3210 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Ruiz et al.
5-(2-{2-[2-(11,17-Dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,-
12,13,14,15,16,17-dodecahydro-3H-cyclopenta[r]phenanthren-17-
yl)-2-oxoethoxycarbonyl]methyl}phenylazo)-2-hydroxybenzoic Acid
(5a). To a solution of tert-butyl ester 15a 2 (0.1 g, 0.000 155 mol)
in dichloromethane (1 mL), trifluoroacetic acid (1 mL) was added.
The mixture was left at room temperature for 4 h. After completion
the solvents and trifluoroacetic acid were evaporated using a stream
of nitrogen to afford the product as a orange oil. The crude product
was purified by flash chromatography using dichloromethane/ethyl
acetate (50:50) to yield the product as orange crystals (0.078 g,
78%): mp 177 ( 1 °C. MS: 665.2503 calculated mass 665.2475.
1H NMR δ (CDCl3): 8.48 (1H, s), 8.10 (1H, d, J ) 9.04 Hz), 7.75
(1H, d, J ) 7.52 Hz), 7.46 (4H, m), 7.08 (1H, d, J ) 9 Hz),
6.24 (1H, d, J ) 10 Hz), 6.00 (1H, s), 5.03 (2H, d, J ) 17.56 Hz),
4.35 (1H, s), 4.31 (2H, d, J ) 8.52 Hz), 2.68-0.8 (19H). 13C NMR
δ (MeOD): 205.18, 187.15, 172.99, 171.82, 164.11, 158.38, 156.35,
149.62, 144.93,133.49, 133.36, 130.90, 129.97, 127.39, 127.0,
126.20, 125.88, 120.57, 117.01, 114.64, 88.75, 68.91, 67.95, 55.40,
50.91, 44.19, 38.19, 36.09, 33.70, 32.75, 31.31, 27.66, 22.95, 19.72,
15.36. Contained ∼5% ethyl acetate with signals at 1.26 (t), 2.05
(s), 4.12 (q). This could not be removed by heating to 150 °C and/
or vacuum.
of IBD with reduced steroid side effects. The design could be
applied to target other hydroxyl-bearing therapeutics to the
colon.
Experimental Section
Chemistry. Melting points were obtained using a Stuart melting
point SMP11 melting point apparatus. Spectra were obtained using
a Perkin-Elmer 205 FT infrared Paragon 1000 spectrometer. Band
positions are given in cm-1. Solid samples were obtained by KBr
1
disk; oils were analyzed as neat films on NaCl plates. H and 13C
spectra were recorded at 27 °C on a Bruker Advance II 600 MHz
spectrometer (600.13 MHz for 1H, 150.91 MHz for 13C) and Bruker
DPX 400 MHz FT NMR spectrometer (400.13 MHz for 1H, 100.16
MHz for 13C), in either CDCl3 or CD3OD (tetramethylsilane as
internal standard). For CDCl3, 1H NMR spectra were assigned
relative to the TMS peak at 0.00 δ, and 13C NMR spectra were
assigned relative to the middle CDCl3 triplet at 77.00 ppm. For
1
CD3OD, H and 13C NMR spectra were assigned relative to the
center peaks of the CD3OD multiplets at 3.30 δ and 49.00 ppm,
respectively. Coupling constants were reported in hertz (Hz). For
1H NMR assignments, chemical shifts are reported: shift values
(number of protons, description of absorption (s ) singlet, d )
doublet, t ) triplet, q ) quartet, m ) multiplet), coupling constant(s)
where applicable, proton assignment). High resolution mass spec-
trometry (HRMS) was performed on a Micromass mass spectro-
photometer (EI mode) at the Department of Chemistry, Trinity
College. HPLC was performed on a reverse phase 250 mm × 4.6
mm Waters Spherisorb ODS-2, 5 µm column using a Waters
Alliance 2695 chromatograph equipped with an autosampler,
column oven, and dual wavelength detector. The flow rate was 1
mL/min with a mobile phase consisting of 40% phosphate buffer,
pH 2.5, and 60% acetonitrile at time 0 and grading to 85%
acetonitrile at 4 min. Injection volume was 20 µL, and areas were
determined at 254 nm. The isocratic HPLC method was aqueous
phosphate buffer solution, pH 2.5, 40% and acetonitrile 60%. Flow
rate was 1 mL/min. Another HPLC method using gradient mobile
phase allowed better resolution at lower concentration and better
peak shapes: column, Xbridge 48 4.6 mm × 250 mm, 5 µm. The
mobile phase was (A) 0.5% NH4HCOO- pH 10.0 or (B) acetoni-
trile, with gradient 10-90% B over 20 min. Flow rate was 1.5
mL/min, and the column temperature was room temperature. Flash
chromatography was performed on Merck Kieselgel 60 particle size
0.040-0.063 mm. Thin layer chromatography (TLC) was performed
on silica gel Merck F-254 plates. Compounds were visually detected
by absorbance at 254 nm and/or vanillin staining. Test compounds
20, 5a, and 5b were >98% by HPLC.
