Synthesis and biological evaluation of 16E-arylidenosteroids as cytotoxic and anti-aromatase agents

Article abstract of DOI:10.1248/cpb.59.327

Taking into consideration the structural requirements for cytotoxicity and aromatase inhibition, several new 16E-arylidenosteroidal derivatives have been prepared and evaluated for their cytotoxic and aromatase inhibitory activity. The new steroidal analo

Full text of DOI:10.1248/cpb.59.327

March 2011
Regular Article
Chem. Pharm. Bull. 59(3) 327—331 (2011)
327
Synthesis and Biological Evaluation of 16E-Arylidenosteroids as Cytotoxic
and Anti-aromatase Agents
Ranju BANSAL, ,a Sheetal GULERIA,a Sridhar THOTA,a Rolf Wolfgang HARTMANN,b and
*
b
Christina ZIMMER
a University Institute of Pharmaceutical Sciences, Sector-14, Panjab University; Chandigarh–160014, India: and
b Pharmaceutical and Medicinal Chemistry, Saarland University; D-66041, Saarbrücken, Germany.
Received September 2, 2010; accepted December 27, 2010; published online January 5, 2011
Taking into consideration the structural requirements for cytotoxicity and aromatase inhibition, several new
16E-arylidenosteroidal derivatives have been prepared and evaluated for their cytotoxic and aromatase in-
hibitory activity. The new steroidal analogues 3, 5—8 and 11 exhibited significant cytotoxic effects when screened
against three cancer cell lines, MCF-7 (breast), NCl-H460 (lung) and SF-268 central nervous system (CNS) at
100 mM and sensible cytotoxic effects subsequently in sixty cancer cell lines derived from nine cancers types
(leukemia, lung, colon, CNS, melanoma, ovarian, renal, prostate and breast cancers). The imidazolyl substituted
steroidal derivatives 5 and 7 exhibited strong inhibition of the aromatase enzyme with 16-[4-{3-(imidazol-1-
yl)propoxy}-3-methoxybenzylidene]-5-androstene-3b,17b-diol (7) displaying 13 times more potency in compari-
son to aminoglutethimide.
Key words aromatase inhibitory activity; 16E-arylidenosteroid; dehyroepiandrosterone; cytotoxic activity; cancer cell line;
breast cancer
New targets are being focused by medical chemist with the 16E-arylidenosteroids possessing an imidazole group to ob-
aim to provide new specific and potent drugs for the treat- tain dual cytotoxic as well as aromatase inhibitory effects.
ment of cancer. Mammary tumors represent the most wide-
spread type of malignant neoplasm and most common cause Results and Discussion
of cancer in women between the ages of 30—541) and is the
The synthetic routes to the preparation of various new
second leading cause of cancer deaths in women today (after steroidal derivatives have been outlined in Charts 1—3. Base
lung cancer). About half of these malignancies require a catalyzed aldol condensation of dehydroepiandrosterone
source of estrogens for their growth and development. Estro- (DHA) with 4-(3-chloropropoxy)-3-methoxybenzaldehyde
gens are biosynthesized from androgens by the microsomal
cytochrome P-450 enzyme system termed aromatase.2,3) Inhi-
bition of aromatase is an important approach to reducing
growth stimulatory effects of estrogens in estrogen-depend-
ent breast cancer.4) Effective aromatase inhibitors have been
developed as therapeutic agents for controlling estrogen-de-
pendent breast cancer. Aromatase inhibitors (AIs) can reduce
estrogen production by more than 90% and in addition AIs
lack estrogen-agonist activity unlike tamoxifen, the most
widely used antiestrogen for the management of breast can-
cer.5) Recent clinical data have also shown that these in-
hibitors have greater efficacy than tamoxifen in late-stage
disease and preliminary data indicate that this efficacy ex-
tends to early disease. Aromatase inhibitors therefore almost
certainly replace tamoxifen as the hormonal agents of choice
for the treatment of breast cancer.5,6)
Despite the success of the third-generation steroidal and
nonsteroidal AIs, they still have some major side effects,
such as the increase of bone loss. For this reason, it is impor-
tant to search for other potent and specific molecules with
lower side effects. Taking into consideration the significance
of azole groupings of many specific and potent P450 in-
hibitors including aromatase,7) we have introduced imidazole
group in androstane nucleus in the present study.
