropwise, and the mixture was stirred at 20 °C for 1.5 h,
after which EtOAc (20 mL) and saturated NaHCO3 (50 mL) were
added and the organic layer was isolated by extraction with EtOAc
(2 × 50 mL). The combined organic phases were concentrated in
vacuo, and the residue was purified by column chromatography
(EtOAc) to give 1352 mg (92%) of 11 (two diastereoisomers, 1:1)
as a colorless oil. Rf ) 1.0 (EtOAc). 1H NMR (300 MHz, CDCl3):
δ 3.87-3.46 (m, 5H), 2.67-2.61 (m, 2H), 1.20-1.17 (m, 12H),
0.90 (s, 9H), 0.89 (s, 9H), 0.09-0.06 (m, 12H). 13C NMR (75 MHz,
CDCl3): δ 117.7, 73.3, 65.0, 58.6, 43.1 (2C), 26.1 (3C), 26.0 (3C),
24.7 (4C), 20.5, 18.5, 18.3, -4.4, -4.5, -5.2, -5.3. 31P NMR (202
MHz, CDCl3): δ 149.0, 148.5. IR (neat): 2958, 2929, 2883, 2857
cm-1. m/z (M + Na+) 543.32.
(CH2Cl2/MeOH/H2O 65:25:4. 1H NMR (500 MHz, CDCl3/CD3OD
3:1): δ 6.96 (d, J ) 8.7 Hz, 2H), 6.53 (d, J ) 8.7 Hz, 2H), 5.08 (q,
J ) 5.4 Hz, 1H), 3.82-3.75 (m, 2H), 3.70-3.58 (m, 3H),
3.54-3.51 (m, 4H), 3.46-3.36 (m, 8H), 3.29-3.22 (m, 2H), 2.45
(t, J ) 7.7 Hz, 2H), 2.24 (J ) 7.7 Hz, 2H), 1.77 (q, J ) 7.4 Hz,
2H), 1.42 (m, 2H), 1.15 (s, 30H), 0.78 (s, 18H), -0.01 (s, 6H),
-0.05 (s, 6H). 13C NMR (75 MHz, CDCl3/CD3OD 3:1): δ 173.2,
144.1, 130.2, 129.4, 111.8, 72.4 (d, J ) 9.6 Hz), 71.5, 71.5 (d, J )
7.8 Hz), 68.8, 66.6 (d, J ) 5.5 Hz), 64.6, 63.6 (d, J ) 5.4 Hz),
53.3, 40.2, 33.6, 33.3, 31.6, 29.4, 29.4, 29.3, 29.2, 29.1, 26.5, 25.7,
25.6, 25.5, 22.4, 18.0, 17.8, 13.8, -4.9, -5.0, -5.7, -5.7. 31P NMR
(202 MHz, CDCl3/CD3OD 3:1): δ -2.00. IR (neat): 3448, 2926,
2854, 1735, 1617, 1519, 1464, 1360, 1252, 1108 cm-1. m/z (M +
Na+) 1034.57.
