O. Bortolini et al. / European Journal of Medicinal Chemistry 52 (2012) 221e229
227
in 10 mL of dry benzene and a drop of DMF at 0 ꢀC. The reaction
1.02e2.30 (m, 27H), 3.36 (m, 1H, 3
b
-CH), 3.80 (br s, 1H, 7
b
-CH), 4.20
mixture was stirred at 60 ꢀC for 5 h and then evaporated to dryness
under vacuum. Dry benzene (5 mL) was added and the syrup
evaporated in twice to complete remove thionyl chloride. The bile
acid chloride was dissolved in 10 mL of dry THF under nitrogen and
tris(trimethylsilyl) phosphite (3.3 mL, 10 mmol) was added drop-
wise, maintaining the temperature 25e35 ꢀC. The reaction mixture
was then stirred at room temperature for 3 h. After evaporation of
volatile fractions under vacuum, the crude product was hydrolyzed
with methanol (20 mL) for 2 days at room temperature. After
evaporation of solvent under vacuum, the obtained compound was
washed with diethyl ether and a few drops of methanol and
recovered by filtration to give bile acid-derived bisphosphonates
3ae5a, 11 and 12 as white or pale yellow solids.
(m, 1H, 23-CH). 13C NMR (75 MHz, CD3OD)
d: 12.2, 18.7, 21.8, 23.4,
24.6, 29.4, 31.3, 33.8, 34.0, 35.8, 36.2, 36.5, 39.2, 40.4, 40.7, 41.1, 43.1,
43.8, 51.6, 58.2, 69.1, 72.3, 72.8, 77.2 (t, J ¼ 108.3 Hz). 31P NMR
(121.5 MHz, CD3OD)
C24 H43 O10 P2 553.2336, found 553.2351.
d
: 11.59. HRMS of [M ꢁ H]ꢁ ions: calculated for
5.2. General procedure for the synthesis of 24-hydroxy-24,24-
bisphosphonobile acid disodium salts 3be5b and bile acids
disodium salts 6be8b
4 mmol of the bisphosphonobile acids 3ae5a, dissolved in 20 mL
of EtOH/H2O (1:1 v/v), were treated with an aqueous solution of
NaOH (8 mmol). The mixture was stirred for 1 h at r.t. The reaction
mixture was lyophilized giving the expected disodium salts in
5.1.2.1. 3
phonic acid 3a. Yield 97%; mp 200e203 ꢀC. IR:
2939 (CeH), 1189 (P]O),1011 (PeO); 1H NMR (300 MHz, CD3OD)
0.74 (s, 3H, 18-CH3), 0.94 (s, 3H, 19-CH3), 1.04 (d, J ¼ 6.8 Hz, 3H, 21-
CH3), 1.15e2.35 (m, 28H), 3.39 (m, 1H, 3 -CH), 3.82 (br s, 1H, 7
CH), 3.98 (br s, 1H, 12 : 11.6, 16.5,
-CH). 13C NMR (75 MHz, CD3OD)
a,7
a,12
a
-Trihydroxy-5
b
-cholan-24-hydroxyl-24,24-bisphos-
almost quantitative yield. 3b: 31P NMR (121.5 MHz, D2O)
d: 17.80 (d,
n
(cmꢁ1) 3300 (OeH),
J ¼ 32.8 Hz), 18.20 (d, J ¼ 32.8 Hz). 4b: 31P NMR (121.5 MHz, D2O)
d:
d:
19.58 (d, J ¼ 31.5 Hz), 19.90 (d, J ¼ 31.5 Hz). 5b: 31P NMR (161.9 MHz,
D2O)
d
: 18.81 (d, J ¼ 34 Hz), 18.46 (d, J ¼ 34 Hz). An almost identical
b
b
-
procedure was followed for the preparation of the simple bile acids
b
d
sodium salts 6be8b used in biological investigations.