A large number of potent steroidal derivatives with substi-
tution at position 16 have been described in the literature as
potent cytotoxic agents.8—10) Recent work from our labora-
Reagents and reaction conditions: (a) MeOH, KOH, RT; (b) Al(t-BuO)3, cyclohexa-
none, reflux, 5 h; (c) imidazole, fusion 110—120 °C, 5 h; (d) (CH3CO)O/dry pyridine,
steam bath, 2 h.
tory has also demonstrated the effectiveness of 16E-aryli-
denosteroids as potential antitumour agents.11—13) These ob-
servations prompted us to prepare and study some more new Chart 1. Synthetic Protocol of the Compounds 1—6
∗ To whom correspondence should be addressed. e-mail: ranju29in@yahoo.co.in
© 2011 Pharmaceutical Society of Japan

328
Vol. 59, No. 3
(1) was carried out at room temperature to afford 2. The room temperature afforded 3b-pyrrolidino-5-androsten-17b-
aldol product 2 showed prominent infrared vibrational bands ol derivative 10. 1H-NMR spectrum displayed a broad singlet
1
at 3220.0 (O–H) and 1709.1 (CꢀO) cmꢁ1 and H-NMR sig- for N-methylenes of pyrrolidino functionality at d 2.61 ppm.
nals at d 3.77 (t, 2H, –CH2Cl) and 7.38 ppm (s, 1H, vinylic- Further, acetylation of compound 10 with acetic anhydride
H, 16-arylidene). The configuration at C16 with respect to the using dry pyridine as catalyst afforded the 17b-acetoxy de-
carbonyl at C17 has been assigned E in analogy with earlier rivative 11. The characteristic IR band for acetoxy group was
reports.11) Oppenauer oxidation of 5-ene-3-hydroxy chloro present at 1725.6 cmꢁ1.
product 2 resulted in the formation of 4-ene-3-keto analogue
Biological Activity All newly synthesized 16-arylideno
4. Repeated efforts to prepare imidazolyl substituted steroid steroidal derivatives, selected for screening by NCI, exhib-
5 from 4 remained unsuccessful. Therefore an alternative ited significant cytotoxic effects in all the three cell lines at
route was followed to prepare 5, 16-arylidene steroid 2 was 100 mM and sensible cytotoxic effects in sixty cancer cell
first thermally fused with powdered imidazole to obtain the lines as shown in Tables 1 and 2. In general, it is observed
imidazolyl substituted product 3, which on Oppenauer oxida- that 16-arylidene group in steroids represents a good phar-
tion gave target 4-ene-3-keto steroid 5 (Chart 1). Presence of macophore for cytotoxic activity. Although introduction of a
imidazolyl protons at d 6.93 (5-CH), 7.06 (4-CH), 7.48 (2- pyrrolidine group at 3 position of 16-arylidene steroid skele-
1
CH) and a triplet at 3.97 ppm for CH2Nꢂ in the H-NMR ton again resulted in loss of cytotoxicity as is shown by data
spectrum confirmed the formation of 3. A down field shift of of 10 and which is also in agreement with earlier reports
4-CH proton (d 5.76 ppm) was observed for 3-keto steroid 5 from our laboratory,11) its 17-acetoxy 3-pyrrolidinyl counter-
in comparison to 5-CH of 3-hydroxy derivative 3, which res- part 11 displayed good cytotoxic effects. It implies that pres-
onated at d 5.39 ppm. Acetylation of 3 afforded correspon- ence of acetoxy group at 17 position is favorable for activity
ding acetoxy compound 6.
Reduction of compound 3 using sodium borohydride in
methanol at room temperature afforded 3b,17b-diol deriva-
tive 7 (Chart 2).
The broad band for O–H stretching vibration at
3235.9 cmꢁ1 and 1H-NMR signals for 3a-H and 17a-H were
observed at d 3.53 and 4.06 ppm, respectively. The proton of
methine bridge was found at an upfield position in 7 (d
6.45 ppm) in comparison to its parent 17-keto derivative 3 (d
7.38 ppm). Acetylation of 3b,17b-diol derivative 7 afforded
corresponding acetoxy compound 8. The methyl proton of
acetoxy group resonated at d 2.00 ppm and CꢀO stretching
absorption was seen at 1733.7 cmꢁ1 in IR spectra. The me-
thine-bridged proton was found further at an upfield value (d
6.15 ppm) as compared to its 17b-diol counterpart 7 in case
of compound 8.
For the preparation 3-pyrrolidinyl substituted 16-arylidene
steroidal derivatives, 5 was heated under reflux with pyrroli-
Reagents and reaction conditions: (a) pyrrolidine/MeOH; (b) NaBH4; (c)
(CH3CO)O/dry pyridine, steam bath, 2 h.
