Androgen receptor antagonists and anti-prostate cancer activities of some synthesized steroidal candidates

Article abstract of DOI:10.1248/cpb.59.1363

In continuation of our previous work, a novel series of steroid derivatives were synthesized and their androgen receptor (AR) antagonist activities and in vivo antiandrogenic properties were evaluated. Twenty-one heterocyclic derivatives containing a cyan

Full text of DOI:10.1248/cpb.59.1363

November 2011
Regular Article
Chem. Pharm. Bull. 59(11) 1363—1368 (2011)
1363
Androgen Receptor Antagonists and Anti-prostate Cancer Activities of
Some Synthesized Steroidal Candidates
Saleh Abd El-Rahman BAHASHWAN,a Mohamed Abd El-Rahman AL-OMAR,b Essam EZZELDIN,c
Mohamed Mostafa ABDALLA,d Ahmed Abd El-Hameed FAYED,a and Abdel-Galil El-Sayed AMR*, e, f
a Department of Pharmacy, College of Health Science, Taibah University; Madinah 3893, Saudi Arabia: b Pharmaceutical
Chemistry Department, College of Pharmacy, King Saud University; c Drug Bioavailability Laboratory, College of
e
Pharmacy, King Saud University; Drug Exploration & Development Chair (DEDC), College of Pharmacy, King Saud
University; Riyadh 11451, Saudi Arabia: d Research Unit, Univet Pharmaceutical Co.; Balteem 15324, Egypt: and
f Applied Organic Chemistry Department, National Research Center; Dokki, Cairo 12622, Egypt.
Received July 1, 2011; accepted July 14, 2011; published online August 15, 2011
In continuation of our previous work, a novel series of steroid derivatives were synthesized and their andro-
gen receptor (AR) antagonist activities and in vivo antiandrogenic properties were evaluated. Twenty-one hetero-
cyclic derivatives containing a cyanopyrane ring fused to a steroidal moiety were conveniently synthesized and
screened for their antagonistic, antiandrogen and prostate anticancer activities comparable to that of bicalu-
tamide as the reference control. Some of the compounds exhibited better antagonistic, antiandrogen and prostate
anticancer activities than the reference controls. Initially the acute toxicity of the compounds was assayed via the
determination of their LD50. Synthetic steroidal structures fused to a substituted cyanopyrane ring seem to be a
promising approach in the search for novel leads for potent antagonistic, antiandrogen and prostate anticancer
agents.
Key words steroid; androgen; antagonist; antiandrogen; cancer
Prostate cancer has become the most common cancer and pharmacological evaluation of a series of steroid deriva-
among men, and the second leading cause of male cancer tives fused to a substituted cyanopyrane ring as antagonistic,
deaths in the United States.1) Testosterone and 5a-dihy- antiandrogen and prostate anticancer agents.
drotestosterone are androgens that are required for the devel-
opment of both the normal prostate and prostate cancer.2) Results and Discussion
Androgens act through the androgen receptor (AR), which
Chemistry In continuation of our previous work, a se-
belongs to the steroid-receptor superfamily of ligand-depen- ries of steroid derivatives 1—11 (Fig. 2) were synthesized in
dent transcription factors.3,4) Finasteride (Fig. 1)5) is known advance and screened as anti-inflammatory agents.16) Herein,
to inhibit Type 2 5a-reductase and thus blocks the conver- we used these compounds for evaluation as androgen recep-
sion of testosterone to dihydrotestosterone (DHT). The struc- tor antagonists and anti-prostate cancer agents.
tural similarity of finasteride to DHT raises the possibility
Additionally, some new derivatives were synthesized by re-
that finasteride may also interfere with the function of the an- acting 3-hydroxy-10,13-dimethyl-16-(4-methylbenzylidene)
-
drogen receptor (AR). Androgen ablation therapy remains dodecahydro-1H-cyclopenta[a]phenanthren-17(2H)-one
the gold standard for the treatment of advanced prostate can- 1217,18) with ethyl cyanoacetate in the presence of sodium
cer, but unfortunately it is not curative, and eventually the ethoxide to afford the corresponding cyanopyrane 13, which
disease will return as lethal castration-resistant prostate can- was then reacted with p-toluenesulfonylchloride to afford the
cer (CRPC).6) In our previous work, we found that certain corresponding cyclohexene derivative 14. Moffat oxidation
substituted steroidal derivatives showed androgenic, ana- of compound 13 gave the 3-oxo analogue 15 without affect-
bolic, anticancer and anti-inflammatory activities.7—10) Some ing D5-ene. Oppenauer oxidation of 13 with aluminum iso-
new heterocyclic compounds containing nitrogen and sulfur propoxide resulted in 3-oxo-analogue with delocalization of
atoms have been synthesized and used as antitumor,11) an- D5-ene into D4-ene derivative 16. Modified Oppenauer oxida-
timicrobial,12—14) and anti-inflammatory15) agents. In this tion19) of 13 afforded the corresponding D4,6-3-oxo-analogue
paper we describe the results of our studies on the synthesis 17 (Fig. 3).