21.7, 22.8, 26.4, 27.2, 28.1, 28.8, 29.7, 30.3, 34.4, 34.5, 35.1, 36.3, 39.0,
39.6, 41.5, 41.7, 46.0, 46.8, 67.8, 71.5, 72.8, 73.3 (t, J ¼ 108 Hz). 31P NMR
5.3. Hydroxyapatite affinity measurements
(121.5 MHz, CD3OD)
d
: 21.67. HRMS of [M ꢁ H]ꢁ ions: calculated for
C24 H43 O10 P2 553.2336, found 553.2333.
In a 15 mL cap vial, magnetically stirred at 25 ꢀC, approximately
20 mg of clodronate 1, 20 mg of neridronate 2 and 40 mg of che-
nodeoxycholic acid-containing hydroxyl-bisphosphonate sodium
salt 4b were dissolved in 10 mL of bidistilled water adjusting the pH
to 7.4. HAP was added increasing the amount from 0 mg/mL to
10 mg/mL (Table 1). One mL of this suspension was take-up at 0, 3,
24, 48, 72 and 96 h. These samples were centrifuged (5000 rpm for
50), transferred into a 5 mm NMR tube containing, every time, the
same sealed capillary filled with a solution of H3PO4 in D2O
(secondary standard) [24a] and analyzed via 31P NMR.
5.1.2.2. 3
nic acid 4a. Yield 95%; mp 150e153 ꢀC. IR:
2955 (CeH), 1255 (P]O), 1065 (PeO); 1H NMR (300 MHz,
CD3OD) : 0.74 (s, 3H, 18-CH3), 0.95 (s, 3H, 19-CH3), 1.03 (d,
J ¼ 6.8 Hz, 3H, 21-CH3), 1.1e2.38 (m, 28H), 3.38 (m, 1H, 3 -CH), 3.83
(br s, 1H, 7 : 10.8, 14.0, 17.7, 20.4,
-CH). 13C NMR (75 MHz, CD3OD)
a,7
a
-Dihydroxy-5
b
-cholan-24-hydroxy-24, 24-bisphospho-
n
(cmꢁ1) 3400 (OeH),
d
b
b
d
22.0, 23.2, 27.8, 28.8, 29.9, 30.4, 32.6, 34.5, 34.8, 35.1, 36.3, 39.0,
39.4, 39.7, 42.2, 50.1, 55.9, 67.7, 71.4, 73.1 (t, J ¼ 110,2 Hz). 31P NMR
(121.5 MHz, D2O)
C24 H43 O9 P2 537.2387, found 537.2390.
d
: 20.83. HRMS of [M ꢁ H]ꢁ ions: calculated for
5.4. Cytotoxicity screening
5.1.2.3. 3
nic acid 5a. Yield 96%; mp 182e185 ꢀC. IR:
2955 (CeH), 1255 (P]O), 1065 (PeO). 1H NMR (300 MHz, CD3OD)
: 0.73 (s, 3H, 18-CH3), 0.99 (s, 3H, 19-CH3), 1.02 (d, J ¼ 6.8 Hz, 3H,
21-CH3), 1.1e2.25 (m, 28H), 3.50 (m, 2H, 3 -, 7
-CH). 13C NMR
(75 MHz, CD3OD) : 11.3, 17.8, 21.0, 22.57, 26.6, 28.2, 28.8, 29.6, 30.3,
a
,7
b
-Dihydroxy-5
b
-cholan-24-hydroxy-24, 24-bisphospho-
5.4.1. Cell cultures
n
(cmꢁ1) 3400 (OeH),
The L929 cells (mouse fibroblasts, ATCC CCLl, NCTC clone 929)
were maintained in complete minimum essential medium (MEM)
with Earle’s salts, containing 10% fetal calf serum (FCS), 20 mM
d
b
a
glutamine, 1% non-essential amino acids penicillin (100 U mLꢁ1
)
d
and streptomycin (l00 m
g/mLꢁ1) at 37 ꢀC in a humidified atmo-
33.8, 34.7, 36.2, 36.6, 37.2, 39.3, 40.2, 42.6, 43.1, 43.3, 55.2, 56.2, 70.6,
sphere of 5% CO2 95% air. Confluent cells were detached by trypsin
and 0.8 ꢃ 103 cells in 0.2 mL of culture medium were seeded in flat-
bottomed 96 microplate wells (Costar, Cambridge, MA, USA). The
cells were incubated for 24 h at 37 ꢀC in a humidified atmosphere of
5% CO2 95% air in order to obtain a complete cell adhesion. Twenty-
four hours after cell seeding, the medium was aspirated from the
monolayer and replaced with 0.2 mL of complete MEM containing
serial dilution (from 10ꢁ4 to 10ꢁ10 M) of the sodium salts of bile
acid-derived hydroxyl-bisphosphonates (3b, 4b, 5b) and ner-
idronate 2. Stock solutions (10ꢁ2 M) were obtained dissolving
compounds in phosphate buffer saline (PBS) at pH 7.4. As controls,
cultures were added with 0.2 mL of complete MEM (negative
control), and methanol fixed cells provided the positive controls.