Chart 3. Synthetic Protocol of the Compounds 9—11
dine in methanol to yield an unstable dienamine 9 as shown
in Chart 3, which upon sodium borohydride reduction at
Table 1. Growth Percentage at 10ꢁ4 M Concentration in 3-Cell Line in
Vitro Cytotoxocity Screening
Growth percentage
Compound
(code)
S. No.
NSC No.
Non-small
cell lung
(NCI-H460)
Breast
(MCF-7)
CNS
(SF-268)
1
2
3
4
5
6
7
3a)
(DPJ-RG-1151)
5a)
728325
728324
730461
730478
730479
728326
730460
9
8
0
0
1
1
(DPJ-RG-1177)
6a)
4
1
7
(DPJ-RG-1196)
7a)
0
0
16
11
108
0
(DPJ-RG-1219)
8a)
1
0
(DPJ-RG-1227)
10
83
0
76
0
(DPJ-RG-1179)
11a)
Reagents and reaction conditions: (a) NaBH4; (b) (CH3CO)O/dry pyridine,
steam bath, 2 h.
(DPJ-RG-1195)
Chart 2. Synthetic Protocol of the Compounds 7 and 8
a) 3-Cell line actives.

March 2011
329
Table 2. Mean Log Dose Response Parameters such as GI50, TGI and
LC50 of the 60-Cell Line Assay
Experimental
Chemistry Melting points were determined on a Veego melting point
apparatus and are uncorrected. IR (wavenumber in cmꢁ1) spectra were taken
on a Perkin-Elmer spectrum RX 1 FT-IR spectrophotometer model using
S.
Compound No.
(code)
Mean log10
GI50 (M)
Mean log10
TGI (M)
Mean log10
LC50 (M)
1
No.
KBr pellets. H-NMR spectra were recorded on Bruker AC-300F, 300 MHz
using deuterated-chloroform (CDCl3) or deuterated dimethylsulfoxide
(DMSO-d6) containing tetramethylsilane as internal standard (chemical shift
in d, ppm). Elemental analyses were carried out on a Perkin-Elmer-2400
CHN elemental analyzer. Plates for thin layer chromatography (TLC) were
prepared with silica gel G according to method of Stahl (E. Merck) using
ethyl acetate as solvent and activated at 110 °C for 30 min. Iodine was used
to develop the TLC plates. Anhydrous sodium sulphate was utilized as dry-
ing agent. All solvents were freshly distilled and dried prior to use according
to standard procedures. All solvents were freshly distilled and dried prior to
use according to standard procedures.
1
2
3
4
5
6
3
ꢁ4.74
ꢁ5.31
ꢁ5.02
ꢁ5.53
ꢁ5.10
ꢁ6.49
ꢁ4.36
ꢁ4.73
ꢁ4.62
ꢁ4.90
ꢁ4.70
ꢁ5.40
ꢁ4.09
ꢁ4.26
ꢁ4.38
ꢁ4.47
ꢁ4.42
ꢁ4.69
(DPJ-RG-1151)
5
(DPJ-RG-1177)
6
(DPJ-RG-1196)
7
(DPJ-RG-1219)
8
(DPJ-RG-1227)
11
Synthesis of 16-[4-(3-Chloropropoxy)-3-methoxybenzylidene]-17-oxo-
5-androsten-3b-ol (2) (DPJ-RG-1150) 1-Bromo-3-chloropropane (6.57
mmol) was added to a stirred and refluxing suspension of vanillin (6.57
mmol) and anhydrous potassium carbonate (2 g) in ethyl methyl ketone
(100 ml). The reaction mixture was further refluxed for 6 h with continuous
stirring. The completion of the reaction was monitored by TLC. The reac-
tion mixture was cooled, filtered and the excess of solvent was removed
under reduced pressure to obtain an oily residue of 4-(3-chloropropoxy)-3-
methoxybenzaldehyde (1),14) which was used as such for further reaction.
A mixture of dehydroepiandrosterone (DHA) (2.60 mmol), above ob-
tained oily residue 1 and sodium hydroxide (1 g) in methanol (10 ml) was
stirred at room temperature for 2 h and the completion of reaction was moni-
tored by TLC. The product was precipitated by addition of cold water and
the precipitate obtained was filtered, washed with water, dried and crystal-
lized from methanol to yield 2.