5-(2-{2-[2-(11,17-Dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,-
12,13,14,15,16,17-dodecahydro-3H-cyclopenta[r]phenanthren-17-
yl)-2-oxoethoxycarbonyl]ethyl}phenylazo)-2-hydroxybenzoic Acid
tert-Butyl Ester (16b). To a solution of azo carrier (15b) (1.21 g,
3.3 mmol), prednisolone (3) (1 equiv, 1.18 g, 3.3 mmol), and PPh3
(3 equiv, 2.59 g, 9.9 mmol) in dry tetrahydrofuran (50 mL), DIAD
(3 equiv, 1.9 mL, 9.9 mmol) was added dropwise over 12 min after
the reaction temperature reached 40 °C. The mixture was stirred at
40 °C for an hour and then left overnight at room temperature under
a nitrogen atmosphere. TLC (DCM) analysis showed completion.
The solvent was removed under reduced pressure to afford the
product as an orange oil. This was flash-columned using DCM/
ethyl acetate (50:50). The triphenylphosphine oxide was removed
using a second flash chromatography [hexane (200 mL), hexane/
ethyl acetate (70:30)] to yield the product as orange crystals (0.92
g, 40%): mp 117 ( 1 °C. 1H NMR δ (CDCl3): 8.40 (1H, d, J ) 2
Hz), 8.10 (1H, dd, J ) 8.52 and 2 Hz), 7.68 (1H, d, J ) 8.04 Hz),
7.44 (3H, m), 7.33 (1H, t, J ) 6.52 Hz), 7.09 (1H, d, J ) 8.52
Hz), 6.27 (1H, d, J ) 10.04 Hz), 6.01 (1H, s), 5.00 (1H, d, J )
17.56 Hz), 4.88 (1H, d, J ) 17.56 Hz), 4.39 (1H, s), 3.49 (2H, t,
J ) 9.52), 2.82 (2H, t, J ) 8.04 Hz), 2.66-0.9 (19H), 1.69 (9H,
s). 13C NMR δ (CDCl3): 204.8, 186.5, 172.5, 170.7, 169, 163.7,
156.7, 149.5, 145.1, 139.3, 130.5, 130.0, 129.26, 127.0, 126.9,
125.7, 121.6, 118.9, 114.9, 113.3, 89.20, 83.2, 69.6, 67.7, 54.9,
50.8, 47.2, 43.8, 38.9, 35.5, 33.8, 33.5, 31.6, 30.7, 27.7, 26.7, 23.4,
20.5, 16.4. HRMS: found (M - Na)+ ) 735.3283, required (M -
Na)+ ) 735.3258.
