Novel steroidal vinyl fluorides as inhibitors of steroid C17(20) lyase

Article abstract of DOI:10.1016/S0968-0896(01)00354-6

20-Fluoro-17(20)-pregnenolone derivatives were designed as enol mimics of pregnenolone. All of the targeted, novel fluoroolefins were potent inhibitors of C₁₇₍₂₀₎ lyase. Copyright

Full text of DOI:10.1016/S0968-0896(01)00354-6

Bioorganic & Medicinal Chemistry 10 (2002) 929–934
Novel Steroidal Vinyl Fluorides as Inhibitors of
Steroid C17(20) Lyase
y
{
,
Joseph P. Burkhart, Philip M. Weintraub,* Cynthia A. Gates, Robert J. Resvick,
Roy J. Vaz, Dirk Friedrich, Michael R. Angelastro, Philippe Bey and Norton P. Peet *
x,
Aventis Pharmaceuticals, Route 202-206, Bridgewater, NJ 08807-0800, USA
Received 5 September 2001; accepted 25 September 2001
Abstract—20-Fluoro-17(20)-pregnenolone derivatives were designed as enol mimics of pregnenolone. All of the targeted, novel
fluoroolefins were potent inhibitors of C17(20) lyase. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
cancer,1
2ꢀ15
since androgens and oestrogens, respec-
tively, are required for the growth of these tumors.
Steroid C17a hydroxylase/C17(20) lyase (EC1.14.99.9/EC
4
.1.2.30), the enzyme responsible for converting proges-
terone and pregnenolone to androstenedione and dehy-
droepiandrosterone, respectively, is found in the testes,
A variety of C17(20) lyase inhibitors of different
mechanistic types and structural classes have been
1
6
described. These categories include steroidal mechan-
17ꢀ19
1
ovaries, adrenals, and placenta. Two distinct steps
comprise this conversion, for example, an initial 17a-
ism-based inhibitors,
0ꢀ26
steroidal C -heterocyclic
17
inhibitors, nonsteroidal heterocyclic inhibitors,
and other inhibitors which do not precisely fall into
2
27ꢀ38
hydroxylation and a subsequent cleavage of the C
1
7(20)
2
,3
39ꢀ44
bond, to give the C17-keto androgens. Inhibition of
C17(20) lyase would, therefore, lead to the cessation of
androgen production and also, indirectly, shut down
estrogen production, since estrogens arise from andro-
gens via the action of the enzyme aromatase. Mechan-
istic work on this intriguing enzyme has been performed
these categories.
Fluoroolefins represented by
structures 8 and 9 (Scheme 2) were proposed as (Z) and
(E) enol mimics, respectively, of pregneolone. Vinyl
halides have previously been designed to mimic an eno-
4
5
lized ketone intermediate. This report describes the
synthesis of these and related vinyl fluorides and C17(20)
lyase inhibition data which was generated for these de
novo designed inhibitors.
4
ꢀ7
by several investigators.
6
A mechanism proposed by
Corina et al. is shown in Scheme 1. A molecular model
8ꢀ10
for this enzyme has been developed,
based on
homology with P450cam. This model is comprised of two
orthogonal lobes, for example, a C17a hydroxylase lobe
and a C17(20) lyase lobe, which share a common heme at
the connecting elbow. The human enzyme has been
expressed and purified in bacteria. The inhibition of
lyase is a potential therapeutic approach for the treat-
ment of prostate cancer and hormone-dependent breast
Chemistry
Synthesis of the targeted vinyl fluorides are detailed in
Schemes 3 and 4. Dehydroepiandrosterone (6) was sily-
lated with tert-butyldimethylsilyl chloride to give the
protected alcohol 10 in 88% yield. Treatment of 10 with
triethyl 2-fluorophosphonoacetate gave a mixture of E-
and Z-isomers 11, which were inseparable by chroma-
tography. Reduction of this mixture with diisobutylalu-
minum hydride gave a new mixture of primary alcohols
which was separable, by virtue of the primary alcohols
which rendered the geometric isomers more polar and
allowed them to be differentiated on silica gel. The
major material was E-isomer 12, which was isolated in
1
1
*Corresponding authors. Fax:+1-908-231-3605; e-mail: philip.wein-
traub@aventis.com; npeet@arqule.com
y
Lilly Research Labs, Eli Lilly and Company, Indianapolis, IN 46285,
USA.
{
Vertex Pharmaceuticals Inc., 130 Waverly St., Cambridge, MA
0
2139-4242, USA.
x
ArQule, 19 Presidential Way, Woburn, MA 01801-5140, USA.