(DPJ-RG-1195)
Table 3. Aromatase Inhibitory Data of Various Compounds
S. No.
Compound (code)
Inhibition on CYP 19a)
48% inhibition at 36 mM
26% inhibition at 36 mM
IC50ꢀ4.4 mM
RPb)
1
2
3
4
5
3
(DPJ-RG-1151)
4
(DPJ-RG-1176)
5
6.8
(DPJ-RG-1177)
1
6
46% inhibition at 36 mM
IC50ꢀ2.4 mM
Yield: 76.96%. mp: 210—212 °C. H-NMR (CDCl3) d: 0.98 (3H, s, 18-
(DPJ-RG-1196)
7
(DPJ-RG-1219)
CH3), 1.08 (3H, s, 19-CH3), 2.31 (2H, m, –OCH2CH2CH2Cl), 3.53 (1H, m,
3a-H), 3.77 (2H, t, –CH2Cl), 3.89 (3H, s, –OCH3), 4.21 (2H, t, –OCH2–),
5.40 (1H, d, 6-CH), 6.95 (1H, d, Joꢀ8.27 Hz, 5-CH, aromatic), 7.06 (1H, d,
Jmꢀ1.73 Hz, 2-CH, aromatic), 7.16 (1H, dd, Jmꢀ1.73 Hz, Joꢀ8.45 Hz, 6-
CH, aromatic), 7.38 (1H, s, vinylic-H, 16-arylidene). FT-IR nmax (KBr)
cmꢁ1: 3220, 2922, 2829, 1709, 1623, 1593, 1515, 1447, 1325, 1260, 1140,
1094, 1057, 1023, 916, 806. Anal. Calcd for C30H39O4Cl: C, 72.20; H, 7.88.
Found: C, 72.40; H, 8.02.
12.4
a) [1b,2b-3H]testosterone. b) Relative potencyꢀrelative to aminoglutethimide
(RPꢀ1; IC50ꢀ28.5 mM).
and may lead to maintenance of cytotoxic effects even if un-
16-[4-{3-(Imidazol-1-yl)propoxy}-3-methoxybenzylidene]-17-oxo-5-
favorable 3-pyrrolidinyl group is present as is depicted by cy- androsten-3b-ol (3) (DPJ-RG-1151) A mixture of 2 (1 mmol) and pow-
dered imidazole (10 mmol, in excess) was fused at 110—120 °C for 5 h. The
completion of reaction was monitored by TLC. The reaction was quenched
with cold water and solid obtained was filtered, washed with water, dried and
totoxic effects of 11 and also the earlier studies.
Four imidazolyl substituted 16-arylideno steroids were
screened for aromatase inhibitory activity and the data is pre-
crystallized from ethyl acetate to yield 3.
1
sented in Table 3. It is anticipated that imidazole group pos-
sessing a sterically available N will be able to interact with
the active site of aromatase by complexing the Fe(III) iron of
cytochrome P450. Of these compounds, 4-ene-3-keto 5
(IC50ꢀ4.4 mM) and diol derivative 7 (IC50ꢀ2.4 mM) exhibited
strong inhibition of the enzyme in comparison to 3-hydroxy
(3) and 3-acetoxy (6) substituted steroids. 16-[4-{3-(Imida-
zol-1-yl)propoxy}-3-methoxybenzylidene]-5-androstene-
3b,17b-diol (7) was found to be 13 times more potent in
comparison to aminoglutethimide. It is observed that struc-
tural modifications of steroids lead to changes in three di-
mensional attachments of the compounds with the enzyme
site.
Yield: 71.47%. mp: 199—201 °C. H-NMR (CDCl3) d: 0.98 (3H, s, 18-
CH3), 1.07 (3H, s, 19-CH3), 2.30 (2H, m, –OCH2CH2CH2Nꢂ), 3.54 (1H, m,
3a-H), 3.91 (3H, s, –OCH3), 3.97 (2H, t, –CH2Nꢂ), 4.23 (2H, t, –OCH2–),
5.39 (1H, d, 6-CH), 6.85 (1H, d, Joꢀ8.33 Hz, 5-CH, aromatic), 6.93 (1H, s,
5-CH, imidazole), 7.06 (2H, d, 2-CH, aromatic and 4-CH, imidazole), 7.13
(1H, d, Joꢀ8.26 Hz, 6-CH, aromatic), 7.38 (1H, s, vinylic-H, 16-arylidene)
and 7.48 (1H, s, 2-CH, imidazole). FT-IR nmax (KBr) cmꢁ1: 3215, 2934,
1700, 1593, 1514, 1463, 1329, 1260, 1143, 1066, 917, 832. Anal. Calcd for
C33H42N2O4: C, 74.68; H, 7.98; N, 5.28. Found: C, 74.62; H, 7.82; N, 5.31.
