Novel C-17-Heteroaryl Steroidal CYP17 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2981
was stirred at ca. 80 °C under Ar for 45 min. After cooling to
room temperature, the reaction mixture was poured onto ice-
cold water (250 mL) and the resulting precipitate was filtered,
washed with water and dried to give a crude dirty white solid.
Purification by FCC [petroleum ether/EtOAc, (4:1)] first gave
3â-acetoxy-17-(benzo-2H-1,2,3-triazol-2-yl)-16-formylandrosta-
5,16-diene (7a, 0.3 g, 9.8%) as minor product; mp 248-250
°C; IR (CHCl3) 3023, 2945, 2358, 1725, 1657, 1600, 1375, 1257,
of hydrazine sulfate (7.9 mg, 0.061 mmol) in 0.25 mL of water.
The mixture was stirred at room temperature for 12 h and
then poured into ice water. The resulting precipitate was
filtered, washed with water and dried to give white crystals
of the titled compound 12; mp: 242-244 °C (lit. 204-206 °C);22
1H NMR (300 MHz, CDCl3): δ 0.76 (s, 3H, 18-CH3), 1.05 (s,
3H, 19-CH3), 3.74 (br s, 1H, 3-H) and 5.35 (s, 1H, 6-H).
17-Iodoandrosta-5,16-dien-3â-ol (13). A stirred solution
of iodine (12.16 g, 0.0203 mol) in dry of THF (144 mL) and
dry of Et2O (72 mL) was cooled in an ice bath to 0 °C, and the
solution was treated with 1,1,3,3, tetramethylguanidine (6.72
mL, 6.24 g, 0.054 mol). A solution of compound 12 (3.0 g, 9.9
mmol) in THF (81 mL) was added dropwise to the iodine
solution over 2 h maintaining the reaction temperature at 0
°C. The reaction mixture was then concentrated under vacuum,
cooled in an ice-bath and then dried to under vacuum at room
temperature to afford a yellow solid (13, 3.65 g, 92.4%). mp:
169-171 °C. (lit. 175-176 °C);22 IR (CHCl3) 2935, 1371, 1039,
862, 843, 799, 715, 665, 582, and 566 cm-1; 1H NMR (300 MHz,
CDCl3): δ 0.76 (s, 3H, 18-CH3), 1.05 (s, 3H, 19-CH3), 3.50 (br
s, 1H, 3R-H), 5.35 (s, 1H, 6-H) and 6.14 (s, 1H, 16-H).
1
1032, 728, 656, 584, 564, 540, 526, 506, 498 cm -1; H NMR
(300 MHz, CDCl3) δ 1.11 (s, 3H, 18-CH3), 1.37 (s, 3H, 19-CH3),
2.04 (s, 3H, 3â-OCH3), 4.62 (m, 1H, 3R-H), 5.43 (br s, 1H, 6-H),
7.43 (d, 1H, J ) 2.4 Hz, aromatic-Hs), 7.45 (d, 1H, J ) 2.7 Hz,
aromatic-H), 7.88 (d, 1H, J ) 2.7 Hz, aromatic-H), 7.90 (d, 1H,
J ) 2.4 Hz, aromatic-H) and 10.66 (s, 1H, 16-CHO). HRMS
calcd 482.2414 (C28H33O3N3‚Na+), found 482.2413. Further
elution with the same solvent system afforded the major
product, 3â-acetoxy-17-(benzo-1H-1,2,3-triazol-1-yl)-16-formy-
landrosta-5,16-diene (7b, 2.3 g, 75.4%); mp: 186-188 °C; IR
(CHCl3) 3023, 2948, 1725, 1670, 1604, 1488, 1450, 1374, 1253,
1196, 1032, 846, 824, 720, 658, 619, 548, 527, 504, 497 cm-1
;
1H NMR (300 MHz, CDCl3) δ 1.07 (s, 6H, 18- and 19-CH3),
2.04 (s, 3H, 3â-OCH3), 4.60 (m, 1H, 3R-H), 5.43 (br s, 1H, 6-H),
7.46 (m, 2H, aromatic-Hs), 7.57 (d, 1H, J ) 6.9 Hz, aromatic-
H), 8.15 (d, 1H, J ) 8.4 Hz), aromatic-H), and 9.59 (s, 1H, 16-
CHO). HRMS calcd 482.2414 (C28H33O3N3‚Na+), found 482.2416.