1-O-Hexadecyl-2-(4-(4-(bis(2-chloroethyl)amino)phenyl)butanoyl)-
sn-glycero-3-phospho-(S)-glycerol (2a). Compound 12a (0.42 g, 0.43
mmol) was dissolved in MeCN (9 mL) and cooled to 0 °C. Then
40% aqueous HF (1 mL) was added, and the mixture was allowed
to reach 20 °C while being stirred vigorously for 2 h. The reaction
mixture was then poured into saturated aqueous NaHCO3 (20 mL)
and extracted with CH2Cl2 (3 × 10 mL) and EtOAc (10 mL). The
combined organic extracts were dried over Na2SO4 and concentrated
in vacuo and the residue was purified by column chromatography
(CH2Cl2/MeOH/H2O 65:25:4) to afford 2a (0.19 g, 59%). Rf ) 0.56
(CH2Cl2/MeOH/H2O 65:25:4). 1H NMR (500 MHz, CDCl3:CD3OD
3:1): δ 6.88 (d, J ) 8.6 Hz, 2H), 6.45 (d, J ) 8.6 Hz, 2H), 4.98 (q,
J ) 5 Hz, 1H), 3.84-3.71 (m, 3H), 3.60 (q, J ) 5 Hz, 1H),
3.54-3.51 (m, 4H), 3.46-3.39 (m, 8H), 3.30-3.20 (m, 2H), 2.37
(t, J ) 7.5 Hz, 2H), 2.17 (t, J ) 7.6 Hz, 2H), 1.71 (q, J ) 7.3 Hz,
2H), 1.35 (t, J ) 6.6 Hz, 2H), 1.07 (s, 26H), 0.69 (t, J ) 6.8 Hz,
3H). 13C NMR (75 MHz, CDCl3/CD3OD 3:1): δ 173.2, 144.0,
129.9, 129.2, 111.7, 71.4 (d, J ) 5.4 Hz), 71.3, 70.4 (d, J ) 4.8
Hz), 68.7, 66.1 (d, J ) 4.9 Hz), 63.8 (d, J ) 4.8), 61.8, 53.1, 40.1,
33.5, 33.4, 33.2, 33.0, 31.5, 29.3, 29.2, 29.1, 29.1, 28.9, 26.4, 25.6,
22.2, 13.5. 31P NMR (202 MHz, CDCl3/CD3OD 3:1): δ -0.99. IR
(neat): 3345, 2923, 2853, 1732, 1616, 1519, 1466, 1356, 1248, 1115,
1002 cm-1. m/z (M + H+) 778.36.
1-O-Octadecyl-2-(4-(4-(bis(2-chloroethyl)amino)phenyl)butanoyl)-
sn-glycero-3-phospho-(S)-glycerol (2b). The synthesis was per-
formed as for 2a, starting from 12b (334 mg, 0.33 mmol) and
affording 2b (83 mg, 32%). Rf ) 0.56 (CH2Cl2/MeOH/H2O 65:
25:4). 1H NMR (500 MHz, CDCl3/CD3OD 3:1): δ 6.89 (d, J ) 8.5
Hz, 2H), 6.46 (d, J ) 8.2 Hz, 2H), 4.99 (q, J ) 5 Hz, 1H),
3.84-3.72 (m, 3H), 3.60 (q, J ) 5 Hz, 1H), 3.53 (m, 4H),
3.47-3.41 (m, 8H), 3.30-3.20 (m, 2H), 2.37 (t, J ) 7.5 Hz, 2H),
2.17 (t, J ) 7.5 Hz, 2H), 1.71 (q, J ) 7.5 Hz, 2H), 1.35 (t, J ) 6.5
Hz, 2H), 1.07 (s, 30H), 0.70 (t, J ) 6.8 Hz, 3H). 13C NMR (75
MHz, CDCl3/CD3OD 3:1): δ 173.3, 144.0, 130.0, 129.2, 111.8,
71.5, 71.4 (d, J ) 6.3 Hz), 70.5 (d, J ) 4.8 Hz), 68.7, 66.1, 63.8
(d, J ) 5.6 Hz), 61.9, 53.2, 40.1, 33.5, 33.2, 31.5, 29.3, 29.3, 29.2,
29.1, 29.0, 26.4, 25.6, 22.3, 13.6. 31P NMR (202 MHz, CDCl3/
CD3OD 3:1): δ -0.31. IR (neat): 3332, 2923, 2853, 1733, 1616,
1519, 1466, 1355, 1236, 1115, 1002 cm-1. m/z (M + Na+) 828.37.