Pharmacological Screening All the candidates were
evaluated for their in vitro AR antagonistic activities using a
reporter assay, and the resulting IC50 values are listed in
Table 1. Also, evaluated was the in vivo anti-androgenic ac-
tivity in Castrated Immature Rats, and the resulting % inhibi-
tion values are listed in Table 2. In addition, all the analogues
were evaluated by anti-prostate cancer screening antiandro-
genic bioassay in human prostate cancer cells; the resulting
IC50 values are listed in Table 3.
The target compounds were tested for cytotoxicity against
two human prostate cancer cell lines, LNCaP and PC-3. The
LNCaP cell line is an androgen-dependent human prostate
Fig. 1. Chemical Structure of Finasteride as a 5a-Reductase Inhibitor
cancer cell line that expresses mutant AR, and the PC-3 cell
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: aamr1963@yahoo.com

1364
Vol. 59, No. 11
Fig. 2. Chemical Structures for the Compounds 1—11
Table 1. In Vitro Androgen Receptor (AR) Antagonistic Activities of
Compounds 1—17
Table 2. In Vivo Antiandrogen Activities of Compounds 1—17
Compound No.
% (inhibition)
ED50 (mg/kg)
Compound
No.
Relative potency
to bicalutamide
IC50 (mM)a)
Bicalutamide
75.00ꢀ0.332
76.81ꢀ0.252
76.00ꢀ0.342
83.77ꢀ0.534
83.13ꢀ0.652
80.19ꢀ0.423
78.12ꢀ0.732
85.88ꢀ0.272
85.32ꢀ0.664
88.00ꢀ0.663
87.97ꢀ0.754
77.71ꢀ0.342
84.16ꢀ0.755
82.71ꢀ0.743
86.16ꢀ0.344
87.45ꢀ0.745
87.89ꢀ0.644
77.11ꢀ0.262
83.99ꢀ0.846
81.21ꢀ0.335
85.98ꢀ0.454
87.16ꢀ0.845
87.67ꢀ0.535
1.60
1.51
1.58
1.07
1.09
1.29
1.31
0.87
0.98
0.63
0.65
1.39
1.00
1.19
0.77
0.71
0.66
1.44
1.01
1.21
0.81
0.74
0.69
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
6
Bicalutamide
0.890ꢀ0.31ꢁ10ꢂ1
0.741ꢀ0.63ꢁ10ꢂ3
0.832ꢀ0.74ꢁ10ꢂ3
0.411ꢀ0.33ꢁ10ꢂ4
0.432ꢀ0.44ꢁ10ꢂ3
0.613ꢀ0.52ꢁ10ꢂ3
0.641ꢀ0.63ꢁ10ꢂ3
0.195ꢀ0.19ꢁ10ꢂ4
0.211ꢀ0.23ꢁ10ꢂ4
0.087ꢀ0.81ꢁ10ꢂ5
0.091ꢀ0.72ꢁ10ꢂ5
0.669ꢀ0.74ꢁ10ꢂ3
0.377ꢀ0.24ꢁ10ꢂ4
0.521ꢀ0.52ꢁ10ꢂ3
0.167ꢀ0.27ꢁ10ꢂ4
0.141ꢀ0.34ꢁ10ꢂ5
0.111ꢀ0.56ꢁ10ꢂ5
0.702ꢀ0.85ꢁ10ꢂ3
0.391ꢀ0.22ꢁ10ꢂ4
0.591ꢀ0.51ꢁ10ꢂ3
0.187ꢀ0.18ꢁ10ꢂ3
0.155ꢀ0.63ꢁ10ꢂ5
0.129ꢀ0.45ꢁ10ꢂ5
1
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
6
1.20
1.07
2.17
2.06
1.45
1.39
4.56
4.22
10.23
9.78
1.33
2.36
1.71
5.33
6.31
8.02
1.27
2.27
1.50
4.76
5.74
6.90
7
8
9
7
8
9
10
11
12
13
14
15
16
17
10
11
12
13
14
15
16
17
a) Compounds were tested for their ability to inhibit AR mediated transcriptional
activation using a reporter assay. IC50 values were determined by six experimental runs
in triplicate.