3â-Hydroxy-17-(2-pyrazyl)-androsta-5,16-diene (14). A
mixture of 17-iodoandrosta-5,16-diene-3â-ol (13; 0.5 g, 1.257
mmol) in solution with dry dimethylformamide (DMF, 10 mL)
along with tetrakis(triphenylphosphine)palladium (Pd(PPh3)4)
(71.6 mg, 0.062 mmol) and (2-tributylstannyl)pyrazine (774.6
mg, 2.099 mmol) was heated at 120 °C for 20 h. After cooling,
the mixture was diluted with cold water (50 mL) and extracted
with EtOAc (30 mL x 3). The combined EtOAc extract was
washed with brine and water, dried over Na2SO4 and then
concentrated to give a brownish solid. This crude product was
purified by flash column chromatography [FCC, petroleum
ether/EtOAc/Et3N (3:2:0.15)] to give 14 (66 mg, 15%); mp:
3â-Acetoxy-17-(benzo-1H-1,2,3-triazol-1-yl)androsta-5,-
16-diene (8). A mixture of bis(triphenyphosphine)rhodium(I)
carbonyl chloride (303 mg, 0.438 mmol) and 1,3-bis-(diphe-
nylphosphino)propane (394 mg, 0.954 mmol) in dry xylene (40
mL) was stirred at 80 °C under Ar for 15 min when fine yellow
precipitate formed. Compound 7b (1.71 g, 3.72 mmol) was
added, and the mixture was refluxed under Ar for 18 h and
then concentrated under reduced pressure. The crude product
was purified by FCC [petroleum ether/EtOAc/Et3N, (8.9:1:0.1)]
to give 1.2 g (74.7%) of pure compound 8; mp 184-186 °C. IR
(CHCl3) 3063, 2918, 2389, 2358, 1725, 1458, 1373, 1254, 1069,
1
199-201 °C. H NMR (300 MHz, CDCl3): δ 0.94 (s, 3H, 18-
CH3), 1.08 (s, 3H, 19-CH3), 3.52 (br s, 1H, 3R-H), 5.40 (s, 1H,
6-H), 6.77 (s, 1H, 16-H), 8.35(s, 1H, pyrazine-H), 8.48(s, 1H,
pyrazine-H), 8.70 (s, 1H, pyrazine-H). HRMS calcd 350.2358
(C23H30ON2), found 350.2354.
1031, 843, 809, 786, 692, 646, 560, 535, 528, 512, 494 cm-1
;
1H NMR (300 MHz, CDCl3) δ 1.10 (s, 3H, 18-CH3), 1.25 (s,
3H, 19-CH3), 2.04 (s, 3H, 3â-OCH3), 4.64 (m, 1H, 3R-H), 5.43
(br s, 1H, 6-H), 6.01 (s, 1H, 16-H), 7.40 (t, 1H, J ) 7.8 Hz,
aromatic-H), 7.51 (t, 1H, J ) 7.8 Hz, aromatic-H), 7.67 (d, 1H,
J ) 8.1 Hz, aromatic-H), and 8.10 (d, 1H, J ) 8.1 Hz, aromatic-
H). HRMS calcd 454.2465 (C27H33O2N3‚Na+), found 454.2469.
3â-Hydroxy-17-(5-pyrimidyl)-androsta-5,16-diene (15).
Reaction of 13 (0.645 g, 1.623 mmol) as described above for
14, but using (5-tributylstannyl)pyrimidine (1.0 g, 2.710 mmol)
dissolved in 10 mL of dry DMF along with (Pd(PPh3)4) (92.88
mg, 0.0804 mmol) and (5-tributylstannyl)pyrimidine (1.0 g,
2.710 mmol) and following purification [FCC, petroleum ether/
EtOAc/Et3N (3:2:0.15)] gave 3â-hydroxy-17-(5-pyrimidyl)-an-
drosta-5,16-diene 15 (44 mg, 10%); mp: 231-233 °C (lit. 240-
242 °C);19 1H NMR (300 MHz, CDCl3): δ 1.05 (s, 3H, 18-CH3),
1.08 (s, 3H, 19-CH3), 3.83 (br s, 1H, 3R-H), 5.39 (s, 1H, 6-H),
7.26 (s, 1H, 16-H), 8.73 (s, 2H, 41-H and 61-H) and 9.07 (s,
1H, 21-H). HRMS calcd 350.2358 (C23H30ON2), found 350.2348.