1-O-Hexadecyl-2-(4-(4-(bis-(2-chloroethyl)amino)phenyl)butanoyl)-
sn-glycero-3-(2-cyanoethylphospho)-(S)-2,3-di-O-tert-butyldimeth-
ylsilylglycerol (12a). To a solution of 6a (0.79 g, 1.8 mmol) and 11
(1.3 g, 2.5 mmol) in CH2Cl2 (10 mL) was added molecular sieves
(3 Å). After the mixture was stirred for 30 min, 1H-tetrazole in
acetonitrile (5.5 mL, 0.45 M, 2.5 mmol) was added and the mixture
stirred for another 30 min before 5.5 M tert-butyl hydroperoxide
in decane (0.50 mL, 2.8 mmol) was added and the mixture stirred
for 1 h before being concentrated in vacuo. The residue was purified
by column chromatography (EtOAc/heptane 1:1) to yield 1.63 g
of an oil. 31P NMR showed two signals at -0.66 and -0.82 in the
1:1 ratio set by the amidite. The product was subsequently treated
with DDQ (0.45 g, 2.2 mmol) in CH2Cl2 (10 mL) and water (0.6
mL) for 2 h before Na2SO3 was added and the mixture diluted with
CH2Cl2. The mixture was filtered and concentrated in vacuo before
purification by column chromatography (CH2Cl2, then EtOAc/
heptane 1:1) afforded 1.16 g. 31P NMR showed two signals at -0.10
and -0.18 ppm. The deprotected compound was dissolved in
CH2Cl2 (12 mL) together with chlorambucil (0.70 g, 2.3 mmol),
and EDCI (0.59 g, 3.1 mmol) and DMAP (0.38 g, 3.1 mmol) were
added. After being stirred for 2 h, the mixture was concentrated in
vacuo and purified by column chromatography (EtOAc/heptane 1:1)
and the resulting 1.36 g product dissolved in CH2Cl2 (10 mL) and
treated with DBU (0.20 mL, 1.3 mmol) for 30 min. The reaction
mixture was concentrated in vacuo and the residue was purified by
column chromatography (CH2Cl2/MeOH 9:1) to give the phospho-
1
lipid (0.96 g, 54%). Rf ) 0.73 (CH2Cl2/MeOH/H2O 65:25:4). H
NMR (500 MHz, CDCl3/CD3OD 3:1): δ 6.90 (d, J ) 8.5 Hz, 2H),
6.47 (d, J ) 8.5 Hz, 2H), 5.00 (q, J ) 5 Hz, 1H), 4.08 (m, 1H),
3.83-3.76 (m, 2H), 3.71-3.59 (m, 3H), 3.55-3.53 (m,
4H), 3.47-3.38 (m, 8H), 3.30-3.21(m, 2H), 2.38 (t, J ) 7.5 Hz,
2H), 2.19 (t, J ) 7.5 Hz, 2H), 1.73 (q, J ) 7.3 Hz, 2H), 1.36 (m,
2H), 1.09 (s, 26 H), 0.72 (s, 18H), -0.06 (s, 6H), -0.11 (s, 6H).
13C NMR (75 MHz, CDCl3/CD3OD 3:1): δ 173.1, 144.0, 130.0,
129.3, 111.8, 72.4 (d, J ) 9.5 Hz), 71.5, 71.4 (d, J ) 6.3 Hz),
68.8, 66.5 (d, J ) 5.6 Hz), 64.6, 63.5 (d, J ) 5.2 Hz), 53.2, 40.1,
33.5, 33.3, 31.5, 29.3, 29.3, 29.2, 29.1, 29.0, 26.4, 25.6, 25.5, 25.4,
22.3, 17.9, 17.7, 13.6, -5.1, -5.1, -5.8, -5.8. 31P NMR (202 MHz,
CDCl3/CD3OD 3:1): δ -1.82. IR (neat): 3450, 2922, 1732, 1616,
1519, 1463, 1360, 1252, 1102 cm-1. m/z (M + Na+) 1006.53.
1-O-Octadecyl-2-(4-(4-(bis-(2-chloroethyl)amino)phenyl)butanoyl)-
sn-glycero-3-(2-cyanoethylphospho)-(S)-2,3-di-O-tert-butyldimeth-
ylsilylglycerol (12b). The synthesis was performed as for 12a,
starting from 6b (650 mg, 1.40 mmol) and 11 (1.0 g, 1.9 mmol),
affording 810 mg (57%) of 12b as a colorless oil. Rf ) 0.73
Acknowledgment. We thank the Danish Council for Stra-
tegic Research (NABIIT Program) for financial support.
MEMPHYSsCenter for Biomembrane Physics is supported by
the Danish National Research Foundation.
Supporting Information Available: Analytical and spectral data
for all synthesized compounds, experimental procedures for the
synthesis of 5a, 5b, 6a, 6b, 7a, 7b, 9, and 10, prodrugs stability
data, Mosher ester analysis data of 10, further MALDI-TOF MS
and HPLC data for sPLA2 degradation experiments, and alkylating
assay data. This material is available free of charge via the Internet
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