Radiosynthesis of Combretastatin A-1
Journal of Natural Products, 2009, Vol. 72, No. 3 419
(1H, d, J ) 12.0 Hz, H-5′′), 6.45 (3H, m, H-2′, H-6′, H-2), 6.43 (1H,
d, J ) 12.0 Hz, H-1), 4.68 (1H, sept, J ) 6.5 Hz, C-2′′ OCH(CH3)2),
4.42 (1H, sept, J ) 6.5 Hz, C-3′′ OCH(CH3)2), 3.78 (3H, s, C-4′′ OCH3),
3.58 (6H, s, C-3′, C-5′ OCH3), 1.29 (6H, d, J ) 6.5 Hz, C-2′′ OCH-
(CH3)2), 1.28 (6H, J ) 6.5 Hz, C-3′′ OCH(CH3)2), 0.99 (9H, s, C-4′
OSi(CH3)2C(CH3)3), 0.11 (6H, s, OSi(CH3)2C(CH3)3); 13C NMR
(CDCl3, 125 MHz) δ 153.6 (C, C-4′′), 151.1 (C, C-3′, C-5′), 150.4 (C,
C-2′′), 140.3 (C, C-3′′), 133.3 (C,C-1′), 129.8 (C, C-4′), 129.2 (CH,
C-1), 125.6 (CH, C-2), 125.4 (CH, C-6′′), 124.8 (C, C-1′′), 106.3 (CH,
C-5′′), 106.1 (CH, C-2′, C-6′), 75.1 (CH, C-3′′ OCH(CH3)2), 74.8
(CH, C-2′′ OCH(CH3)2), 55.8 (CH3, C-4′′ OCH3), 55.4 (CH3, C-3′, C-5′
OCH3), 25.8 (CH3, C-4′ OSi(CH3)2C(CH3)3), 22.5 (CH3, C-2′′ OCH-
(CH3)2), 22.4 (CH3, C-3′′ OCH(CH3)2), 18.7 (C, C-4′ OSi(CH3)2C-
(CH3)3), -4.7 (CH3, C-4′ OSi(CH3)2C(CH3)3); HREIMS m/z 516.2936
δ 6.76 (1H, d, J ) 8.6 Hz, H-6′′), 6.59 (1H, d, J ) 12.2 Hz, H-2), 6.54
(3H, m, H-1, H-2′, H-6′), 6.38 (1H, d, J ) 8.8 Hz, H-5′′), 5.40 (2H, s,
C-2′′, C-3′′ OH), 3.86 (3H, s, C-4′′ OCH3), 3.83 (3H, s, C-4′ OCH3),
3.67 (6H, s, C-3′, C-5′ OCH3); 13C NMR (CDCl3, 125 MHz) δ 152.8
(C, C-3′, C-5′), 146.3 (C, C-4′′), 141.6 (C, C-2′′), 137.3 (C, C-4′), 132.6
(C, C-3′′), 132.5 (C, C-1′), 130.3 (CH, C-1), 124.0 (CH, C-2), 120.3
(CH, C-6′′), 117.8 (C, C-1′′), 105.9 (CH, C-2′, C-6′), 102.9 (C, C-5′),
60.9 (CH3, C-4′ OCH3), 56.2 (CH3, C-4′′ OCH3), 55.8 (CH3, C-3′, C-5′
OCH3); HPLC retention time 21.39 min.
(Z)-1-[3′,4′,5′-Trimethoxyphenyl]-2-[2′′,3′′-di[([bis[(benzyl)oxy-
]]phosphoryl)oxy]-4′′-methoxyphenyl]ethene (16).18,23 N-Chlorosuc-
cinimide (0.430 g, 3.24 mmol) was dissolved in anhydrous CH3CN
(10 mL). The reaction mixture was then heated to 40 °C and stirred at
this temperature for 5 min. The heat source was removed, and dibenzyl
phosphite (DBP) (0.71 mL, 3.24 mmol) was added dropwise. The
reaction mixture was then stirred for 3 h at rt.
