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S. Ahmed et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1343–1346
sodium bicarbonate (NaHCO3) solution (50 mL), extracted
into dichloromethane (DCM) (2Â50 mL), washed with water
(3Â30 mL) and dried over anhydrous magnesium sulfate
(MgSO4). The mixture was filtered and the solvent removed
under vacuum to give a yellow oil, which was purified using
flash chromatography to give (1) (0.12g, 16.0%) as a pure
white solid [mp 146–149 ꢁC (literature 165 ꢁC, Hedayatullah,
1975); Rf 0.31 diethyl ether/petroleum ether 40–60 ꢁC (50/50)].
(Film) cmÀ1: 3343.7, 3222.2 (NH), 1693.9 (C¼O) 1401.2,
1162.5 (S¼O). dH (CDCl3): 8.12(2H, d, J=9 Hz, ArH), 7.80
(2H, d, J=9 Hz, ArH), 7.79 (2H, d, J=9 Hz, ArH), 7.44 (2H,
d, J=9 Hz, ArH), 5.03 (2H, s, NH2), 4.41 (2H, q, J=7 Hz,
CH2–), 1.42(3H, t, J=7 Hz, CH2CH3). dc (d6-Acetone): 199.7
(C¼O), 154.2, 135.0, 131.4, 130.3, 122.7, 115.2 (C-Ar), 32.0
(OCH2), 8.2(CH ). MS m/z 321 (M+).
3
12. ES assay: The total assay volume was 1 mL. 3H-estrone
sulfate (25 mL, 20 mM/tube; 300,000 dpm) and the inhibitors
(50 mM/tube) dissolved in ethanol were added to a 10 mL assay
tube, and the ethanol removed with a stream of nitrogen. Tris–
HCl buffer (0.05 M, pH 7.2, 0.2 mL) was added to each tube.
Placental microsomes were then diluted with Tris–HCl buffer
(115 mg/mL). The microsomes and assay tubes were pre-incu-
bated for 5 min at 37 ꢁC in a shaking water bath prior to the
addition of the microsomes (0.8 mL) to the tubes. After 20 min
incubation (at 37 ꢁC), toluene (4 mL) was added to quench the
assay, and the tubes placed on ice. The quenched samples were
vortexed for 45 s and centrifuged (3000 rpm, 10 min). 1 mL of
toluene was removed and added to 5 mL scintillation cocktail
(TRITONX). The aliquots were counted for 3 min. All sam-
ples were run in triplicate. Control samples with no inhibitor
were incubated simultaneously. Blank samples were obtained
by incubating with boiled microsomes.
13. Irreversible ES assay: The irreversible inhibition was
determined using EMATE (10 mM), COUMATE (100 mM), 6
(700 mM) and 8 (700 mM). Placental microsomes (18 mg/mL,
55 mL) were incubated with each of the inhibitors (25 mL in
ethanol, removed with a stream of nitrogen) in Tris–HCl buf-
fer (50 mM, pH 7.2, 945 mL) at 37 ꢁC for 10 min. A control
tube with no inhibitor was incubated simultaneously (100%
tubes). An aliquot (100 mL) in triplicate, was taken from each
sample and tested for ES activity using the procedure above,
except that 900 mL of Tris–HCl buffer was added to the assay
tubes. A second aliquot (100 mL) in triplicate, was subjected to
dialysis at 4 ꢁC for 16 h, with regular changes of Tris–HCl
buffer. The microsomes were then removed from the dialysis
tubing and tested for ES activity as described above.
n
max (Film) cmÀ1: 3421.3, 3301.9 (NH), 1382.0, 1177.9 (S¼O).
dH (CDCl3) 7.85–7.15 (9H, m, ArH), 7.12(2H, s, NH 2). dC
(CDCl3): 1129.603, 128.734, 127.529, 123.402 (CAr). MS m/z
found: MNH+4 267.0798, (C12H11NO3S)NH+4 requires
267.0803.
10. Ethyl-(40-hydroxy)-1,10-biphenyl-4-carboxylate (5): Conc.
H2SO4 (1 mL) was added to a suspension of 4-hydroxy-
biphenyl carboxylic acid (0.5 g, 2.34 mmol) in ethanol (20 mL)
and the solution refluxed for 1 h. After cooling to room tem-
perature, NaOH (ꢀ15 mL) was added to neutralise the solu-
tion. The resulting mixture was allowed to stand for 15 min,
before being poured into a cool beaker, and made up to
500 mL with water. The white precipitate was filtered, and
dried (80 ꢁC), to give (5) (0.46 g, 81.23%) as a white solid [mp
141–143 ꢁC; Rf 0.63 diethyl ether/petroleum ether 40–60 ꢁC
(70/30)]. nmax (Film) cmÀ1: 3335.9 (OH), 1681.4 (C¼O). dH
(CDCl3): 8.00 (2H, d, J=8 Hz, ArH), 7.60 (2H, d, J=8 Hz,
ArH), 7.53 (2H, d, J=8 Hz, ArH), 6.95 (2H, d, J=8 Hz,
ArH), 5.81 (1H, s, OH), 4.41 (2H, q, J=7 Hz, CH2CH3), 1.42
(3H, t, J=7 Hz, CH2CH3). dC (CDCl3): 167.2(C ¼0), 157.3,
145.6, 132.5, 130.1, 128.6, 128.4, 126.4, 115.9 (CAr), 61.1
(OCH2CH3), 14.3 (OCH2CH3). GCMS tR 19.223 m/z 242 (M+).
11. Ethyl-40-[(aminosulfonyl)oxy]-1,10-biphenyl-4-carboxylate
(6): Compound 6 was synthesised following the same proce-
dure as for compound 1 except that NaH (60% dispersion in
mineral oil, 0.05 g, 1.25 mmol) was added to a stirred solution
of 60 (0.2g, 0.83 mmol) in DMF (10 mL). Aminosulfonyl
chloride in toluene (10 mL, ꢀ10 mmol) was added after
30 min. Removal of the solvent under vacuum produced a
solid, which was purified using flash chromatography to give 6
(0.08 g, 30.0%) as a pure white solid [mp 171.2–173.5 ꢁC; Rf
0.30 petroleum ether 40–60 ꢁC: ethyl acetate (65/35)]. nmax
14. Ahmed, S.; James, K.; Owen, C. P.; Patel, C. K.; Samp-
son, L. J. Steroid Biochem. Mol. Biol. In press.