Synthesis and antineoplastic activity of O-alkylated derivatives of 7-hydroximinoandrost-5-ene steroids

Article abstract of DOI:10.1002/ardp.200900258

Varied positioning of the hydroximino group on the parental steroid skeleton results in remarkable changes in the antineoplastic activity profile of the compounds. Here, the compound 7-oximino-5-androstene and its O-alkylated derivatives have been prepared and screened for cytotoxic and aromatase inhibitory activity. The steroidal 7-oximino ether derivatives exhibited insignificant cytotoxic effects when screened against three cancer cell lines, MCF-7 (breast), NCl-H460 (lung), and SF-268 (CNS) at 100 μM. However, the imidazolyl-substituted steroidal oxime ethers displayed moderate inhibition of cytochrome P450 aromatase.

Full text of DOI:10.1002/ardp.200900258

Arch. Pharm. Chem. Life Sci. 2010, 343, 377–383
R. Bansal et al.
377
Full Paper
Synthesis and Antineoplastic Activity of O-Alkylated
Derivatives of 7-Hydroximinoandrost-5-ene Steroids
Ranju Bansal1, Sheetal Guleria1, Christina Ries2, and Rolf W. Hartmann2
1 University Institute of Pharmaceutical Sciences, Sector-14, Panjab University, Chandigarh, India
2 Pharmaceutical and Medicinal Chemistry, Saarland University, Saarbrꢀcken, Germany
Varied positioning of the hydroximino group on the parental steroid skeleton results in remark-
able changes in the antineoplastic activity profile of the compounds. Here, the compound 7-ox-
imino-5-androstene and its O-alkylated derivatives have been prepared and screened for cyto-
toxic and aromatase inhibitory activity. The steroidal 7-oximino ether derivatives exhibited
insignificant cytotoxic effects when screened against three cancer cell lines, MCF-7 (breast), NCl-
H460 (lung), and SF-268 (CNS) at 100 lM. However, the imidazolyl-substituted steroidal oxime
ethers displayed moderate inhibition of cytochrome P450 aromatase.
Keywords: Antineoplastic activity / Aromatase inhibitory activity / Hydroximino steroids / Oxime ethers /
Received: October 26, 2009; Accepted: December 11, 2009
DOI 10.1002/ardp.200900258
The reported aromatase inhibitory and cytotoxic activ-
ity of various steroidal oximes, their ether derivatives,
and the importance of the 2-alkylaminoethyl side chain
of tamoxifen [8] for antitumor activity, prompted us to
prepare some more steroidal-oxime ethers. The current
study involves the exploitation of the C-7 position of the
steroid skeleton, in which O-alkylated derivatives of 7-
hydroximino-5-androstene have been prepared and eval-
uated for cytotoxic and aromatase inhibitory activity.
Introduction
Hydroximino steroids represent a distinct class of anti-
neoplastic agents [1, 2]. Recent work from our laboratory
has also demonstrated the effectiveness of 16E-arylide-
nosteroids and their hydroximino derivatives as poten-
tial antitumor agents [3, 4]. Various steroidal oxime
ethers derivatized with a 2-alkylaminoethyl side chain,
an important structural component of several antitumor
agents, in the androstene and estrane series have also
been reported by us as potent cytotoxics [5]. Studies have
revealed that the location of the hydroximino group on
the parent steroid skeleton greatly affects the cytotoxic-
ity profile of the compounds. Recently, it has been
reported that the presence of an oxime group on ring B
and a hydroxyl on ring A or B resulted in higher cytotox-
icity than other structural motifs [6].
Results and discussion
Chemistry
The synthesis of the oxime-steroid system was based on
the methodology developed by Singh et al. [9] as shown in
Fig. 1. For this purpose, dehydroepiandrosterone was
reduced using sodium borohydride in methanol at room
temperature to afford 3b,17b-diol derivative 1, which,
upon subsequent acetylation with acetic anhydride and
dry pyridine, afforded 3b,17b-diacetoxyandrost-5-ene 2.