1
(calcd for C29H44O6Si, 516.2907); E-isomer 2: H NMR (CDCl3, 500
MHz) δ 7.29 (1H, d, J ) 8.6 Hz, H-6′′), 7.27 (1H, d, J ) 16.5 Hz,
H-2), 6.87 (1H, d, J ) 16.5 Hz, H-1), 6.71 (2H, s, H-2′, H-6′), 6.68
(1H, d, J ) 8.8 Hz, H-5′′), 4.60 (1H, sept, J ) 6.0 Hz, C-2′′
OCH(CH3)2), 4.46 (1H, sept, J ) 6.0 Hz, C-3′′ OCH(CH3)2), 3.85 (3H,
s, C-4′′ OCH3), 3.83 (6H, s, C-3′, C-5′ OCH3), 1.30 (12H, d, J ) 6.0
Hz, C-2′′, C-3′′ OCH(CH3)2), 1.02 (9H, s, C-4′ OSi(CH3)2C(CH3)3),
0.14 (6H, s, C-4′ OSi(CH3)2C(CH3)3); 13C NMR (CDCl3, 125 MHz) δ
153.7 (C, C-4′′), 151.7 (C, C-3′, C-5′), 150.0 (C, C-2′′), 140.4 (C, C-3′′),
134.1 (C, C-1′), 131.0 (C, C-4′), 127.3 (CH, C-1), 125.8 (CH, C-2),
122.4 (CH, C-6′′), 119.8 (C, C-1′′), 107.3 (C, C-5′′), 103.6 (CH, C-2′,
C-6′), 75.5 (CH, C-2′′ OCH(CH3)2), 75.1 (CH, C-3′′ OCH(CH3)2), 55.9
(CH3, C-4′′ OCH3), 55.7 (CH3, C-3′, C-5′ OCH3), 25.8 (CH3, C-4′
OSi(CH3)2C(CH3)3), 22.6 (CH3, C-2′′ OCH(CH3)2), 22.5 (CH3, C-3′′
OCH(CH3)2), 18.7 (C, C-4′ OSi(CH3)2C(CH3)3), -4.6 (CH3, C-4′
OSi(CH3)2C(CH3)3); HREIMS m/z 516.2893 (calcd for C29H44O6Si,
516.2907).
In a separate 100 mL dry round-bottom flask, equipped with a stir
bar, was charged CA1 (3) (360 mg, 1.080 mmol) followed by anhydrous
CH3CN (10 mL) and DMAP (13 mg, 0.10 mmol). The temperature of
the reaction mixture was maintained between 10 and 20 °C, and
anhydrous Et3N (0.45 mL, 3.24 mmol) was added. The reaction mixture
was then cooled to 0 °C, and the dibenzyl chlorophosphate solution
was added slowly over a period of 5 to 10 min. The brown-colored
reaction mixture was then warmed to rt and stirred for 16 h. The solvent
was evaporated completely under reduced pressure using a rotary
evaporator, followed by the addition of toluene (∼15 mL). The solvent
(toluene) was evaporated under reduced pressure, and additional toluene
(15 mL) was added. The precipitated succinimide byproduct was filtered
and washed with more toluene. The combined filtrates were washed
with 0.5 M KH2PO4 (2 × 10 mL), followed by 0.5 M NaOH (2 × 5
mL), and finally with brine. The organic phase was dried over anhydrous
Na2SO4, filtered, and concentrated to dryness. The crude reaction
mixture was purified by flash column chromatography (silica gel, 40:
60 EtOAc-hexanes) to obtain the tetrabenzyl phosphate derivative of
CA1 16 (0.39 g, 0.46 mmol, 42% yield) as an off-white oil, Rf 0.22
(Z)-1-[3′,4′,5′-Trimethoxyphenyl]-2-[2′′,3′′-di[(isopropyl)oxy]-4′′-
methoxyphenyl]ethene (15). The Z-isomer of cold CA1 precursor 1
(1.40 g, 2.71 mmol) was dissolved in anhydrous CH3CN (20 mL), and
the solution was cooled to 0 °C. CH3I (0.67 mL, 10.8 mmol) was added
