L. Gil et al. / Bioorg. Med. Chem. 7 (1999) 901±919
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acid 14 (210 mg, 48%). H NMR (CDCl3): d 9.65 (1H,
stirred at room temperature for 4 h and diluted with
brine, extracted with EtOAc (4Â10 mL). The extracts
were combined, concentrated in vacuo and the residue
was puri®ed by circular chromatography (EtOAc/hex-
br. s), 7.98 (2H, d, J=8.1 Hz), 7.26 (20H, s), 7.13 (2H,
d, J=8.1 Hz), 4.95 (8H, m), 3.31 (2H, td, J=16.7,
6.4 Hz), 2.82 (1H, tt, J=24.0, 6.4 Hz). 13C NMR
(CDCl3): d 170.0, 144.6 (t, J=7.6 Hz), 135.9, 135.8,
130.1, 129.0, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 68.3
(dd, J=19.6, 6.5 Hz), 39.4 (t, J=133.2 Hz), 31.2 (br. s).
ane=80/20) to furnish the corresponding acid (110 mg,
1
94%). 17: IR (neat) 3680±3200, 3000±2840, 1740 cm
.
1H NMR (CDCl3): d 7.79 (2H, d, J=8.0 Hz), 7.25 (20H,
m), 7.13 (2H, d, J=8.0 Hz), 5.68 (2H, 2m), 5.37 (3H,
m), 4.92 (8H, m), 4.07 (1H, m), 3.26 (2H, td, J=16.6,
6.2 Hz), 2.84 (1H, tt, J=24.0, 6.2 Hz), 2.68 (1H, br. dd,
J=18.4, 7.4 Hz), 2.41±1.22 (19H, m), 1.09 (9H, s), 0.86
(3H, m); 13C NMR (CDCl3): d 214.8, 176.7, 165.2, 144.4
(t, J=7.6 Hz), 135.8 (dd, J=9.0, 6.9 Hz), 131.5, 129.3,
128.6, 128.5, 128.2, 74.9, 72.0, 68.3, 54.5, 53.2, 39.2 (t,
J=132.9 Hz), 46.2, 34.5, 33.4, 31.6, 31.1, 26.6, 25.1,
24.9, 24.6, 22.5, 14.0. MS (FAB, NaI) m/z (relative
intensity): 1027 (M+Na+, 31), 671 (100). HRMS
(FAB, NaI): calcd for C57H67P2O12 (MH+): 1005.4108;
found: 1005.4106.
PGE2-TBDPS ester bisphosphonate conjugate 17.
Freshly distilled oxalyl chloride (1.5 equiv) was added
to a solution of the acid 14 (177 ng, 0.264 mmol) and
DMF (10 mL, 0.132 mmol) in dichloromethane (1 mL) at
0 ꢀC. After stirring 10 min the volatiles were evaporated
under high vacuum and the residue acid chloride 15
used directly. IR (neat): 1770, 1740 cm 1. Compound 15
was dissolved in dichloromethane (100 mL), cooled to
20 ꢀC and pyridine 50 mL was added followed by
PGE2-TBDPS in pyridine (350 mL) and dichloro-
methane (100 mL). After stirring 10 min at 10 ꢀC and
0.5 h at 0 ꢀC. A solution of saturated ammonium chlo-
ride was added and the mixture was extracted with
EtOAc acetate (3Â5 mL). The organic extracts were
washed with brine, dried over magnesium sulfate and
evaporated to dryness. The residue was puri®ed by
HPLC (ZORBAX, 21±5Â25 cm, 20 mL/min EtOAc:
hexane, (80/20) as eluant). The ®rst fraction corre-
sponded to the C-11 regioisomer (16b) (66.4 mg; 18%).