0
968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00354-6

930
J. P. Burkhart et al. / Bioorg. Med. Chem. 10 (2002) 929–934
Scheme 1. Proposed mechanism for C17(20) lyase.
Treatment of 12 with tetrabutylammonium fluoride
removed the silyl protecting group to give diol 14 in
95% yield. Hydrogenolysis of the allylic alcohol gave
fluoroolefin 15 in 76% yield; removal of the silyl pro-
tecting group, again with tetrabutylammonium fluoride,
gave fluoroolefin 9 (E-isomer) in 94% yield. In similar
fashion were prepared the fluoroolefins (Z-isomers) 16
and 8, also shown in Scheme 4. Compounds 8, 9, 14,
and 16, which were the designed enol mimics of preg-
nenolone, are novel compounds.
Scheme 2. Vinyl fluoride C17(20) pregnenolone enol mimics.
5
7% yield. The minor Z-isomer 13, was isolated in 16%
yield. Unequivocal assignment of these regioisomers
was determined using high-resolution NMR techniques,
for example, DQF-COSY and HMQC for resonance
Enzymology
1
19
assignments, and NOESY and heteronuclear H– F
4
Percent inhibition values for fluoroolefins 14, 9, 16, and
8 were evaluated against cynomolgous monkey testi-
cular C17(20) lyase at given concentrations, with and
without preincubation of enzyme with inhibitor, are
presented in Table 1. Methods used for determination
of the IC50 values in Table 1 are detailed in the Experi-
mental.
6
NOE difference experiments for determination of
fluoro olefin geometry.
The major isomer 12 from the Horner–Emmons vinyl
homologation reaction was then manipulated as shown
in Scheme 4 to produce two additional E-vinyl fluorides.
2 2 2 2
Scheme 3. Reagents: (a) TBDMS-Cl, DMAP, TEA, DMF; (b) (EtO) P(O)CHFCO Et, LHMDS, THF; (c) DIBAL-H, CH Cl .

J. P. Burkhart et al. / Bioorg. Med. Chem. 10 (2002) 929–934
931
Results and Discussion
have collected to date is insufficient to support a
hypothesis. However, it is clear that this study supports
the concept of designing enol mimics of ketone sub-
strates as enzyme inhibitors.
From Table 1, it is evident that the 20-fluoro-17(20)-ene
pregnenolones 14, 9, 16, and 8 are potent inhibitors of
cynomolgous monkey testicular C17(20) lyase. From a
comparison of the % inhibition values for the com-
pounds with and without preincubation, it appears that
neither of the E-isomers (14 and 9) is time-dependent.
Allyl alcohol 16 is the most potent inhibitor, and it is
not clear whether this Z-isomer is displaying time-
dependency from the data which was generated, since
all values at both concentrations used are close to 100%
inhibition. However, fluoroolefin 8, also a Z-isomer but
less potent than 16, is clearly displaying time-depen-
dence.
Experimental
General
Melting points were determined with a Thomas-Hoover
capillary melting apparatus and are uncorrected. TLC
analyses were performed with Merck DC-F254 silica gel
plates, with visualization by alkaline permanganate and
UV light. Flash chromatography was performed with
Merck silica gel 60 (0.040–0.063 mm). NMR spectra
were recorded on Unity 400 or Gemini-300 spectro-
1
13
meters in CDCl , unless otherwise stated. H and
3
C
Conclusion
NMR signals are reported in ppm from tetra-
methylsilane (s, d, t, m and br for singlet, doublet, tri-
plet, multiplet and broad, respectively) and coupling
constants are reported in hertz (Hz). Infrared spectra
were recorded on a Perkin-Elmer Model 1800 or Matt-
son Galaxy 5020 FT-IR spectrophotometer. Mass spec-
tral data were collected at 70 eV on a Finnigan MAT
4600, MAT TSQ-700 or VG Analytical Ltd ZAB2-SE
mass spectrophometer and computerized for HRMS.
Combustion analyses performed using a Perkin-Elmer
Model 2400 elemental analyzer fell within ꢁ0.4% of the
calculated values. The organic extracts were dried over
magnesium sulfate or sodium sulfate prior to evapora-
tion in vacuum on a rotary evaporator. Anhydrous sol-
vents and starting materials were purchased from
Aldrich Chemical Company and used as obtained with
the following exception: dehydroepiandrosterone was
purchased from Proquina.