In Vitro Assay of CYP17. The in vitro CYP17 inhibitory
activities of the compounds were evaluated using our rapid
acetic acid releasing assay (AARA), utilizing intact P450c17-
expressing E. coli as the enzyme source.24,25 It involves the
use of [21-3H]-17R-hydroxypregnenolone as the substrate, and
CYP17 activity was measured by the amount of tritiated acetic
acid formed during the cleavage of the C-21 side chain of the
substrate. We have firmly established that the method is
comparable in terms of accuracy and reliability to the HPLC
analysis procedure used by researchers in the field.24,25 IC50
values were obtained directly from plots relating percentage
inhibition versus inhibitor concentration over appropriate
ranges. Each compound was tested at a minimum of five
different concentrations. The assays were performed in trip-
licate, and the IC50 values reported are the mean of triplicate
experiments. The standard deviations were (5% of the mean
values.
3â-Hydroxy-17-(benzo-1H-1,2,3-triazol-1-yl)androsta-
5,16-diene (9). The method followed that described for
compound 5 but using 3â-acetoxy-17-(benzo-1H-1,2,3-triazol-
1-yl)androsta-5,16-diene (8; 700 mg, 1.62 mmol). Recrystalli-
zation from EtOAc/MeOH give the title compound 9 (600 mg,
95%); mp 241-244 °C; IR (CHCl3) 3603, 2937, 2859, 1609,
1488, 1451, 1373, 1287, 1243, 1069, 1040, 1007, 953, 845, 805,
715, 665, 618, 570, 553, 517 cm-1; 1H NMR (300 MHz, CDCl3)
δ 1.09 (s, 3H, 18-CH3), 1.24 (s, 3H, 19-CH3), 3.55 (m, 1H, 3R-
H), 5.41 (br s, 1H, 6-H), 6.06 (s, 1H, 16-H), 7.40 (t, 1H, J ) 7.8
Hz, aromatic-H), 7.52 (t, 1H, J ) 7.8 Hz, aromatic-H), 7.67 (d,
1H, J ) 8.1 Hz, aromatic-H), and 8.10 (d, 1H, J ) 8.1 Hz,
aromatic-H). HRMS calcd 412.2359 (C25H31ON3‚Na+), found
412.2365.
17-(Benzo-1H-1,2,3-triazol-1-yl)androsta-4,16-dien-3-
one (10). The method followed that described for compound
6 but using â-hydroxy-17-(benzo-1H-1,2,3-triazol-1-yl)and-
rosta-5,16-diene (9; 500 mg, 1.28 mmol). Purification of the
crude product by FCC [CH2Cl2/EtOH, (50:1)] afforded the titled
compound 10 (420 mg, 84.4%); mp: 280-283 °C; IR (CHCl3)
2944, 1658, 1450, 1070, 8444, 825, 721, 624, 589, 564, 554,
1
541, 521 cm-1; H NMR (300 MHz, CDCl3) δ 1.26 (s, 3H, 18-
CH3), 1.27 (s, 3H, 19-CH3), 5.77 (s, 1H, 4-H), 6.01 (s, 1H, 16-
H), 7.40 (t, 1H, J ) 7.8 Hz, aromatic-H), 7.52 (t, 1H, J ) 7.8
Hz, aromatic-H), 7.67 (d, 1H, J ) 7.8 Hz, aromatic-H), and
8.10 (d, 1H, J ) 8.1 Hz, aromatic-H). HRMS calcd 410.2203-
(C25H29ON3‚Na+), found 410.2185.
Human 5R-Reductase Type 1 and 2 Assay. The inhibi-
tory activities of compounds and finasteride as reference were
determined using the DU145 cell line (for human type 1
enzyme) and human prostate homogenate (BPH tissue for type
2 enzyme) according to the procedure described by Hartmann
and colleagues.27 The percent inhibition values at a concentra-
Dehydroepiandrosterone-17 Hydrozone (12). Dehy-
droepiandrosterone (11, 3.5 g, 12.2 mmol) was dissolved in
ethanol (60 mL), and the resulting solution was treated with
hydrazine hydrate (2.37 mL, 0.049 mol) followed by a solution