November 2011
1365
Fig. 3. Synthetic Routes for Compounds 13—17
line is an androgen-independent human prostate cancer cell
line that does not express functional AR. All the tested com-
pounds exhibited significant cytotoxicity in either LNCaP or
PC-3 cells.
Here we discuss the anti-androgenic activities of the tested
compounds in vivo and in vitro, aiming to reach the proper
mechanism of action. Firstly we discuss the in vitro AR an-
tagonistic activities, followed by quantification of the in vivo
anti-androgenic activities.
Anti-androgen ability plays an important role in lowering
the androgen level, contributing to the healing from prostate
cancer. Therefore we screened these anti-androgenic com-
pounds for their anti prostate cancer activities, aimed at find-
ing a new lead drug that combine both anti-androgenic and
anti prostate cancer activity, a property long sought that can
revolutionize of prostate cancer therapy.
First of all we have two categories of lead steroidal com-
pounds, the 5a-androstane-16-arylidenes 1a, b (as bio-isoster
for 5a-DHT) and their unsaturated forms androst-5-en-16-
arylidenes 6 and 12, as AR antagonistic and anti-androgenic
starting lead compounds. The descending order of activity is
in the following order 6, 12, 1a and b, which means that the
unsaturated analogues are more active than the reduced 5a-H
analogues (sp2 atoms in the androsten cage provide more ac-
tivity than the sp3 atoms in the androstan cage). Also the at-
Table 3. Anti-prostate Cancer Activities of Compounds 1—17
Cytotoxicity IC50 (mM)
Compound
No.
PC-3
LNCaP
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
6
7
8
9
10
11
12
13
14
0.722ꢀ0.44ꢁ10ꢂ5
0.733ꢀ0.53ꢁ10ꢂ5
0.584ꢀ0.73ꢁ10ꢂ5
0.595ꢀ0.31ꢁ10ꢂ5
0.646ꢀ0.42ꢁ10ꢂ5
0.677ꢀ0.33ꢁ10ꢂ5
0.507ꢀ0.36ꢁ10ꢂ5
0.517ꢀ0.43ꢁ10ꢂ5
0.217ꢀ0.12ꢁ10ꢂ5
0.227ꢀ0.43ꢁ10ꢂ5
0.687ꢀ0.64ꢁ10ꢂ5
0.537ꢀ0.62ꢁ10ꢂ5
0.617ꢀ0.61ꢁ10ꢂ5
0.487ꢀ0.54ꢁ10ꢂ5
0.446ꢀ0.62ꢁ10ꢂ5
0.415ꢀ0.31ꢁ10ꢂ5
0.714ꢀ0.54ꢁ10ꢂ5
0.543ꢀ0.82ꢁ10ꢂ5
0.623ꢀ0.51ꢁ10ꢂ5
0.493ꢀ0.25ꢁ10ꢂ5
0.473ꢀ0.73ꢁ10ꢂ5
0.423ꢀ0.23ꢁ10ꢂ5
0.822ꢀ0.11ꢁ10ꢂ5
0.681ꢀ0.22ꢁ10ꢂ5
0.684ꢀ0.21ꢁ10ꢂ5
0.483ꢀ0.23ꢁ10ꢂ5
0.492ꢀ0.15ꢁ10ꢂ5
0.634ꢀ0.26ꢁ10ꢂ5
0.645ꢀ0.35ꢁ10ꢂ5
0.426ꢀ0.15ꢁ10ꢂ5
0.447ꢀ0.34ꢁ10ꢂ5
0.215ꢀ0.12ꢁ10ꢂ5
0.224ꢀ0.12ꢁ10ꢂ5
0.663ꢀ0.34ꢁ10ꢂ5
0.453ꢀ0.23ꢁ10ꢂ5
0.492ꢀ0.27ꢁ10ꢂ5
0.282ꢀ0.32ꢁ10ꢂ5
0.263ꢀ0.12ꢁ10ꢂ5
0.242ꢀ0.22ꢁ10ꢂ5
0.672ꢀ0.43ꢁ10ꢂ5
0.472ꢀ0.12ꢁ10ꢂ5
0.612ꢀ0.16ꢁ10ꢂ5
0.292ꢀ0.23ꢁ10ꢂ5
0.272ꢀ0.23ꢁ10ꢂ5
0.252ꢀ0.32ꢁ10ꢂ5
0.619ꢀ0.61ꢁ10ꢂ5
15
16
17
Bicalutamide

1366
Vol. 59, No. 11
Table 4. Acute Toxicity (LD50) of the Synthesized Compounds
taching groups that have an -I affect on the arylidene moiety
play an important role in increasing the activity. Regarding
anti-prostate cancer activity, the same occurred.