In summary, we have designed novel 20-fluoro-17(20)-
ene pregnenolones which are potent inhibitors of cyno-
molgous monkey testicular C17(20) lyase. These fluor-
oolefins were designed as mimics of the enol tautomers
of pregnenolone (13). Compound 8 was clearly time-
dependent, and additional compounds in this set may
also have a time-dependent component. Although it is
interesting to speculate on possible reasons for this
time-dependent behavior, the limited data which we
Table 1. Inhibition of cynomolgus monkey testicular C17(20) lyase
with novel 20-fluoro-17,20-ene pregnenolone analogues
Compd Isomer
X
F
Y
Concn Preincubation Inhibition
(min)
(mM)
(%)
1
1
8
9
6
Z
E
Z
E
CH OH
2
10
1
0
0
100
96
100
94
85
63
87
61
78
49
94
72
88
54
94
60
1
1
10
1
0
40
40
0
4
CH
2
OH
F
0
1
1
10
1
0
40
40
0
F
CH
3
0
1
1
10
1
0
40
40
0
CH
3
F
0
1
1
0
40
40
3.
Scheme 4. Reagents: (a) TBAF, THF; (b) SO pyridine complex,
LiAlH , THF.
4

932
J. P. Burkhart et al. / Bioorg. Med. Chem. 10 (2002) 929–934
(
3ꢀ)-3-[(1,1-Dimethylethyl)dimethylsilyl]oxy]androst-5-
15%) of ethyl acetate in hexane gave 12 (3.80 g, 57%) as
ꢂ
en-17-on-3-ol (10). To a stirred solution of dehy-
droepiandrosterone (7, 20.0 g, 69.3 mmol) in anhydrous
DMF (350 mL) under nitrogen was added t-butyldi-
methylsilyl chloride (11.0 g, 72.8 mmol), 4-dimethyl-
aminopyridine (0.42 g, 3.47 mmol) and triethylamine
a white solid: mp 144–147 C. TLC R 0.37, ethyl ace-
f
1
tate/ hexane (15:85); H NMR (400 MHz) d 5.34–5.29
(m, 1H, vinyl), 4.29 (ddd, 1H, J=21.4, 13.4, 6.4 Hz, 0.5
CH O), 4.20 (ddd, 1H, J=21.4, 13.4, 6.4 Hz, 0.5
2
CH O), 3.53–3.44 (m, 1H, OCH), 1.01 (s, 3H, 19-Me),
2
(
10.6 mL, 76.3 mmol). The resultant suspension was
0.91 (d, 3H, J=1.1 Hz, 18-Me), 0.89 (s, 9H, Si-t-Bu),
1
3
stirred at room temperature for 3 days and then poured
into rapidly stirred water (1.5 L). The resultant suspen-
sion was filtered and the white solid crystallized from
0.054 (s, 6H, 2ꢃSiMe); C NMR d 150.8 (d, J=241.3
Hz, 20-C), 141.7, 131.9 (d, J=14.7 Hz, 17-C), 120.7,
72.6, 58.7 (d, J=31.6 Hz, 21-C), 56.5, 50.1, 42.9, 42.6
(d, J=5.6 Hz, 13-C), 37.4, 37.2, 36.7, 32.1, 31.8, 31.5,
31.4, 26.1 (d, J=4.6 Hz, 16-C), 26.0, 23.9, 21.3, 19.4,
aqueous acetone to give 10 (24.6 g, 88%) as a white
ꢂ
crystalline solid: mp 146–148 C. TLC R 0.78, ethyl
f
1
19
acetate/hexane (1:1); H NMR d 5.38–5.32 (m, 1H,
vinyl), 3.55–3.43 (m, 1H, OCH), 1.03 (s, 3H, Me), 0.89
[s, 9H, C(Me) ], 0.88 (s, 3H, Me), 0.06 (s, 6H, 2ꢃSiMe);
MS (CI, CH ) m/z (rel intensity) 403 (MH , 3), 401 (5),
3
C, 74.57;H, 10.51. Found: C, 74.89; H, 10.84.
18.6 (d, J=3.4 Hz, 18-Me), 18.2; F NMR d ꢀ114.32
+
(br t, J=24 Hz); MS (CI, CH ) m/z (rel intensity) 449
4
(MH , 3), 448 (5), 447 (19), 431(10), 429 (5), 391 (32),
299 (100), 297 (17). Anal. calcd for C H FO Si:
C,72.27; H, 10.11. Found: C, 72.18; H, 10.28.