General Procedure for the Synthesis of Compounds 4 and 5 Com-
pounds 2 and 3 (2 mmol) were dissolved in a mixture of cyclohexanone
(10 ml) and dry toluene (150 ml). Traces of moisture were removed by
azeotropic distillation. The distillation was continued at a slow rate while
adding a solution of aluminium isopropoxide (1 g) in dry toluene (15 ml)
drop wise. The reaction mixture was refluxed for 5 h and allowed to stand at
room temperature overnight. The slurry was filtered and the residue was
washed thoroughly with dry toluene. The combined filtrate and the washings
were steam distilled until the removal of organic solvents was affected. The
Conclusion
It is concluded that 16-arylidene steroids represent an im- solid obtained was filtered, washed with water, dried and treated with diethyl
ether and n-hexane to furnish the corresponding 4-ene-3-keto steroids 4 and
5.
portant pharmacophore for anticancer activity. Suitable struc-
tural modifications in steroid skeleton may lead to com-
pounds with dual cytotoxic and antiaromatase properties.
Further, the study provides new evidence showing the rela-
tionship between the chemical structure and biological func-
tion.
16-[4-{3-(Chloropropoxy)}-3-methoxybenzylidene]-4-androstene-3,17-
dione (4) (DPJ-RG-1176) Yield: 70.28%. mp: 165—167 °C. 1H-NMR
(CDCl3) d: 0.96 (3H, s, 18-CH3), 1.24 (3H, s, 19-CH3), 2.32 (2H, m,
–OCH2CH2CH2Cl), 3.76 (2H, t, –CH2Cl), 3.85 (3H, s, –OCH3), 4.14 (2H, t,
–OCH2–), 5.65 (1H, s, 4-CH), 6.84 (1H, d, Joꢀ8.39 Hz, 5-CH, aromatic),
6.95 (1H, d, Jmꢀ1.14 Hz, 2-CH, aromatic), 7.04 (1H, d, Joꢀ8.51 Hz, 6-CH,
aromatic), 7.26 (1H, s, vinylic-H, 16-arylidene). FT-IR nmax (KBr) cmꢁ1
2939, 1709, 1665, 1614, 1594, 1511, 1464, 1421, 1328, 1262, 1141, 1095,
:

330
Vol. 59, No. 3
1030, 934, 864, 807. Anal. Calcd for C30H37O4Cl: C, 72.49; H, 7.50. Found: 73.52; H, 7.95; N, 5.01.
C, 72.35; H, 7.62.
16-[4-{3-(Imidazol-1-yl)propoxy}-3-methoxybenzylidene]-5-an-
16-[4-{3-(Imidazol-1-yl)propoxy}-3-methoxybenzylidene]-4-an-
drostene-3,17-dione (5) (DPJ-RG-1177) Yield: 75.28%. mp: 107—
109 °C. H-NMR (CDCl3) d: 1.01 (3H, s, 18-CH3), 1.25 (3H, s, 19-CH3),
2.26 (2H, m, –OCH2CH2CH2Nꢂ), 3.92 (3H, s, –OCH3), 3.97 (2H, t, –OCH2CH2CH2Nꢂ), 3.89 (3H, s, –OCH3), 3.95 (2H, t, –CH2Nꢂ), 4.23 (2H,
–CH2Nꢂ), 4.24 (2H, t, –OCH2–), 5.76 (1H, s, 4-CH), 6.85 (1H, d, t, –OCH2–), 4.61 (1H, m, 3a-H), 5.37 (1H, s, 17a-H), 5.40 (1H, d, 6-CH),
Joꢀ8.30 Hz, 5-CH, aromatic), 6.94 (1H, s, 5-CH, imidazole), 7.06 (2H, m,
drostene-3b,17b-diol Diacetate (8) (DPJ-RG-1227) Yield: 65.64%. mp:
1
163—165 °C. H-NMR (CDCl3) d: 0.80 (3H, s, 18-CH3), 1.05 (3H, s, 19-
1
CH3), 2.04 (3H, s, 3b-OCOCH3), 2.22 (3H, s, 17b-OCOCH3), 2.26 (2H, m,
6.15 (1H, s, vinylic-H, 16-arylidene), 6.80 (1H, d, Joꢀ8.08 Hz, 5-CH, aro-
2-CH, aromatic and 4-CH, imidazole), 7.13 (1H, dd, Jmꢀ1.27 Hz, matic), 6.89 (2H, s, 2-CH, aromatic and 5-CH, imidazole), 6.93 (1H, dd,
Joꢀ8.20 Hz, 6-CH, aromatic), 7.39 (1H, s, vinylic-H, 16-arylidene) and 7.50
Jmꢀ1.78 Hz, Joꢀ8.86 Hz, 6-CH, aromatic), 7.06 (1H, s, 4-CH, imidazole),
(1H, s, 2-CH, imidazole). FT-IR nmax (KBr) cmꢁ1: 2936, 1712, 1666, 1622, 7.54 (1H, s, 2-CH, imidazole). FT-IR nmax (KBr) cmꢁ1: 2938, 1733, 1595,
1595, 1512, 1463, 1328, 1260, 1230, 1141, 1094, 1027, 917, 810.6. Anal. 1512, 1443, 1371, 1239, 1141, 1034, 804. Anal. Cald for C37H48N2O6: C,
Calcd for C33H40N2O4: C, 74.97; H, 7.63; N, 5.30. Found: C, 74.82; H, 7.66; 72.05; H, 7.84; N, 4.54. Found: C, 72.14; H, 7.72; N, 4.66.