5-(2-{2-[2-(11,17-Dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,-
12,13,14,15,16,17-dodecahydro-3H-cyclopenta[r]phenanthren-17-
yl)-2-oxoethoxycarbonyl]methyl}phenylazo)-2-hydroxybenzoic Acid
tert-Butyl Ester (16a). Prednisolone 3 (1.1 equiv, 1.18 g, 0.0013
mol) and triphenylphosphine (3 equiv, 0.88 g, 0.0033 mol) in dry
THF (50 mL) were added to a solution of 15a (0.40 g, 0.0011 mol)
followed by DIAD (3 equiv, 0.66 mL, 0.0033 mol) dropwise over
12 min at 40 °C. The mixture was stirred at 40 °C for an hour and
left overnight at room temperature under nitrogen atmosphere.
Solvents were removed under reduced pressure to afford the product
as an orange oil. This was purified by flash chromatography using
dichloromethane/ethyl acetate (50:50). After this column the
triphenylphosphine oxide was removed and a second flash column
was performed to yield the product as orange crystals (0.33 g, 43%):
5-(2-{2-[2-(11,17-Dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,-
12,13,14,15,16,17-dodecahydro-3H-cyclopenta[r]phenanthren-17-
yl)-2-oxoethoxycarbonyl]ethyl}phenylazo)-2-hydroxybenzoic Acid
(5b). To a solution of 16b (0.9 g, 1.26 mmol) in DCM (3 mL) was
added trifluoroacetic acid (3 mL), and the mixture was left at room
temperature for 4 h. The solvents and trifluoroacetic acid were
evaporated using nitrogen to afford the product as an orange oil.
The crude product was purified by flash chromatography using
dichloromethane/ethyl acetate (50:50), (100 mL), ethyl acetate (100
mL), and acetone/ethyl acetate (70:30) to yield the product as orange
1
crystals (0.7 g, 84%): mp 177 ( 1 °C. H NMR δ (MeOD): 8.44
(1H, s), 8.07 (1H, d, J ) 9.03 Hz), 7.65 (1H, d, J ) 7.53 Hz), 7.43
(3H, m), 7.31 (1H, t, J ) 7.53 Hz), 7.07 (1H, d, J ) 9.04 Hz),
6.26 (1H, d, J ) 10.04 Hz), 5.99 (1H, s), 4.88 (2H, d, J ) 17.56
Hz), 4.36 (1H, s), 3.44 (2H, t, J ) 7.53 Hz), 2.80 (2H, t, J ) 8.03
Hz), 2.64-0.89 (19H). 13C NMR δ (MeOD): 205.4, 187.2, 173.1,
172.1, 164.0, 161.3, 158.4, 149.6, 144.9, 139.1, 129.8, 128.6, 126.5,
125.9, 124.5, 120.6, 117.6, 116.8, 114.5, 88.7, 68.9, 67.5, 55.4,
50.9, 44.2, 38.4, 35.2, 33.7, 32.8, 31.3, 30.8, 26.3, 22.9, 19.7, 15.45.
MS: found (M - H)+ ) 657.2814, requires (M - H)+ ) 657.2812;
found (M)- ) 655.2662, requires (M)- ) 655.2656. Contained
1
mp 125 ( 1 °C. H NMR δ (CDCl3): 11.49 (1H, s), 8.44 (1H, s),
8.05 (1H, d, J ) 9 Hz), 7.75 (1H, d, J ) 7.52 Hz), 7.40 (3H, m),
7.24 (1H, d, J ) 10.04), 7.06 (1H, d, J ) 9 Hz), 6.25 (1H, s), 5.98
(1H, s), 4.27 (2H, s), 2.68-0.84 (19H), 1.67 (9H, s). 13C NMR δ
(CDCl3): 204.40, 186.26, 171.36, 170.10, 169.09, 163.85, 156.25,
149.53, 145.07, 133.25, 130.98, 130.53, 129.89, 127.85, 127.17,
125.52, 121.79, 118.14, 115.14, 113.26, 89.17, 83.30, 69.51, 67.98,
54.86, 50.86, 47.26, 43.68, 38.85, 36.44, 33.92, 33.58, 31.56, 30.71,
27.78, 23.36, 20.56, 16.37.