3
+
4
27 45
2
87 (9), 345 (18), 271 (100). Anal. calcd for C H SiO :
2
2
5
42
Also obtained was 13 (1.10 g, 16%) as a white solid: mp
ꢂ
(3ꢀ) - [(1,1 - Dimethylethyl)dimethylsilyl]oxy] - 20 - fluoro-
pregna-5,17(20)-dien-21-oic acid, ethyl ester (13). To a
stirred solution of triethyl 2-fluoro-2-phosphonoacetate
174–176 C. TLC R 0.29, ethyl acetate/hexane (15:85);
f
1
H NMR d 5.36–5.31 (m, 1H, vinyl), 4.15 (dd, 2H,
J=21.2, 6.1 Hz, CH O), 3.56–3.44 (m, 1H, OCH), 1.03
(s, 3H, 19-Me), 0.94 (s, 3H, 18-Me), 0.91 (s, 9H, Si-t-
2
(
0.76 mL, 3.75 mmol) in THF (15 mL) under nitrogen
was added lithium hexamethyldisilazane (3.50 mL of a
.0 M solution in THF, 3.50 mM). After 1 h, a solution
1
3
Bu), 0.075 (s, 6H, 2ꢃSiMe); C NMR d 149.9 (d,
J=245.0 Hz, 20-C), 141.8, 129.5 (d, J=13.7 Hz, 17-C),
120.7, 72.6, 59.9 (d, J=30.8 Hz, 21-C), 55.6, 50.1, 44.2,
42.8, 37.3, 36.7, 36.1 (d, J=6.3 Hz, 16-C), 32.0, 31.8,
31.2, 25.9, 25.8 (d, J=6.5 Hz, 15-C), 24.9, 21.0, 19.4,
1
of 10 (1.01 g, 2.50 mL) in THF (5 mL) was added and
the reaction mixture was heated to reflux. After 2.5 h, the
reaction mixture was allowed to cool to room temperature
and concentrated. The residue was partitioned between
1
9
18.3, 16.9, 16.88; F NMR d ꢀ128.10 (t, J=21.1 Hz);
+
Et O (40 mL) and 0.4 M aqueous hydrochloric acid (40
2
MS (CI, CH ) m/z (rel intensity) 449 (MH , 2), 448 (4),
4
mL). The layers were separated and the organic layer was
washed with 0.5 M aqueous hydrochloric acid (20 mL),
saturated aqueous sodium bicarbonate (20 mL), and brine
447 (15), 431 (10), 429 (5), 391 (26), 317 (47), 299 (100)
297 (15). Anal. calcd for C H FO Si: C, 72.27; H,
10.11. Found: C, 72.06; H, 10.22.
2
7
45
2
(
20 mL). Drying, filtration, and concentration gave
crude 11. Flash chromatography (6ꢃ17 cm column) elut-
(3ꢀ,17E)-20-Fluoropregna-5,17(20)-diene-3,21-diol (14).
To compound 12 (307 mg, 0.68 mmol) under nitrogen
was added tetrabutylammonium fluoride (3.0 mL of a
1.0 M solution in THF, 3.0 mmol) and the resultant
solution was stirred at room temperature for 23 h. The
reaction solution was added dropwise to vigorously
stirred water (50 mL) and the resultant suspension was
ing with ethyl acetate/hexane (2:98) gave 11 (mixture of E
and Z isomers, 0.83 g, 67%) as a white solid. TLC R 0.41
f
1
9
and 0.51, ethyl acetate/hexane (3:97); F NMR d ꢀ121.59
+
(
z (rel intensity) 491 (MH , 97), 475 (59), 445 (24), 433
s, E isomer) and ꢀ135.49 (s, Z isomer); MS (CI, CH ) m/
4
(65), 359 (100), 339 (24). Anal. calcd for C H FO Si:
29 47 3
C, 70.97: H, 9.65. Found: C, 71.32; H, 10.02.
filtered and dried to give 14 (218 mg, 95%) as a white
ꢂ
solid: mp 214–216 C. TLC R 0.15, ethyl acetate/hexane
f
1
(
3ꢀ,17E) - 3 - [(1,1 - Dimethylethyl)dimethylsilyl]oxy] - 20 -
fluoropregna-5,17(20)-diene-3,21-diol (20) and (3ꢀ,17Z)-
(45:55). H NMR (DMSO-d ) d 5.30–5.25 (m, 1H,
6
vinyl), 4.92 (t, 1H, J=5.5 Hz, OH), 4.60 (d, 1H, J=4.8
Hz, OH), 4.15–3.89 (m, 2H, CH O), 3.35–3.19 (m
3
5
-[(1,1-dimethylethyl)dimethylsilyl]oxy]-20-fluoropregna-
,17(20)-diene-3,21-diol (13). To a stirred solution of 11
2
OCH), 0.95 (s, 3H, 19-Me), 0.85 (d, 3H, J=1.1 Hz, 18-
1
9
(
7.29 g, 14.9 mmol) in dichloromethane (135 mL) under
Me); F NMR (DMSO-d ) d ꢀ108.66 (dd, J=28.0,
6
ꢂ
+
nitrogen and cooled to ꢀ78 C was slowly added diiso-
butylaluminum hydride (6.55 mL of a 1.0 M solution in
dichloromethane, 65.5 mmol). After 1 h, the reaction
was quenched with a solution of glacial acetic acid (3.8
mL) in dichloromethane (9 mL) and the reaction mix-
ture was poured into dichloromethane (250 mL)/satu-
rated aqueous potassium sodium tartrate (250 mL). The
resultant emulsion was filtered through a Celite pad (3
cm), the filtrates transferred to a separatory funnel, and
the layers separated. The organic layer was washed with
saturated aqueous potassium sodium tartrate (130 mL),
saturated aqueous sodium bicarbonate (250 mL), and
brine (100 mL). Drying, filtration, and concentration
gave crude product. Flash chromatography (two equal
batches, 8ꢃ20 cm column) eluting with a gradient (10–
24.1 Hz), MS (CI, CH ) m/z (rel intensity) 335 (MH ,
4
4), 334 (9), 333 (18), 317 (100), 299 (93), 297 (28). Anal.