N, 5.49.
16-[4-{3-(Imidazol-1-yl)propoxy}-3-methoxybenzylidene]-3-pyrro-
lidino-3,5-androsta-dien-17-one (9) (DPJ-RG-1178) Pyrrolidine (1 ml)
was added to a refluxing solution of 5 (1.89 mmol) in methanol (10 ml). The
reaction mixture was further refluxed for 1 h and chilled on ice. The crys-
talline material obtained was filtered, washed with methanol and dried to af-
16-[4-{3-(Imidazol-1-yl)propoxy}-3-methoxybenzylidene]-3b-pyrro-
lidino-5-androsten-17b-yl Acetate (11) (DPJ-RG-1195) Yield: 59.71%.
mp: 95—97 °C. 1H-NMR (CDCl3) d: 0.77 (3H, s, 18-CH3), 1.03 (3H, s, 19-
CH3), 2.21 (1H, s, –OCOCH3), 2.28 (2H, m, –OCH2CH2CH2Nꢂ), 2.62 (4H,
br s, –N-(CH2)2–, pyrrolidine), 3.88 (3H, s, –OCH3), 3.95 (2H, t, –CH2Nꢂ),
4.22 (2H, t, –OCH2–), 5.37 (2H, s, 6-CH and 17a-H), 6.15 (1H, s, vinylic-
H, 16-arylidene), 6.80 (1H, d, Joꢀ8.09 Hz, 5-CH, aromatic), 6.89 (2H, s, 2-
ford 9.
1
Yield: 45.45%. mp: 148—150 °C. H-NMR (CDCl3) d: 1.07 (3H, s, 18- CH, aromatic and 5-CH, imidazole), 6.92 (1H, m, 6-CH, aromatic), 7.05
CH3), 1.14 (3H, s, 19-CH3), 2.30 (2H, m, –OCH2CH2CH2Nꢂ), 2.77 (4H, (1H, s, 4-CH, imidazole), 7.49 (1H, s, 2-CH, imidazole). FT-IR nmax (KBr)
br s, –N-(CH2)2–, pyrrolidine), 3.60 (3H, s, –OCH3), 3.97 (2H, s, –CH2Nꢂ), cmꢁ1: 2937, 1725, 1599, 1512, 1463, 1374, 1240, 1142, 1040, 964, 807.
4.27 (2H, t, –OCH2–), 4.97 (2H, m, 4-CH and 6-CH) and 6.97—7.54 (7H, Anal. Calcd for C39H53N3O4: C, 74.60; H, 8.51; N, 6.69. Found: C, 74.65; H,
m, 2-CH, 5-CH, 6-CH, aromatic; 2-CH, 4-CH, 5-CH, imidazole and vinylic-
H,16-arylidene). FT-IR nmax (KBr) cmꢁ1: 2938, 1711, 1656, 1623, 1595,
1513, 1460, 1419, 1376, 1328, 1260, 1143, 1096, 1027, 914, 852, 809.4.
8.42; N, 6.74.
Antineoplastic Activity The synthesized compounds were screened at
National Cancer Institute, Bethesda, U.S.A. for in vitro and in vivo antineo-
General Procedure for the Synthesis of Compounds 7 and 10 To a plastic activity.
stirred suspension of requisite keto steroid 3 and 9 (1.88 mmol) in methanol
3-Cell Line Assay The compounds 3, 5—8, 10, 11 were selected by
(100 ml) at room temperature, sodium borohydride (1.5 g) was added in Drug Synthesis and Chemistry Branch, National Cancer Institute, based in
small fractions over a period of 2 h. The reaction mixture was further stirred general, on the basis of degree of novelty of the structure and computer
for 6 h. Solvent was removed under reduced pressure and iced water was modeling techniques for anticancer screening. Firstly, they were assayed
added to it. The precipitate obtained was filtered, washed with water, dried using one dose (10ꢁ4 M) primary anticancer in vitro assay against tumor in
and crystallized from methanol to yield 7 and 10, respectively.
the 3-cell line panel consisting of MCF-7 (breast), NCI-H460 (lung) and SF-
268 central nervous system (CNS) (Table 1) and then were passed on for
evaluation in the full panel of 60-cell lines over a 5-log dose range.