Allylic oxidation of 2 using t-butyl chromate reagent [10]
and further treatment with hydroxylamine hydrochlo-
ride in CCl4 furnished the 7-oxo derivative 3 and 7-ox-
imino-5-androstene-3b,17b-diol diacetate 4, sequentially
(Fig. 1). A downfield 1H-NMR signal at d = 6.46 ppm (6-CH)
for compound 4, because of the influence of hydroxyl,
Furthermore, this group of steroids and 7-substituted
androstenedione derivatives also show a high affinity for
human placental aromatase and function as competitive
inhibitors of this enzyme [7].
Correspondence: Ranju Bansal, University Institute of Pharmaceutical
Sciences, Panjab University, Chandigarh-160014, India.
E-mail: ranju29in@yahoo.co.in
Fax: +91 172 254-1142
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378
R. Bansal et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 377–383
Figure 1. Synthetic routes to compounds 6a–h.
indicates the Z configuration of the hydroximino group The structures were assigned on the basis of spectro-
at C7 with respect to the vinylic proton of C6 because of scopic and elemental analyses data. Resonances for 5a at
the influence of hydroxyl in accordance with the earlier d = 2.49 (m, 4H, -N-(CH2)2-, piperidine), 5b at 2.53 (m, 4H,
reports [6, 9].
-N-(CH2)2-, morpholine), 5c at 1.04 (t, 6H, -N-(CH2CH3)2), 5d
Alkylation of hydroximino derivative 4 using hydro- at 2.30 (s, 6H, -N-(CH3)2), 5e at 2.54 (m, -N-(CH2)2-, pyrroli-
chlorides of various dialkylamino ethyl chlorides, 1- dine), and 5f at 3.60 ppm (t, 2H, -CH2Cl) were indicative of
bromo-3-chloropropane, and cyclopentyl bromide in the substitution of the side chain in these compounds. A
refluxing ethyl methyl ketone in the presence of anhy- multiplet at d = 4.15 ppm (-OCH2-) was observed in the
drous potassium carbonate afforded oxime ethers 5a–g. NMR of all these O-alkylated steroids 5a–f. The cyclopen-
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Arch. Pharm. Chem. Life Sci. 2010, 343, 377–383
Derivatives of 7-Hydroximinoandrost-5-ene Steroids
379
Table 1. Aromatase inhibitory data of various compounds.
Compound
4
R
R1
Inhibition on CYP 19 §
RP &
H
OCOCH3
4% at 36 lM
5a
5b
5c
OCOCH3
OCOCH3
OCOCH3
8% at 36 lM
11% at 36 lM
14% at 36 lM
5d
5f
OCOCH3
OCOCH3
3% at 36 lM
9% at 36 lM
5h
6a
OCOCH3
OH
IC50 = 3.1 lM
12% at 36 lM
9.6
6b
6c
OH
OH
9% at 36 lM
14% at 36 lM
6d
6f
OH
OH
8% at 36 lM
8% at 36 lM
6h
OH
IC50 = 12 lM
2.5
§ [1b,2b-3H ] Testosterone; & relative potency = relative to aminoglutethimide (RP = 1, IC50 = 28.5 lM).
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tyloxy derivative 5g exhibited a one-proton multiplet for atives 5h and 6h exhibited modest inhibition of the
-O-CH- at d = 4.56–4.73 ppm. The imidazolyl derivative 5h enzyme and were found to be more potent as compared
was obtained by thermally fusing 5f with an excess of to aminoglutethimide, even up to approximately ten
imidazole. To study the impact of incorporation of a times more in the case of diacetoxy oxime ether 5h (IC50
=
hydroxyl group on ring A on cytotoxicity, 3b,17b-diacet- 3.1 lM). It is expected that compounds 5h and 6h interact
oxy derivatives 5a–h were subjected to alkaline hydroly- with the active site of aromatase by complexing the Fe(III)
sis to afford their corresponding 7-substituted hydrox- iron of cytochrome P450 with one of the imidazole nitro-
imino-5-androstene-3b,17b-diol counterparts 6a–h. These gens as previously described [13, 14]. These imidazolyl-
hydrolyzed steroids exhibited infrared O-H stretching substituted steroidal derivatives produced only about
vibrations near 3400–3200 cm–1. An upfield shift was one fifth of the aromatase inhibition in comparison to a
observed for the two-proton NMR multiplet of 3a-H and steroidal aromatase inhibitor, formestane (4-hydroxyan-
17a-H in case of diol derivatives 6a–h, where signals were drost-4-ene-3,17-dione, 4-OHA IC50 = 0.6 lM) [15] and a
observed at d = 3.65 ppm in comparison to their diacet- very weak inhibition in contrast to an imidazolic non-
oxy counterparts 6a–h (d = 4.65). The downfield chemical steroidal inhibitor fadrozole (IC50 = 40 nM).