The second fraction was the desired C-15 regioisomer
16a (156.4 mg, 42%). Ethyl acetate elution provided the
bis-acylated product (20 mg) and recovered PGE2-
TBDPS (55.3 mg, 31.4%). C-15 isomer 16b: IR (neat)
3400, 3060±2860, 1740±1720 cm 1. 1H NMR (CDCl3): d
7.78 (2H, d, J=8.2 Hz), 7.66, 7.64 (4H, 2d, J=7.9 Hz),
7.43 7.18 (26H, m), 7.11 (2H, d, J=8.2 Hz), 5.67, 5.39
(4H, 2m), 5.28 (1H, m), 4.93 (8H, m), 4.05 (1H, m), 3.28
(2H, td, J=16.6, 6.4 Hz), 2.72 (1H, tt, J=24.0, 6.4 Hz),
2.71 (1H, m), 2.44 (2H, t, J=7.5 Hz), 2.41±1.22 (19H,
PGE2 bisphosphonate conjugate 18. In a 3 mL boron-
silicate test tube a solution of the acid 17 (36 mg,
0.036 mmol) in EtOH (420 mL) and EtOAc (80 mL)
under nitrogen was immersed in a 20 ꢀC water bath. To
the solution was added Pd/C (5% Pd content, 5.7 mg,
0.036 mmol) followed by 1,4-cyclohexadiene (136 mL,
1.44 mmoL) and the resultant mixture was stirred at
room temperature for 4.5 h and transferred to a 1.5 mL
plastic Eppendorf vial and centrifuged. The supernatant
was separated and the residue rinsed twice with ethanol
(1 mL). The supernatants were combined, neutralized
with 0.5 N ammonium acetate (144 mL, 0.072 mmol)
and concentrated. The crude product (ꢁ90% pure by
1H NMR) could be puri®ed in two ways: (1) by C18
mini-columns (6 mL Varian Bond Elute) using water
(5 mL), 30% MeOH/water (5 mL), 60% MeOH/water
(5 mL) and MeOH (5 mL). The desired product eluted
with the 30% MeOH/water fraction. The fraction was
lyophilized to aord the compound 18 (21 mg, 76%); as
a light-yellow ¯uy powder. (2) by HPLC using
Waters PrepPak mbondapak1 C18 column (25Â100 mm,
10 mL/min, gradient composition: 0.5 N NH4OAc/
CH3CN=90/10 to 70/30 in 10 min and 70/30 for 10 min,
UV detection: 254 nM). The fractions thus obtained
were lyophilized to give the desired product, 18. 1H
NMR (D2O): d 7.82 (2H, d, J=7.9 Hz), 7.37 (2H, d,
J=7.9 Hz), 5.65 (2H, m), 5.37 (2H, m), 5.19 (1H, m),
4.05 (1H, m), 3.03 (2H, m), 2.65 (1H, dd, J=18.8,
7.5 Hz), 2.42±1.16 (21H, m), 0.71 (3H, m). 13C NMR
(D2O): d 222.1, 180.0, 169.5, 133.0, 130.4, 130.3, 77.2,
72.1, 53.2, 42.1, 40.4, 35.1, 34.6, 34.5, 32.4, 32.1, 31.8,
25.2, 25.1, 22.9, 14.3. MS (FAB, NaI) m/z (relative
intensity): 667 (M+Na+, 4), 645 (2), 399 (4), 311 (11),
293 (14), 177 (60), 136 (100). HRMS (FAB, NaI): calcd
for C29H43P2O12 (MH+): 645.2231; found: 645.2230.
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m), 1.09 (9H, s), 0.86 (3H, m). H NMR (CD3COCD3):
d 7.85 (2H, d, J=8.3 Hz), 7.73, 7.72 (4H, 2d, J=7.7 Hz),
7.48±7.26 (28H, m), 5.87 and 5.78 (2H, 2dd, J=15.5,
7.8 Hz and J=15.5, 6.5 Hz, respectively), 5.52, 5.40 (3H,
m), 5.03 (8H, m), 4.31 (1H, d, J=5.1 Hz), 4.16 (1H, m),
3.33 (2H, td, J=16.4, 6.6 Hz), 3.05 (1H, tt, J=23.7,
6.6 Hz), 2.67 1.20 (19H, m), 1.10 (9H, s), 0.87 (3H, m);
13C NMR (CD3COD3): d 214.3, 173.0, 165.9, 145.7 (t,
J=7.8 Hz), 137.5 (dd, J=9.0, 6.9 Hz), 136.0, 130.1,
129.2, 75.5, 72.4, 68.5 (m), 54.8, 53.9, 39.8 (t, J=
131.1 Hz), 47.5, 35.9, 35.4, 33.9, 32.3, 27.3, 25.8, 25.7,
23.2, 19.6, 14.3. MS (FAB, NaI) m/z (relative intensity):
1265 (M+Na+, 24), 1243 (13), 1192 (17), 819 (13), 761
(16), 671 (100), 581 (62). HRMS (FAB, NaI): calcd for
C73H85P2SiO12 (MH+) 1243.5286; found 1243.5287. C-11
isomer 16b: IR (neat) 3400, 3060±2860, 1740±1720 cm
1
.
1H NMR (CDCl3): d 7.75 (2H, d, J=8.3Hz), 7.64 (4H,
m), 7.46±7.18 (26H, m), 7.11 (2H, d, J=8.3 Hz), 5.62±
5.21 (5H, 4m), 5.28 (1H, m), 4.93 (8H, m), 4.06 (1H, d,
J=6.6 Hz), 4.00 (1H, m), 3.27 (2H, td, J=16.7, 6.5 Hz),
3.00 (1H, dd, J=18.3, 6.8 Hz), 2.79±1.11 (19H, m), 1.09
(9H, s), 0.81 (3H, br. t, J=6.7 Hz).
Preparation of 4-mercaptobutane-1,1-diphosphonic acid
(21)
3-acetylthiopropyliodide (19). To a solution of 1,3-diio-
dopropane (10 g, 33.8 mmol) in 10 mL of anhydrous
DMF at 0 ꢀC under nitrogen was added, via a cannula
over 15 min, a solution of potassium thioacetate (1.3 g,
PGE2 bisphosphonate ester conjugate 17. A solution
of PGE2-TBDPS ester conjugate 16 (145mg,
0.117 mmol) in THF (4 mL) and 0.2 N HCl (1 mL) was