and the reaction mixture stirred for 10 min at 0 °C. To this solution
was added tetrabutylammonium fluoride (TBAF) (2.98 mL, 2.98 mmol),
and the resultant deep yellow colored reaction mixture was stirred for
10 min at 0 °C. H2O (10 mL) was added, and the product was extracted
in EtOAc (3 × 50 mL). The combined organic phases were washed
with brine, dried over anhydrous Na2SO4, and filtered. White crystals
of tetrabutylammonium hydroxide byproduct precipitated. These crystals
were filtered and washed with additional ethyl acetate. The combined
filtrates were then evaporated under reduced pressure to obtain the crude
product, which was subjected to flash column chromatography (silica
gel, 4:96 EtOAc-hexanes) to afford product 15 (1.01 g, 2.42 mmol,
89% yield) as a colorless oil, Rf 0.26 (90:10 hexanes-EtOAc); 1H NMR
(CDCl3, 500 MHz) δ 6.94 (1H, d, J ) 8.6 Hz, H-6′′), 6.64 (1H, d, J
) 12.3 Hz, H-2), 6.49 (3H, m, H-2′, H-6′, H-5′′), 6.43 (1H, d, J )
12.3 Hz, H-1), 4.70 (1H, sept, J ) 6.2 Hz, C-2′′ OCH(CH3)2), 4.43
(1H, sept, J ) 6.2 Hz, C-3′′ OCH(CH3)2), 3.82 (3H, s, C-4′′ OCH3),
3.79 (3H, s, C-4′ OCH3), 3.64 (6H, s, C-3′, C-5′ OCH3), 1.29 (12H, d,
J ) 6.2 Hz, C-2′′, C-3′′ OCH(CH3)2); 13C NMR (CDCl3, 125 MHz) δ
153.7 (C, C-4′′), 152.6 (C, C-3′, C-5′), 150.3 (C, C-2′′), 140.3 (C, C-4′),
136.8 (C, C-3′′), 132.8 (C, C-1′), 128.7 (CH, C-1), 126.6 (CH, C-2),
125.1 (C, C-1′′), 124.7 (CH, C-6′′), 106.4 (CH, C-5′′), 106.0 (C, C-2′,
C-6′), 75.0 (CH, C-2′′ OCH(CH3)2), 74.8 (CH, C-3′′ OCH(CH3)2), 60.8
(CH3, C-4′ OCH3), 55.8 (CH3, C-4′′ OCH3), 55.7(CH3, C-3′, C-5′
OCH3), 22.5 (CH3, C-2′′ OCH(CH3)2), 22.4 (CH3, C-3′′ OCH(CH3)2);
anal. C 69.25%, H 7.78%, calcd for C24H32O6, C 69.21%, H 7.74%;
HPLC retention time 19.73 min.
1
(EtOAc-hexanes, 50:50); H NMR (CDCl3, 300 MHz) δ 7.24 (20H,
m, C-2′′, C-3′′ OP(O)(OCH2C6H5)2), 7.00 (1H, d, J ) 8.7 Hz, H-6′′),
6.67 (1H, d, J ) 8.5 Hz, H-5′′), 6.64 (1H, d, J ) 12.0 Hz, H-2), 6.51
(1H, d, J ) 12.0 Hz, H-1), 6.46 (2H, s, H-2′, H-6′), 5.09 (8H, m, C-2′′,
C-3′′ OP(O)(OCH2C6H5)2), 3.79 (3H, s, C-4′′ OCH3), 3.76 (3H, s, C-4′
OCH3), 3.62 (6H, s, C-3′, C-5′ OCH3); 13C NMR (CDCl3, 75 MHz) δ
152.8 (C, C-3′, C-5′), 151.6 (C, C-4′′), 137.2 (C, C-2′′), 135.9, 135.8
(C, C-2′′, C-3′′ OP(O)(OCH2C6H5)2), 135.7 (C, C-4′), 135.6 (C, C-3′′),
132.0 (C, C-1′), 131.6 (CH, C-1), 128.4, 128.3, 128.0, 127.8 (CH, C-2′′,
C-3′′ OP(O)(OCH2C6H5)2), 126.9 (CH, C-2), 124.6 (CH, C-6′′), 124.4
(C, C-1′′), 109.3 (CH, C-5′′), 106.2 (CH, C-2′, C-6′), 70.0, 69.9, 69.8,
69.6 (CH2, C-2′′, C-3′′ OP(O)(OCH2C6H5)2), 60.8 (CH3, C-4′ OCH3),
56.4 (CH3, C-4′′ OCH3), 55.9 (CH3, C-3′, C-5′ OCH3); 31P NMR
(CDCl3, 122 MHz) δ -5.3, -5.4; HPLC retention time 38.13 min.