16-[4-{3-(Imidazol-1-yl)propoxy}-3-methoxybenzylidene]-5-an-
drostene-3b,17b-diol (7) (DPJ-RG-1219) Yield: 84.68%. mp: 197—
1
198 °C. H-NMR (CDCl3) d: 0.72 (3H, s, 18-CH3), 1.05 (3H, s, 19-CH3),
60-Cell Line Assay The 3-cell line actives meaning the compounds,
2.25 (2H, m, –OCH2CH2CH2Nꢂ), 3.53 (1H, m, 3a-H), 3.89 (3H, s, which reduced the growth of any one of the cell lines to approximately 32%
–OCH3), 3.95 (2H, t, –CH2Nꢂ), 4.06 (1H, s, 17a-H), 4.23 (2H, t, –OCH2), or less, were assayed in vitro against a panel consisting of 60 human tumor
5.38 (1H, d, 6-CH), 6.45 (1H, s, vinylic-H, 16-arylidene), 6.81 (1H, m, 5-
cell lines, derived from nine cancer types (leukemia, lung, colon, CNS,
CH, aromatic), 6.93 (3H, m, 2-CH, 6-CH, aromatic and 5-CH, imidazole), melanoma, ovarian, renal, prostate and breast cancers), using five concentra-
7.07 (1H, s, 4-CH, imidazole), 7.54 (1H, s, 2-CH, imidazole). FT-IR nmax tions at 10-fold dilutions, the highest being 10ꢁ4 M. A 48 h continuous drug
(KBr) cmꢁ1: 3235, 2928, 1599, 1514, 1463, 1410, 1323, 1258, 1231, 1167, protocol was used and a sulforhodamine B (SRB) protein assay was used to
1140, 1079, 1052, 949, 915, 798. Anal. Calcd for C33H44N2O4: C, 74.40; H, estimate the cell viability or growth.15,16) Mean log dose response parameters
8.33; N, 5.26. Found: C, 74.27; H, 8.39; N, 5.39.
such as GI50 (drug concentration resulting in a 50% reduction in the net pro-
tein increase), TGI (drug concentration of total growth inhibition) and LC50
(concentration of drug resulting in a 50% reduction in the measured protein
16-[4-{3-(Imidazol-1-yl)propoxy}-3-methoxybenzylidene]-3b-pyrrol-
idino-5-androsten-17b-ol (10) (DPJ-RG-1179) Yield: 54.62%. mp:
1
249—251 °C. H-NMR (CDCl3) d: 0.72 (3H, s, 18-CH3), 1.03 (3H, s, 19- at the end of the drug treatment as compared to that at the beginning) are
CH3), 2.28 (2H, m, –OCH2CH2CH2Nꢂ), 2.61 (4H, br s, –N-(CH2)2–, pyrrol- summarized in Table 2. Two standard drugs, meaning that their activities
idine), 3.88 (3H, s, –OCH3), 3.94 (2H, t, –CH2Nꢂ), 4.04 (1H, s, 17a-H), against the cell lines are well documented, were tested against each cell line:
4.22 (2H, t, –OCH2–), 5.36 (1H, d, 6-CH), 6.45 (1H, s, vinylic-H, 16-aryli- NSC 19893 (5-Fluorouracil) and NSC 123127 (Adriamycin).
dene), 6.80 (1H, d, Joꢀ8.78 Hz, 5-CH, aromatic), 6.93 (3H, m, 2-CH, 6-CH,
In general, a compound is selected for in vivo studies if its mean
log10 GI50ꢃꢁ6 in 60-cell line assay. The total pattern of activity of the com-
aromatic and 5-CH, imidazole), 7.05 (1H, s, 4-CH, imidazole), 7.49 (1H, s,
2-CH, imidazole). FT-IR nmax (KBr) cmꢁ1: 3184, 2930, 2784, 1600, 1513, pounds is also taken into consideration by the Biological Evaluation Com-
1462, 1382, 1325, 1254, 1135, 1075, 1029, 949, 913, 796. Anal. Calcd for mittee for Cancer Drugs to select the compound for further in vivo evalua-
C37H51N3O3: C, 75.86; H, 8.78; N, 7.17. Found: C, 75.92; H, 8.98; N, 7.22.