calcd for C H FO : C, 75.41; H, 9.34. Found: C,
75.61; H, 9.50.
2
1
31
2
(3ꢀ,17E) - 3 - [(1,1 - Dimethylethyl)dimethylsilyl]oxy] - 20 -
fluoropregna-5,17(20)-dien-3-ol (15). To a stirred solu-
tion of 12 (1.35 g, 3.00 mmol) in THF (30 mL) under
nitrogen and cooled in an ice water bath was added
sulfur trioxide pyridine complex (0.84 g, 5.27 mmol).
The resultant suspension was stirred at ice bath tem-
perature for 3 h, and then stored in a refrigerator over-
night. To the stirred suspension was carefully added
lithium aluminum hydride (0.80 g, 21.1 mmol) in por-
tions. The reaction was quenched by cautiously adding

J. P. Burkhart et al. / Bioorg. Med. Chem. 10 (2002) 929–934
933
1
0
.6 mL of water, 0.6 mL of 1.0 N aq NaOH, and finally,
0.27, ethyl acetate/hexane (1:3). H NMR d 5.37–5.33
(m, 1H, vinyl), 3.59–3.46 (m, 1H, OCH), 3.49 (s, 0.6H,
MeOH solvate), 1.79 (dt, 3H, J=17.2, 1.4 Hz, 21-Me),
another 0.6 mL of water. The resultant suspension was
diluted with diethyl ether (80 mL) and stirred vigorously
for several minutes. The suspension was filtered and the
filtrate was concentrated to give crude 15. Flash chro-
matography (5ꢃ14 cm column) eluting with ethyl ace-
tate/hexane (5:95) gave 15 (0.99 g, 76%) as a white
solid. A portion of 15 was crystallized from aqueous
1
9
1.02 (s, 3H, 19-Me), 0.89 (s, 3H, 18-Me); F NMR d
ꢀ110.27 (q of q, J=17.2, 2.0 Hz); MS (CI, CH ) m/z
4
+
(rel intensity) 319 (MH , 7), 318 (15), 317 (28), 301
.
(100), 299 (39), 281 (8). Anal. calcd for C H FO 0.2-
MeOH: C, 78.38; H,9.87. Found: C, 78.40; H, 9.82.
2
1
31
ꢂ
acetone to give a white crystalline solid: mp 128–130 C.
1
TLC R 0.52, ethyl acetate/hexane (2:98). H NMR d
f
Enzymes and assays
5
(
.35-5.31 (m, 1H, vinyl), 3.55–3.42 (m, 1H, OCH), 1.92
dt, 3H, J=18.9, 1.9 Hz, 21-Me) 1.01 (s, 3H, 19-Me)
Compounds were tested for inhibition of cynomolgous
monkey C17(20) lyase in vitro using microsomal pre-
parations of the enzyme from testicular tissue. Testes
were removed from anesthetized animals and flash fro-
zen in liquid nitrogen. Microsomes were isolated as
0
0
.89 (s, 9H, Si-t-Bu), 0.86 (d, 3H, J=1.3 Hz, 18-Me),
1
9
.06 (s, 6H, 2ꢃSiMe); F NMR d ꢀ95.86 (q, J=18.9
+
Hz); MS ( CI,CH ) m/z (rel intensity) 433 (MH , 9),
4
432 (10), 431 (38), 417 (43) 413 (70), 375 (55), 301 (100).
Anal. calcd for C H FOSi: C, 74.94; H, 10.48. Found:
C, 75.16; H, 10.46.