Compound No.
LD50 (mg/kg)
Fusion of a heterocyclic cyano pyrone ring system onto
ring D in [17,16-C] resulted in increasing both AR antago-
nistic and anti-androgenic activities. Here the descending
order of activity is 7, 13, 2a and b, concluding that the hete-
rocyclic fused ring systems play an important role in increas-
ing the tested activities, but only to a certain extent. Regard-
ing the anti-prostate cancer activity, the same thing occurred,
but activity increases within 15—30%. Removal of a mole-
cule of water from ring A of the steroidal cage lead to forma-
tion of the androstene derivatives 3a, b, 8 and 14. The de-
scending order of activity is 8, 14, 3a and b. Here the activity
is reduced by about 20—25% from their original derivatives
(3b-hydroxyl essential for high anti-androgenic activity). Re-
garding the anti-prostate cancer activity, the same effects
occurred but the activity decreased from 5—15%.
2a
2b
3a
3b
4a
4b
5a
5b
7
8
9
10
11
13
14
15
4.186
2.514
1.711
1.880
3.813
2.681
2.684
2.751
3.111
1.250
3.111
2.868
3.068
2.962
1.750
3.055
2.821
2.870
1.618
16
17
Moffat oxidation of the 3b-hydroxyl in ring A of the
steroidal cage leads to derivatives 4a, b, 9 and 15 that have 3-
fold higher activity than their starting 3b-hydroxy deriva-
Prednisolone
tives, and in the same time as the dehydrated derivatives. The of the synthesized compounds. The dose that killed 50% of
order of activity is 9, 15, 4a and b. Thus, the 3-oxo (sp2) the animals was calculated according to Austen and Brockle-
function plays an important role in increasing anti-andro- hurst.20) (Table 4).
genic activity. Regarding the anti-prostate cancer activity, the
same effects occurred but the increase in activity was to very Conclusion
limited extent, 12—22%. Also, Oppenour oxidation of the
All the tested compounds showed potent in vitro (via an-
3b-hydroxyl in ring A of compounds 7 and 13 lead to com- drogen receptor antagonist) and in vivo anti-androgenic oral
pounds 10 and 16, which had about 2.5 times higher activity activities. Also these compounds showed potent anti-prostate
than their starting compounds confirming the essential role cancer activity. The descending order of activity is 5a, b, 11,
of the 3-oxo function in increasing the activity. The Oppe- 17, 10, 16, 9, 15, 4a, b, 7, 13, 2a, b, 8, 14, 3a, b, 6, 12, 1a, b.
nour oxidation products were more active than the normal
Moffat oxidation products, leading to the conclusion that the vivo anti-androgenic and anti-prostate cancer activities, while
enone system is more active than the normal 3 ketones. compound 12 had the least potent activities and in between,
Compound 5a had high potent in vitro AR antagonistic, in
Regarding anti-prostate cancer activity the same effects the descending order of potency is 5a, b, 11, 17, 10, 16, 9,
had occurred but to a very narrow extent. Dichlorodi- 15, 4a, b, 7, 13, 2a, b, 8, 14, 3a, b, 6, 12, 1a, b. The reduction
cyanoquinone (DDQ)-treatment of 4a, b lead to the forma- of a double bond decreases the activity, so we sought the
tion of the 1,4-diene-3-ones analogues 5a, b, the most potent double bonds essential for high activity, especially for C-4
compounds (i.e. increasing the sp2 hybridization or the unsat- where sp2 hybridization is essential for high potency. Com-
uration or the degree of enone system that diminishes the pounds 1a and b, 6, 12, and 3a and b showed the least po-
probability of keto-enol form formation increases the anti- tency (the lowest is 1), which may be due to the sp3 hy-
androgenic effects). Regarding anti-prostate cancer activity, bridization of C-4 reaching that of the 5-a androgenic char-
the same effects occurred, but the activity increased 2-fold in acters. A heterocyclic ring system fused with ring D resulted
PC-3, and to lower extent in LNCaP.