(Z)-1-[3′,4′,5′-Trimethoxyphenyl]-2-[2′′,3′′-di[(monopotassium)-
phosphate]-4′′-methoxyphenyl]ethene (4).23 The tetrabenzyl phosphate
derivative of CA1 16 (0.32 g, 0.38 mmol) was dissolved in anhydrous
CH3CN (10 mL), and the reaction contents were cooled to -10 °C.
Trimethlysilyl bromide (TMSBr) (freshly distilled over CaH, 0.25 mL,
1.89 mmol) was added dropwise to the reaction mixture. The reaction
mixture was stirred for 1.5 h at -10 °C to afford the tetra-TMS-
phosphate ester derivative of CA1, which was added dropwise to a
well-stirred solution of KOMe (260 mg, 3.78 mmol) in dry MeOH
(4.5 mL) at -10 °C. The reaction mixture was gradually allowed to
reach rt and then stirred for additional 15 min at rt. The solvents were
evaporated under reduced pressure (the temperature of the H2O bath
was maintained below 35 °C) to obtain a dry off-white-colored powder,
which was dissolved in deionized H2O (2 mL). The pH of the resultant
solution was then carefully titrated to 4.75 to 4.85 using 1 M HCl
(subsequently switched to 0.1 M HCl once the pH of the reaction
mixture reached ∼5.5) with good stirring. The reaction mixture was
then filtered, and to the filtrate was added anhydrous EtOH (7 mL).
CA1P 4 precipitated out as a white solid. The reaction mixture was
stirred for 5 min. The solid separated was filtered, washed with
anhydrous EtOH (2-4 mL), and dried (filter funnel, 15 to 20 min) to
(Z)-1-[3′,4′,5′-Trimethoxyphenyl]-2-[2′′,3′′-dihydroxy-4′′-methox-
yphenyl]ethene (Z-CA1, 3).4,18 The Z-isomer 15 (1.01 g, 2.42 mmol)
was dissolved in anhydrous CH2Cl2 (20 mL), and the solution cooled
to 0 °C. TiCl4 (1.16 mL, 10.06 mmol) was added to the reaction mixture,
and the dark brown colored reaction mixture was stirred for 40 min at
0 °C. The reaction mixture was quenched with H2O and extracted with
CH2Cl2 (2 × 50 mL). The combined organic phases were rinsed with
brine and dried over Na2SO4. Removal of the solvent under reduced
pressure followed by purification using flash chromatography (silica
gel capped with Florisil, 40:60 EtOAc-hexanes) afforded pure Z-CA1
3 as a pale yellow oil, which was crystallized with hexanes-EtOAc
(50:50), yielding Z-CA1 3 (0.36 g, 1.08 mmol, 45% yield) as tan-colored
crystals, Rf 0.15 (EtOAc-hexanes, 40:60); 1H NMR (CDCl3, 500 MHz)
1
yield CA1P 4 (0.10 g, 0.16 mmol, 45% yield): H NMR (D2O, 300
MHz) δ 6.87 (1H, d, J ) 8.7 Hz, H-6′′), 6.74 (1H, d, J ) 11.9 Hz,
H-2), 6.68 (1H, d, J ) 8.7 Hz, H-5′′), 6.67 (2H, s, H-2′, H-6′), 6.64
(1H, d, J ) 11.9 Hz, H-1), 3.83 (3H, s, C-4′′ OCH3), 3.74 (3H, s, C-4′