Sample and control cultures were tested in triplicate. The plates
were incubated for 24, 48 and 72 h.
70.7, 73.1 (t, J ¼ 109.5 Hz). 31P NMR (121.5 MHz, CD3OD)
d: 21.56 (d,
J ¼ 41.6 Hz), 21.59 (d, J ¼ 41.6 Hz). HRMS of [M ꢁ H]ꢁ ions: calcu-
lated for C24 H43 O9 P2 537.2387, found 537.2399.
5.1.2.4. 3
nic acid (bisphosphonate of hyodeoxycholic acid) 11. Yield 96%; mp
207e210 ꢀC. IR: (cmꢁ1) 3400 (OeH), 2950 (CeH), 1255 (P]O),
1068 (PeO). 1H NMR (300 MHz, CD3OD)
: 0.70 (s, 3H, 18-CH3), 0.93
(s, 3H, 19-CH3), 0.98 (d, J ¼ 6.8 Hz, 3H, 21-CH3), 1.1e2.25 (m, 28H),
3.51 (m, 1H, 3 -CH), 4.01 (m, 1H, 6
-CH). 13C NMR (75 MHz, CD3OD)
: 12.5, 19.0, 21.9, 24.1, 25.3, 29.2, 29.9, 30.2, 31.1, 31.6, 35.5, 36.2,
a,6a-Dihydroxy-5b-cholan-24-hydroxy-24, 24-bisphospho-
n
d
b
b
d
36.8, 36.9, 37.6, 41.3, 41.4, 43.9, 49.8, 57.4, 57.68, 68.7, 72.3, 74.5 (t,
J ¼ 108.9 Hz). 31P NMR (121.5 MHz, CD3OD)
: 21.40 (d, J ¼ 40.6 Hz),
d
21.71 (d, J ¼ 40.6 Hz). HRMS of [M ꢁ H]ꢁ ions: calculated for C24 H43
O9 P2 537.2387, found 537.2394.
5.4.2. Neutral-red uptake assay
5.1.2.5. 3
bisphosphonic acid (bis- phosphonate of phocaecholic acid) 12. Yield
93%; mp 212e215 ꢀC. IR: (cmꢁ1) 3410 (OeH), 2950 (CeH), 1255
(P]O), 1068 (PeO). 1H NMR (300 MHz, CD3OD)
: 0.72 (s, 3H, 18-
CH3), 0.94 (s, 3H, 19-CH3), 1.01 (d, J ¼ 6.8 Hz, 3H, 21-CH3),
a
,7
a
,23(R)-Trihydroxy-5
b
-cholan-24-hydroxy-24,24-
At the end points, the mediumwas aspirated from the monolayer
and replaced with 0.1 mL per well of 0.4% neutral-red diluted 1:80 in
complete MEM. After incubation for 2 h at 37 ꢀC the dye-containing
mediumwasdiscarded, thewells werewashed twicewith saline and
0.1 mL of extractant solution (50% ethanol in acetic acid, 1%) were
n
d