in an increase in the activities. Replacement of the sp3 of C-1
Modified Oppenour oxidation of both compounds 7 and 13 with sp2 via oxidation of the 3-b hydroxyl into the 3-oxo
leads to the formation of derivatives 11 and 17 with 3-times function increases the anti-androgenic activity. The sp2 hy-
higher activity, which also proves that the conjugation of bridization for both C-3 and C-4 are essential for high activ-
enone is essential for high anti-androgenic activity, but that ity. It is also possible that as the keto-enol form in ring C in-
the conjugation in ring A provides the most active com- creases the activity decreases (compounds 10, 16 are more
pounds. In conclusion, different types of oxidation leads to active than 4a, b). Next, we introduced a substitution group
the following descending activity of the highly active anti- onto the phenyl ring; here the halide with a high inductive
androgenic products 5a, b, 11, 17, 10 and 16.
effect greatly increases the activity, higher than the methyl
DDQ provides more active products than those resulting ones.
from modified Oppenour, while the latter provides more ac-
Structure–Activity Relationship Conjugated 1,4-diene-
tive products than normal Oppenour, whereas, Oppenour 3-one provided more activity than conjugated 4,6-diene-3-
modified products provided more active products than Mof- one.
fat. Regarding anti-prostate cancer activity the same effect
As the degree of unsaturation decreased led to the activity
occurred, but the activity increased 2-fold in LNCaP and to decreased.
lower extent in PC-3.
Decreasing the degree of extended conjugation decreased
Determination of Acute Toxicity (LD50) The LD50 was activity (c.f., derivatives 10, 16 with extended conjugated
determined using rats injected with different increasing doses enone systems were more active than 9, 15 which contained

November 2011
1367
rated under reduced pressure. The formed residue was finally crystallized
no enone extended conjugation).
from acetic acid/water to give the corresponding oxo-compound 15. Yield
3-Oxo function increased the activity more than the 3-hy-
droxyl one, while the latter increased the activity more than
the dehydrated one.
A heterocyclic fused ring system markedly increased the
activities.
Regarding substitution on the aryl moiety, both chloride
and fluoride atoms provided higher activity than the methyl
group.
1
58%; mp 184—86 °C; IR (KBr, cmꢂ1): 2235 (CN), 1725 (CꢃO). H-NMR
(DMSO-d6) d: 0.75 (s, 3H, CH3, C-19), 0.79 (s, 3H, CH3, C-18), 0.96—1.02
(m, 1H, CH), 1.26—1.32 (m, 4H, 2CH2), 1.44—1.62 (m, 6H, 3CH2), 1.68—
1.92 (m, 2H, CH2), 2.10 (m, 1H, CH), 2.25 (s, 3H, CH3), 2.30—2.42 (m,
2H, CH2), 2.47 (m, 1H, CH), 5.56 (m, 1H, CH-olefinic, C-6), 7.22—7.35
(m, 4H, Ar-H). MS m/z (%): 453 (Mꢄ, 32), corresponding to the molecular
formula C30H31NO3 and at 229 (100, base peak). Elemental analysis: Calcd:
C, 79.44; H, 6.89; N, 3.09. Found: C, 79.36; H, 6.82; N, 3.00.