shift of H-6 at d = 6.4 ppm in proton NMR spectra of all
In light of the above findings, it is concluded that sub-
these alkoximino derivatives, confirmed the Z configura- stitution of 7-oximino and its further derivatization to
tion with respect to the double bond in concordance oxime-ethers in androstane series of steroids results in
with earlier reports [6, 9].
compounds with insignificant cytotoxic activity, how-
ever, selection of suitable substituents in the side chain
may result in molecules with improved aromatase inhib-
itory activity.
Antineoplastic activity
Three-cell line assay
Out of the numerous synthesized steroidal oximino
derivatives, compounds 5a–5c, 5f, 6a–6d, 6f, and 6h were
selected for screening by the Drug Synthesis and Chemis-
try Branch, National Cancer Institute, Bethesda, Mary-
land, USA, based, in general, on the degree of novelty of
the structure and modeling techniques for the antineo-
plastic activity using in-vitro three-cell lines (MCF-7
(breast), NCl-H460 (lung) and SF-268 (CNS)) one-dose pre-
screen. In the current protocol, each cell line was inocu-
lated and pre-incubated on a microtiter plate. Test agents
were then added and the culture incubated for 48 h. End-
point determinations were made with alamar blue.
Results for each test agent were reported as the % growth
of the treated cells when compared to the untreated con-
trol cells [11, 12].
In comparison to our earlier reported work [5], the ster-
oidal 7-oximino ether derivatives exhibited insignificant
cytotoxic effects in all the three cell lines at 100 lM. This
is in agreement with the earlier observation [6] that the
parent steroids with a hydroximino group located at
different positions show remarkable difference in their
cytotoxicities. The study provided new evidence showing
the relationship between the chemical structure and bio-
logical function.
Experimental
General
Melting points were determined on a Veego melting point appa-
ratus (Veego, Mumbai, India) and are uncorrected. IR (wavenum-
ber in cm–1) spectra were taken on a Perkin-Elmer spectrum RX 1
FT-IR spectrophotometer (Perkin-Elmer, USA) model using KBr
pellets. 1H-NMR spectra were recorded on a Bruker AC-300F
(Bruker AG, Switzerland), 300 MHz using deuterated-chloroform
(CDCl3) or deuterated dimethylsulphoxide (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 chromatogra-
phy (TLC) were prepared with silica gel G according to the
method of Stahl (E. Merck, Germany) using ethyl acetate as sol-
vent and activated at 1108C for 30 min. Iodine was used to
develop the TLC plates. Anhydrous sodium sulphate was utilized
as drying agent. All solvents were freshly distilled and dried
prior to use according to standard procedures.
Compound 4 was prepared according to Ref. [9].
General method for the preparation of compounds 5a–g
To a solution of 7-oximino-5-androstene-3b,17b-diol diacetate (4,
1.0 g, 2.48 mmol) in ethyl methyl ketone (100 mL), anhydrous
K2CO3 (2.48 mmol) was added. The reaction mixture was refluxed
with stirring for 3 h. Respective hydrochloride of alkylami-
noethyl chloride (0.45 g, 2.48 mmol) was added and it was fur-
ther refluxed for 7–12 h with continuous stirring. The comple-
tion of the reaction was monitored by TLC. The slurry was
cooled, filtered, and excess of solvent was removed under
reduced pressure to obtain an oily residue. Iced water was added
to it and it was allowed to stand overnight. The solid product
obtained was filtered, washed with water, dried, and recrystal-
lized to afford the corresponding alkylaminoethyloximino
Aromatase-inhibitory activity
As is evident from Table 1, the 7-oxime steroid 4, its O-
alkylated diacetoxy derivatives 5a–f and diols 6a–f with-
out the presence of a basic nitrogen either did not show
or only exhibited marginal inhibition of the aromatase
enzyme. However, both the imidazolyl substituted deriv-
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Arch. Pharm. Chem. Life Sci. 2010, 343, 377–383
Derivatives of 7-Hydroximinoandrost-5-ene Steroids
381
derivatives 5a–e. Compounds 5f and 5g were obtained on treat-
ment of 7-oximino-5-androstene-3b,17b-diol diacetate 4 with 1-
bromo-3-chloropropane and cyclopentyl bromide, respectively,
using the same procedure. The compounds 5a–g were recrystal-
lized using petroleum ether.