4
7
described previously. The compound to be tested was
dissolved in dimethyl sulfoxide and diluted in 0.05 M
potassium phosphate buffer, pH 7.4, to give the desired
concentrations of test compound; this contributes 0.1%
v/v dimethyl sulfoxide to the final mix. Assays con-
tained 0.05 M potassium phosphate, pH 7.4, an
NADPH regenerating system (1 mM NADPH, 5 mM
glucose-6-phosphate, 1 IU/mL glucose-6-phosphate
dehydrogenase), test compound, substrate and micro-
somal protein in a total volume of 0.2 mL. Control
assays contained all components, including dimethyl
sulfoxide, but no test compound. All assays were per-
formed in duplicate. The reaction was initiated by the
2
7
45
(3ꢀ,17E)-20-Fluoropregna-5,17(20)-dien-3-ol (9). In a
fashion analogous to the preparation of 14 was pre-
ꢂ
pared 9 (299 mg, 94%) as a white solid: mp 129–133 C.
1
TLC R 0.25, ethyl acetate/hexane (1:3). H NMR d
f
5
(
.38–5.34 (m, 1H, vinyl), 3.60–3.47 (m, 1H, OCH), 1.92
dt, 3H, J=18.9, 1.9 Hz, 21-Me), 1.02 (s, 3H, 19-Me),
1
9
0
J=18.8 Hz); MS (CI, CH ) m/z (rel intensity) 319
.86 (d, 3H, J=1.2 Hz, 18-Me); F NMR d ꢀ95.79 (q,
4
+
(
(
MH , 9), 318 (17), 317 (33), 301 (100), 299 (65), 281
9). Anal. calcd for C H FO:C, 79.20; H, 9.81. Found:
C, 79.10, H, 9.81.
2
1
31
3
addition of substrate, 7- H-17a-hydroxypregnenolone
(11.2 mCi/mmol; 0.20 mCi per assay) plus unlabeled
(
3ꢀ,17Z)-20-Fluoropregna-5,17(20)-diene-3,21-diol (16).
17a-hydroxypregnenolone dissolved in dimethyl sulfox-
ide, contributing 2.5% v/v to the final assay mix, and
phosphate buffer, yielding a final concentration of 0.05
mM 17a-hydroxypregnenolone (ca. equal to the Km
value) to the other assay components. The complete
In a fashion analogous to the preparation of 14 was
prepared 16 (299 mg, 94%) as a white solid: mp 198–
2
NMR d 5.29–5.25 (m, 1H, vinyl), 4.91 (t, 1H, J=5.7 Hz,
OH), 4.60 (d, 1H, J=4.5 Hz, OH), 3.88 (dd, 1H,
J=23.1, 5.7 Hz, CH O), 3.34–3.19 (m, 1H, OCH), 0.95
ꢂ
03 C. TLC R 0.19, ethyl acetate/hexane (45:55). H
f
1
ꢂ
assay was incubated at 34 C for 6 min. Each assay was
terminated by addition of 5 mL of chloroform:methanol
(2:1). Carrier steroids representing substrates and pro-
ducts (17a-hydroxypregnenolone, dehydroepiandroster-
one, and androst-5-ene-3b,17b-diol) and 0.8 mL of
distilled, deionized water were also added at this time.
The steroids were extracted by the method of Moore
2
1
9
(
ꢀ
s, 3H, 19-Me), 0.86 (s, 3H, 18-Me); F NMR d
123.18 (t, J=23.1 Hz); MS (CI, CH ) m/z (rel inten-
4
+
sity) 335 (MH , 4), 334 (6), 333 (15) 317 (100) 299 (46).
Anal. calcd for C H FO : C, 75.41; H, 9.34. Found:
C, 75.37; H, 9.43.
2
1
31
2
4
8
and Wilson. The organic phase containing the steroids
was evaporated using nitrogen gas, the residues were
dissolved in 18% tetrahydrofuran (v/v) in hexane, and
the steroids were separated by HPLC on a Si60 (5 mm)
column (250ꢃ4 mm) using a gradient of 18–22% tetra-
hydrofuran (v/v) in hexane. Radioactivity in the steroid
peaks was measured using a Radiometric Model HS or
Model A515 Flo-One detector.
(
3ꢀ,17Z) - 3 - [(1,1 - Dimethylethyl)dimethylsilyl]oxy] - 20 -
fluoropregna-5,17(20)-dien-3-ol (17). Compound 17
0.59 g, 91%), a white solid, was prepared in an analo-
(
gous fashion to compound 15. Crystallization from
ꢂ
acetone gave a white crystalline solid: mp 138–140 C.