2-Oxo-3-cyano-6-(4-chlorophenyl)androst-4-ene[17,16-c]pyran-3-one
(16) (Oppenauer Oxidation) To a solution of compound 13 (6.9 mmol) in
a mixture of cyclohexanone (50 ml)/dry benzene (45 ml), freshly distilled
aluminum isopropoxide (2 g, 9.7 mmol) in benzene (5 ml) was added. The
reaction mixture was refluxed for 16 h. The reaction mixture was treated
dropwise with water (4 ml) and the precipitated aluminum salt was collected
by filtration. The filtrate was evaporated under reduced pressure and the ob-
tained residue was crystallized from methanol to give the corresponding oxi-
dized derivative 16. Yield 65%; mp 142—143 °C; IR (KBr, cmꢂ1): 2236
Experimental
Chemistry Melting points were determined on open glass capillaries
using an Electrothermal IA 9000 SERIES digital melting point apparatus
(Electrothermal, Essex, U.K.), and are uncorrected. Elemental analyses were
performed with all final compounds on an Elementar, Vario EL, Microana-
lytical Unit, National Research Centre, Cairo Egypt, and the results were
found within ca. 0.4% of the theoretical values. Analytical data were ob-
tained from the Microanalytical Unit, Cairo University, Egypt. The IR spec-
tra (KBr) were recorded on a FT IR-8201 PC spectrophotometer (Shimadzu,
Tokyo, Japan). The 1H-NMR spectra were measured with a JEOL FTGNM-
EX 270, 270 MHz instrument (JEOL, Tokyo, Japan) in DMSO-d6, and
chemical shifts were recorded in (d, ppm) relative to tetramethylsilane
(TMS). The mass spectra were run at 70 eV with a Finnigan SSQ 7000 spec-
trometer (Thermo Electron Corporation, Madison, WI, U.S.A.) using EI, and
the values of m/z are indicated in Dalton. TLC (Silica gel, aluminum sheets
60F254, Merck, Darmstadt, Germany) followed the reactions. The starting
material 12 was prepared according to the published procedure.17,18)
2-Oxo-3-cyano-6-(4-chlorophenyl)androst-5-ene[17,16-c]pyran-3b-ol
(13) A mixture of 12 (3.90 g, 10 mmol) and ethyl cyanoacetate (1.27 ml,
12 mmol) in 25 ml sodium ethoxide (920 mg sodium metal in 25 ml absolute
ethanol) was refluxed for 7 h. The reaction mixture was poured into ice
water, and the obtained solid was filtered off, washed with water, dried, and
crystallized from ethanol to give the corresponding pyrano-steroidal deriva-
tives 13. Yield 72%; mp 210—212 °C; IR (KBr, cmꢂ1): 3524—3412 (OH),
1
(CN), 1758 (CꢃO, enone), 1726 (CꢃO, ketone). H-NMR (DMSO-d6) d:
0.75 (s, 3H, CH3, C-19), 0.80 (s, 3H, CH3, C-18), 0.98—1.10 (m, 1H, CH),
1.24—1.32 (m, 4H, 2CH2), 1.45—1.55 (m, 4H, 2CH2), 1.62—1.75 (m, 4H,
2CH2), 1.82 (m, 1H, CH), 1.88—2.05 (m, 2H, CH2), 2.22 (s, 3H, CH3),
2.32—2.42 (m, 1H, CH), 5.73 (s, 1H, CH-olefinic, C-4), 7.23—7.38 (m, 4H,
Ar-H). MS m/z (%): 453 (Mꢄ, 100, base peak), corresponding to the molec-
ular formula C30H31NO3. Elemental analysis: Calcd: C, 79.44; H, 6.89; N,
3.09. Found: C, 79.35; H, 6.84; N, 3.01.
3-Oxo-3-cyano-6-(4-chlorophenyl)androst-4,6-diene[17,16-c]pyrane-3-
one (17) The compound was prepared according to the Wettstein method
(Modified Oppenauer)19,21) using compound 13 as the starting material.
Yield 68%; mp 152—153 °C (AcOH/H2O); IR (KBr, cmꢂ1): 2236 (CN),
1
1767 (CꢃO, enone), 1724 (CꢃO, ketone). H-NMR (DMSO-d6) d: 0.76 (s,
3H, CH3, C-19), 0.84 (s, 3H, CH3, C-18), 1.02—1.12 (m, 1H, CH), 1.24—
1.34 (m, 2H, CH2), 1.43—1.54 (m, 2H, CH2), 1.64—1.76 (m, 4H, 2CH2),
1.79 (m, 1H, CH), 1.82—2.04 (m, 2H, CH2), 2.15 (s, 3H, CH3), 2.28—2.42
(m, 1H, CH), 5.77 (s, 1H, CH-olefinic, C-4), 6.18 (m, 1H, CH-olefinic, C-7),
6.74 (d, 1H, CH-olefinic, C-6), 7.23—7.28 (m, 4H, Ar-H). MS m/z (%): 451
(Mꢄ, 18), corresponding to the molecular formula C30H29NO3 and at 292
(100, base peak). Elemental analysis: Calcd: C, 79.80; H, 6.47; N, 3.10.
Found: C, 79.75; H, 6.40; N, 3.04.