7-[O-(3-Chloropropyl)oximino]-5-androstene-3ꢀ,17ꢀ-diol
diacetate 5f
Yield: 58.82%, m.p.; 1558C; IR mmax (KBr) cm–1: 2945.5, 2878.1,
1736.5, 1640.6, 1450.8, 1371.7, 1246.2, 1033.4, 953.2, 909.3,
840.0; 1H-NMR (CDCl3): 0.81 (s, 3H, 18-CH3), 1.13 (s, 3H, 19-CH3),
2.03 (s, 6H, 2 -OCOCH3), 2.10 (m, 2H, -OCH2CH2CH2Cl), 3.60 (t, 2H,
-CH2Cl), 4.14 (t, 2H, -OCH2-), 4.64 (m, 2H, 3a-H and 17a-H), 6.43 (s,
1H, 6-CH). Anal. calcd. for C26H38NO5Cl: C, 65.05; H, 7.98; N, 2.92.
Found: C, 65.32; H, 8.18; N, 2.69.
7-[O-{2-(Piperidin-1-yl)ethyl}oximino]-5-androstene-
3ꢀ,17ꢀ-diol diacetate 5a
Yield: 47.06%, m.p.: 156–1578C; IR mmax (KBr) cm–1: 2949.4, 1735.0,
1642.9, 1448.6, 1376.5, 1287.6, 1238.9, 1118.5, 1032.3, 955.9,
1
914.6, 896.1, 845.7, 749.9; H-NMR (CDCl3): 0.83 (s, 3H, 18-CH3),
7-[O-(Cyclopentyl)oximino]-5-androstene-3ꢀ,17ꢀ-diol
1.12 (s, 3H, 19-CH3), 2.04 (s, 6H, 2 -OCOCH3), 2.49 (m, 4H, -N-(CH2)2-,
piperidine), 2.63 (t, 2H, -CH2N), 4.14 (t, 2H, -OCH2-), 4.66 (m, 2H, 3a-
H and 17a-H), 6.45 (s, 1H, 6-CH). Anal. calcd. for C30H46N2O5: C,
70.00; H, 9.01; N, 5.44. Found: C, 70.12; H, 8.98; N, 5.88.
diacetate 5g
Yield: 51.28%, m.p.: 135–1378C; IR mmax (KBr) cm–1: 2948.0,
1
1735.5, 1636.3, 1441.2, 1375.5, 1247.4, 1035.7, 995.5, 912.4; H-
NMR (CDCl3): 0.83 (s, 3H, 18-CH3), 1.12 (s, 3H, 19-CH3), 2.04 (s, 3H,
3b-OCOCH3), 2.05 (s, 3H, 17b-OCOCH3), 4.56–4.73 (m, 3H, -O-CH-,
cyclopentyl, 3a-H and 17a-H), 6.46 (s, 1H, 6-CH). Anal. calcd. for
C28H41NO5: C, 71.31; H, 8.76; N, 2.97. Found: C, 70.62; H, 8.78; N,
2.88.
7-[O-{2-(Morpholin-4-yl)ethyl}oximino]-5-androstene-
3ꢀ,17ꢀ-diol diacetate 5b
Yield: 54.67%, m.p.: 161–1628C; IR mmax (KBr) cm–1: 2949.4, 1735.0,
1642.9, 1448.6, 1376.5, 1287.6, 1238.9, 1118.5, 1032.3, 955.9,
914.6, 896.1, 845.7, 749.9; 1H-NMR (CDCl3): 0.83 (s, 3H, 18-CH3),
1.13 (s, 3H, 19-CH3), 2.04 (s, 6H, 2 -OCOCH3), 2.53 (m, 4H, -N-(CH2)2-,
morpholine), 2.67 (t, 2H, -CH2N), 3.73 (t, 3H, -O-(CH2)2-, morpho-
line), 4.17 (m, 2H, -OCH2-), 4.66 (m, 2H, 3a-H and 17a-H), 6.45 (s,
1H, 6-CH). Anal. calcd. for C29H44N2O6: C, 67.41; H, 8.58; N, 5.42.