1
TLC R 0.52, ethyl acetate/hexane (2:98); H NMR d
f
5
(
.34–5.30 (m 1H, vinyl), 3.54–3.42 (m, 1H, OCH), 1.79
dt, 3H, J=17.2, 1.4 Hz, 21-Me), 1.01 (s, 3H, 19-Me),
1
9
0
.89 (s, 12H, 18-Me and Si-t-Bu), 0.057 (2ꢃSiMe);
+
F
The enzyme activity for each assay was calculated from
the percent conversioon of substrate to products, and the
results were expressed as percent inhibition of control.
NMR d ꢀ110.32 (q of q, J=17.1, 2.0 Hz). MS (CI,
CH ) m/z (rel intensity) 433 (MH , 10), 432 (12), 431
4
(45), 417 (58), 413 (45), 375 (58), 301 (100). Anal. calcd
for C H FOSi: C, 74.94; H, 10.48. Found: C, 75.20;
H, 10.43.
2
7
45
References and Notes
(
3ꢀ,17Z)-20-fluoropregna-5,17(20)-dien-3-ol (8). In a
fashion analogous to the preparation of 14 was pre-
pared 8 (204 mg, 64%) as a white crystalline solid, after
crystallization from methanol: mp 153–155 C. TLC Rf
1. Hall, P. F. J. Steroid Biochem. Mol. Biol. 1991, 40, 527.
2. Nakajin, S.; Hall, P. F.; Onoda, M. J. Biol. Chem. 1981,
256, 6134.
ꢂ

934
J. P. Burkhart et al. / Bioorg. Med. Chem. 10 (2002) 929–934
3
P. F. J. Biol. Chem. 1984, 259, 3971.
. Nakajin, S.; Shinoda, M.; Haniu, M.; Shively, J. E.; Hall, 27. Sergejew, T.; Hartmann, R. W. J. Enzyme Inhib 1996, 8,
113.
4
5
8
6
. Swinney, D. C.; Mak, A. Y. Biochemistry 1994, 33, 2185.
. Mak, A. Y.; Swinney, D. C. J. Am. Chem. Soc. 1992, 114,
309.
28. Wachter, G. A.; Hartmann, R. W.; Sergejew, T.; Grun,
G. L.; Ledergerber, D. J. Med. Chem. 1996, 39, 834.
29. Chan, F. C. Y.; Potter, G. A.; Barrie, S. E.; Haynes, P. B.;
Rowlands, M. G.; Houghton, J.; Jarman, M. J. Med. Chem.
1996, 39, 3319.
30. Ahmed, S.; Smith, J. H.; Nicholls, P. J.; Whomsley, R.;
Cariuk, P. Drug Des. Discov. 1995, 12, 275.
31. Laughton, C. A.; Neidle, S. J. Med. Chem. 1990, 33, 3055.
32. McCague, R.; Rowlands, M. G.; Barrie, S. E.; Houghton,
J. J. Med. Chem. 1990, 33, 3050.
. Corina, D. L.; Miller, S. L.; Wright, J. N.; Akhtar, M. J.
Chem. Soc. Chem. Commun 1991, 782.
. Miller, S. L.; Wright, J. N.; Corina, D. L.; Akhtar, M. J.
Chem. Soc. Chem. Commun 1991, 157.
. Laughton, C. A.; Neidle, S.; Zvelebil, M. J. J. M.; Stern-
berg, M. J. E. Phys. Res. Commun. 1990, 171, 1160.
. Burke, D. F.; Laughton, C. A.; Neidle, S. Anti-Cancer Drug
Des. 1997, 12, 113.
7
8
9
33. Ahmed, S.; Smith, J. H.; Nicholls, P. J.; Whomsley, R.;
Cariuk, P. Drug Des. Discov. 1995, 13, 27.
10. Auchus, R. J.; Miller, W. L. Mol. Endocrin 1999, 13, 1169.
11. Imai, T.; Globerman, H.; Gertner, J. M.; Kagawa, N.;
34. Rowlands, M. G.; Barrie, S. E.; Chan, F.; Jarman, M.;
McCague, R.; Potter, G. A. J. Med. Chem. 1995, 38, 4191.
35. Hartmann, R. W.; Frotscher, M.; Ledergerber, D.;
Wachter, G. A.; Grun, G. L.; Sergejew, T. F. Arch Pharm.
Pharm. Med. Chem. 1996, 329, 251.
36. Hartmann, R. W.; Bayer, H.; Grun, G.; Sergejew, T.;
Bartz, U.; Mitrengo, M. J. Med. Chem. 1995, 38, 2103.
37. Hartmann, R. W.; Wachter, G. A.; Sergejew, T.; Wurtz, R.;
Duerkop, J. Arch Pharm. Pharm. Med. Chem. 1995, 328, 573.