1
2234 (CN), 1722 (CꢃO). H-NMR (DMSO-d6) d: 0.77 (s, 3H, CH3, C-19),
0.82 (s, 3H, CH3, C-18), 0.88—1.05 (m, 1H, CH), 1.23—1.31 (m, 4H,
2CH2), 1.44—1.62 (m, 6H, 3CH2), 1.68—1.82 (m, 2H, CH2), 1.96 (m, 1H,
CH), 2.18 (s, 3H, CH3), 2.28—2.48 (m, 2H, CH2), 2.62 (m, 1H, CH), 3.64
(m, 1H, 3a-CH), 5.66 (m, 1H, CH-olefinic, C-6), 7.32—7.44 (m, 4H, Ar-H),
10.46 (s, 1H, OH, exchangeable with D2O). MS m/z (%): 455 (Mꢄ, 15), cor-
responding to the molecular formula C30H33NO3 and at 279 (100, base
peak). Elemental analysis: Calcd: C, 79.09; H, 7.30; N, 3.07. Found: C,
79.00; H, 7.24; N, 3.00.
Pharmacology Screening. Evaluation of Transcriptional Activity for
Human Androgen Receptor22) (a) Establishment of Chinese Hamster
Ovary (CHO) Cells Stably Transfected with Human Androgen Receptor
Gene and MMTV-Luciferase Reporter Gene or SV40-Luciferase Gene
CHO cells were maintained in alpha-modified Eagle’s medium supple-
mented with 10% fetal bovine serum (FBS). The culture medium of
neomycin-resistant clone cells was supplemented with 10% dextran-coated
charcoal-stripped FBS (DCC-FBS) and 500 mg/ml of neomycin. The CHO
cells were transfected at 40—70% confluence in 10-cm petri dishes with a
total of 20 mg DNA (pMAMneoLUC; MMTV-luciferase reporter plasmid
and pSG5-hAR; human androgen receptor expression plasmid, or SV40-
LUC; SV40-luciferase reporter plasmid containing neomycin resistant gene)
by calcium phosphate mediated transfection. The stable transfected cells
were selected in the culture medium supplemented with neomycin. The
selected clone was designated as AR/CHO#3 (human AR gene and MMTV-
luciferase reporter gene integrated CHO cell) or SV/CHO#10 (SV-40-
luciferase reporter gene integrated CHO cell), respectively.
(b) Activities of the Tested Compounds to Inhibit Androgen Receptor
Mediated Transcription Induced by DHT (AR Antagonistic Activity)
The stable transfected AR/CHO#3 or SV/CHO#10 cells were plated onto
96-well luminoplates (Packard) at a density of 2ꢁ104 cells/well, respec-
tively. Four to eight hours later, the medium was changed to a medium con-
taining DMSO, 0.3 nM of DHT, or 0.3 nM of DHT, and the tested compound.
At the end of incubation, the medium was removed and the cells were lysed
with 20 ml of lysis buffer [25 mM Tris–HCl (pH 7.8), 2 mM dithiothreitol,
2 mM 1,2-cyclo-hexanediamine-tetraacetic acid, 10% glycerol and 1% Triton
X-100]. Luciferase substrate [20 mM Tris–HCl (pH 7.8), 1.07 mM (MgCO3)4
Mg-(OH)2·5H2O, 2.67 mM MgSO4·7H2O, 0.1 mM ethylenediaminetetra-
acetic acid (EDTA), 33.3 mM dithiothreitol, 0.27 mM CoA, 0.47 mM luciferin,
0.53 mM ATP] was added, and luciferase activity was measured with a
ML3000 luminometer (Dynatech Laboratories). AR antagonistic activities
were calculated by the formula below:
2-Oxo-3-cyano-6-(4-chlorophenyl)androst-3,5-diene[17,16-c]pyrane
(14) A mixture of compound 13 (2.24 g, 5 mmol), p-toluene sulfonyl chlo-
ride (0.4 g, 5 mmol), and triethylamine (1 ml) in dry benzene (15 ml) was re-
fluxed for 2 h. The solvent was evaporated under reduced pressure. The ob-
tained residue was solidified with water and the solid formed was filtered
off, washed with water, dried, then dissolved in dry benzene (15 ml), and
potassium tert-butoxide (25 ml, 0.5 N, in dimethyl sulfoxide (DMSO)) was
added. The reaction mixture was heated at 50 °C for 5 h, The formed solid
was filtered off and crystallized from dioxane to give the corresponding oxi-
dized cyanopyrane derivative 14. Yield 75%; mp 168—170 °C; IR (KBr,
cmꢂ1): 2238 (CN), 1726 (CꢃO). 1H-NMR (DMSO-d6) d: 0.76 (s, 3H, CH3,
C-19), 0.82 (s, 3H, CH3, C-18), 0.88—0.96 (m, 1H, CH), 1.18—1.26 (m,
4H, 2CH2), 1.40—1.55 (m, 4H, 2CH2), 1.60—1.76 (m, 2H, CH2), 1.92 (m,
1H, CH), 2.16 (s, 3H, CH3), 2.30—2.42 (m, 2H, CH2), 2.58 (m, 1H, CH),
5.28 (m, 1H, CH-olefinic, C-3), 5.54 (m, 1H, CH-olefinic, C-4), 5.68 (m,
1H, CH-olefinic, C-6), 7.32—7.52 (m, 4H, Ar-H). MS m/z (%): 437 (Mꢄ,
24), corresponding to the molecular formula C30H31NO2 and 277 (100, base
peak). Elemental analysis: Calcd: C, 82.35; H, 7.14; N, 3.20. Found: C,
82.30; H, 7.08; N, 3.12.