General Procedure for the Synthesis of Compounds 6, 8 and 11
tion.
Aromatase Inhibitory Activity. Preparation of Aromatase The en-
A
mixture of respective hydroxyl derivative 3,
7
(0.94 mmol) and 10
zyme was obtained from the microsomal fraction of freshly delivered human
term placental tissue according to the procedure of Thompson and Siiteri.17)
The isolated microsomes were suspended in the minimum volume of phos-
phate buffer (0.05 M, pH 7.4) and stored at ꢁ30 °C as described. No loss of
activity was observed within four months.
Inhibition of Aromatase in Vitro The assay was performed similar to
the described methods18,19) monitoring the enzyme activity by measuring the
3H2O formed from [1b, 2b-3H] testosterone during aromatization. Each in-
cubation tube contained 0.225 mCi of [1b, 2b-3H] testosterone, 5 mM unla-
beled testosterone, 2 mM reduced nicotinamide adenine dinucleotide phos-
phate (NADPH), 20 mM glucose-6-phosphate, 1EU glucose-6-phosphate de-
hydrogenase and inhibitor (0—250 mM) in phosphate buffer (0.05 M, pH 7.4).
The test compound had been dissolved in EtOH and diluted with buffer. The
final ethanol concentration of the control and inhibitor incubation was 2%.
Each tube was preincubated for 5 min at 30 °C in a shaking water bath. Mi-
(0.85 mmol), acetic anhydride (1 ml) and dry pyridine (2 ml, 0.5 ml was used
for 11) was heated in a steam bath for 2 h. The reaction contents were then
poured into cold water and basified with liquid ammonia. The precipitate ob-
tained was filtered, washed with water, dried and crystallized from n-hexane
to afford corresponding acetoxy steroids 6, 8 and 11.
16-[4-{3-(Imidazol-1-yl)propoxy}-3-methoxybenzylidene]-17-oxo-5-
androsten-3b-yl Acetate (6) (DPJ-RG-1196) Yield: 50.04%. mp: 109—
1
111 °C. H-NMR (CDCl3) d: 0.98 (3H, s, 18-CH3), 1.09 (3H, s, 19-CH3),
2.04 (3H, s, –OCOCH3), 2.29 (2H, m, –OCH2CH2CH2Nꢂ), 3.91 (3H, s,
–OCH3), 3.98 (2H, t, –CH2Nꢂ), 4.23 (2H, t, –OCH2–), 4.61 (1H, m, 3a-H),
5.42 (1H, d, 6-CH), 6.85 (1H, d, Joꢀ8.48 Hz, 5-CH, aromatic), 6.93 (1H, s,
5-CH, imidazole), 7.07 (2H, m, 2-CH, aromatic and 4-CH, imidazole), 7.12
(1H, dd, Jmꢀ1.44 Hz, Joꢀ8.26 Hz, 6-CH, aromatic), 7.38 (1H, s, vinylic-H,
16-arylidene), 7.50 (1H, s, 2-CH, imidazole). FT-IR nmax (KBr) cmꢁ1: 2942,
1729, 1628, 1596, 1513, 1465, 1371, 1325, 1248, 1139, 1095, 1029, 915, crosomal protein (0.5 mg) was added to start the reaction. The total volume
812. Anal. Calcd for C35H44N2O5: C, 73.40; H, 7.74; N, 4.89. Found: C, of each incubation was 0.5 ml. The reaction was terminated by withdrawing

March 2011
331
100 ml aliquots at 0, 7, 14 and 21 min and pipetting them into 200 ml of a
cold 1 mM HgCl2 solution. After addition of 200 ml of an aqueous dextran-
coated charcoal (DCC) suspension (2%), the vials were shaken for 20 min
and centrifuged at 1500 g for 5 min to separate the charcoal-adsorbed
steroids. Aliquots of the supernatant were assayed for 3H2O by counting in a
scintillation mixture in a 1209 Rackbeta Wallac liquid scintillation spec-
trometer (Pharmacia LKB, Freiburg, Germany).
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Acknowledgements The authors are thankful to Department of Science
and Technology and Council for Scientific and Industrial Research, India for
providing financial assistance and National Cancer Institute, Bethesda,
Maryland, U.S.A. for screening the compounds for antineoplastic activity.
The generous supply of steroids by Cipla Ltd., India is gratefully acknowl-
edged.
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(2004).
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