38. Al-Hamrouni, A. M.; Ahmadi, M.; Nicholls, P. J.; Smith,
H. J.; Lombardi, P.; Pestellini, V. Pharm. Sci. 1997, 3, 259.
39. Li, J.; Li, Y.; Son, C.; Banks, P.; Brodie, A. J. Steroid
Biochem. Mol. Biol. 1992, 42, 313.
Waterman, M. R. J. Biol. Chem. 1993, 268, 19681.
2. De Coster, R.; Wouters, W.; Bruynseels, J. J. Steroid
Biochem. Mol. Biol. 1996, 56, 133.
1
13. Van Wauwe, J. P.; Janssen, P. A. J. J. Med. Chem. 1989,
32, 2231.
14. Bossche, H. V., Moereels, H. In Design of Enzyme Inhi-
biting Drugs; Sandler, M., Smith, H. J., Eds.; Oxford: Oxford,
1
1
1
5
1
994, p 438
5. Santen, R. J.; Brodie, A. M. H. Clin. Oncol 1982, 1, 77.
6. Njar, V. C. O.; Brodie, A. M. H. Curr. Pharm. Des 1999,
, 163.
7. Angelastro, M. R.; Marquart, A. L.; Weintraub, P. M.;
Gates, C. A.; Laughlin, M. E.; Blohm, T. R.; Peet, N. P.
Bioorg. Med. Chem. Lett. 1996, 6, 97.
40. Curran, T. T.; Flynn, G. A.; Rudisell, D. E.; Weintraub,
P. M. Tetrahedron Lett. 1995, 36, 4761.
1
Bey, P.; Blohm, T. R. Biochem. Biophys. Res. Commun. 1989,
8. Angelastro, M. R.; Laughlin, M. E.; Schatzman, G. L.;
41. Weintraub, P. M.; Burkhart, J. P.; Blohm, T. R. US
Patent 5,120,840 1992, Chem. Abstr. 1992, 117, 49009.
42. Weintraub, P. M.; Gates, C. A.; Blohm, T. R. US Patent
5,143,909, 1992, Chem. Abstr. 1992, 116, 214776.
43. Weintraub, P. M.; Burkhart J. P.; Blohm, T. R. US Patent
5,318,961 1992, Chem. Abstr. 1992, 117, 49009.
44. Njar, C. O.; Klus, G. T.; Johnson, H. H.; Brodie, A. M. H.
Steroids 1997, 62, 468.
45. Li, X.; Singh, M.; Luu-The, V.; Cote, J.; Laplante, S.;
Labrie, F. Bioorg. Med. Chem. 1996, 4, 55.
1
1
62, 1571.
9. Stevens, J. C.; Jaw, J.-Y.; Peng, C.-T.; Halpert, J. Bio-
chem. 1991, 30, 3649.
0. Barrie, S. E.; Potter, G. A.; Goddard, P. M.; Hayes, B. P.;
Dowsett, M.; Jarman, M. J. Steroid Biochem. Molec. Biol.
2
1
2
994, 50, 267.
1. Ling, Y.; Li, J.; Liu, Y.; Kato, K.; Klus, G. T.; Brodie,
A. J. Med. Chem. 1997, 40, 3297.
2. Njar, V. C. O.; Kato, K.; Nnane, I. P.; Grigoryev, D. N.;
Long, B. J.; Brodie, A. M. H. J. Med. Chem. 1998, 41, 902.
3. Burkhart, J. P.; Gates, C. A.; Laughlin, M. E.; Resvick,
R. J.; Peet, N. P. J. Bioorg. Med. Chem. 1996, 4, 1411.
4. Potter, G. A.; Barrie, S. E.; Jarman, M.; Rowlands, M. G.
J. Med. Chem. 1995, 38, 2463.
2
46. For 12: NOE at H16a/H16a (in addition to CH
2
OH) upon
pre-irradiation of fluorine at d 109.5, for 13: NOE at H12a
/
H12b and 18-CH (in addition to CH OH) upon pre-irradia-
2
3
2
tion of fluorine at d 124.0.
47. Schatzman, G. L.; Laughlin, M. E.; Blohm, T. R. Anal.
Biochem. 1988, 175, 219.
2
2
1
2
5. Li, J.; Li, Y.; Son, C.; Brodie, A. M. H. J. Med. Chem.
996, 39, 4335.
6. Njar, V. C. O.; Hector, M.; Hartmann, R. W. Bioorg.
48. Moore, R. J., Wilson, J. D. In Methods in Enzymology:
Hormone Action 36; O’Malley, B. W., Hardman, J. G., Eds.;
Academic: New York, 1975; p 466.
Med. Chem. 1996, 4, 1447.