2-Oxo-3-cyano-6-(4-chlorophenyl)androst-5-ene[17,16-c]pyran-3-one
(15) The appropriate compound 13 (0.92 g, 2 mmol) was dissolved in a
mixture of benzene (3 ml), dimethylsulphoxide (3 ml), pyridine (0.16 ml),
and trifluoroacetic acid (TFA, 0.08 ml). Dicyclohexylcarbodiimide (1.24 g,
6 mmol) was then added and the reaction mixture was kept overnight at
room temperature. Ether (50 ml) was added, followed by oxalic acid (0.54 g,
6 mmol) in methanol (50 ml). After 30 min, water (50 ml) was added and the
obtained dicyclohexyl urea was removed by filtration. The filtrate was ex-
tracted with ether, washed with 5% sodium bicarbonate, then with water.
The ethereal solution was dried over anhydrous sodium sulphate and evapo-
AR antagonistic activity (%)ꢃ100(IꢂX)/(IꢂB)

1368
Vol. 59, No. 11
I: (luciferase activity of AR/CHO#3)/(luciferase activity of SV/CHO#10) in
the presence of 0.3 nM of DHT,
B: (luciferase activity of AR/CHO#3)/(luciferase activity of SV/CHO#10) in
the presence of DMSO,
X: (luciferase activity of AR/CHO#3)/(luciferase activity of SV/CHO#10) in
the presence of 0.3 nM of DHT and the tested compound.
rived data were expressed as relative luciferase activity normalized to the in-
ternal luciferase control. Cells cultured in medium containing DHT (andro-
gen), as a positive control, induced a marked increase in reporter gene
expression. Test compounds capable of significantly suppressing this DHT-
induced reporter gene expression were identified as potential antiandrogens.
The concentration of compounds showing 50% AR antagonistic activity,
or IC50 values, were obtained by nonlinear analysis using a statistical analy-
sis system (SAS).
Acknowledgement The authors extend their appreciation to the Dean-
ship of Scientific Research at King Saud University for funding this work
through the research group project No. RGP-VPP-099.
In Vivo Evaluation of Antiandrogenic Activities in Castrated Imma-
ture Rats22) Andorgen treated male Wistar rats were obtained from the
Animal House Colony, Research Institute of Ophthalmology, Giza, Egypt.
Prepubertal male rats aged 3 weeks were castrated by the scrotal route under
ether anesthesia. Three days after the castration, testosterone propionate (TP,
0.5 mg/kg, subcutaneously (s.c.)) was administered once daily for 5#days,
alone or in combination with the tested compound (10—30 mg/kg, per os
(p.o.)). TP was dissolved in cotton seed oil containing 5% ethanol. The
tested compound was suspended with 0.5% methylcellulose. The rats were
sacrificed by excessive chloroform anesthesia 6 h after final dosing, and both
their ventral prostates and seminal vesicles-coagulate glands were removed
and weighed. The antiandrogenic activity was expressed as a percentage of
inhibition of the TP effect (TP-treated rats were arbitrarily assigned a value
of 0% and vehicle-treated rats a value of 100%).
Anti-prostate Cancer Screening Antiandrogenic Bioassay in Human
Prostate Cancer Cells23) Human prostate cancer LNCaP and PC-3 cells
were maintained in RPMI medium and Dulbecco’s minimum essential
medium (DMEM), respectively. Both media were supplemented with peni-
cillin (25 units/ml), streptomycin (25 mg/ml), and 10% fetal calf serum. For
the androgen receptor transactivation assay, an androgen-dependent reporter
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