Found: C, 67.65; H, 8.98; N, 5.42.
7-[O-{3-(Imidazol-1-yl)propyl}oximino]-5-androstene-
3ꢀ,17ꢀ-diol diacetate 5h
A finely triturated mixture of 5f (1 g, 2.08 mmol) and imidazole
(1.5 g, in excess) was thermally fused at 110–1208C for 5 h with
continuous stirring. The completion of the reaction was moni-
tored with TLC. Water was added to remove excess of imidazole.
The solid product obtained was filtered, dried, and crystallized
from a mixture of acetone and diethyl ether to afford 5h.
Yield: 19.45%, m.p.: 170–1728C; IR mmax (KBr) cm–1: 2947.0,
2871.2, 1730.4, 1632.3, 1456.6, 1378.7, 1231.1, 1187.9, 1069.7,
1021.6, 985.0, 912.8; 1H-NMR (CDCl3): 0.81 (s, 3H, 18-CH3), 1.13 (s,
3H, 19-CH3), 2.05 (s, 6H, 2 -OCOCH3), 2.34 (m, 2H, -OCH2CH2CH2N),
4.03 (m, 4H, -OCH2CH2CH2N), 4.60 (m, 2H, 3a-H and 17a-H), 6.41
(s, 1H, 6-CH), 6.92 (brs, 1H, 5-CH, imidazole), 7.04 (brs, 1H, 4-CH,
imidazole), 7.49 (brs, 1H, 2-CH, imidazole). Anal. calcd. for
C29H41N3O5: C, 68.08; H, 8.08; N, 8.21. Found: C, 68.12; H, 8.34; N,
8.10.
7-[O-(2-Diethylaminoethyl)oximino]-5-androstene-
3ꢀ,17ꢀ-diol diacetate 5c
Yield: 48.16%, m.p.: 124–1268C; IR mmax (KBr) cm–1: 2965.9,
1726.6, 1640.6, 1452.1, 1375.1, 1249.2, 1036.7, 955.7, 914.0,
843.1; 1H-NMR (CDCl3): 0.83 (s, 3H, 18-CH3), 1.04 (t, 6H,
-N(CH2CH3)2), 1.12 (s, 3H, 19-CH3), 2.04 (s, 6H, 2 -OCOCH3), 2.58 (q,
4H, -N(CH2CH3)2), 2.74 (t, 2H, -CH2N), 4.10 (m, 2H, -OCH2-), 4.64 (m,
2H, 3a-H and 17a-H), 6.47 (s, 1H, 6-CH). Anal. calcd. for
C29H46N2O5: C, 69.29; H, 9.23; N, 5.57. Found: C, 69.25; H, 9.82; N,
5.68.
General method for the preparation of compounds 6a–h
To the refluxing solution of 7-[O-(2-alkylaminoethyl)]oximino-5-
androstene-3b,17b-diol diacetate (5a–h, 1.0 mmol) in methanol
(50 mL), potassium hydroxide (0.12 g) was added and the reac-
tion mixture was further refluxed for 45 min. The reaction mix-
ture was concentrated, acidified with glacial acetic acid, and
poured into cold water. The precipitate formed was filtered,
washed thoroughly with water, dried, and crystallized from
diethyl ether to afford 6a–h.
7-[O-(2-Dimethylaminoethyl)oximino]-5-androstene-
3ꢀ,17ꢀ-diol diacetate 5d
Yield: 51.02%, m.p.: 93–958C; IR mmax (KBr) cm–1: 2948.7, 2872.3,
1
1733.0, 1637.3, 1447.4, 1374.5, 1245.9, 1040.9, 927.6, 788.3; H-
NMR (CDCl3): 0.83 (s, 3H, 18-CH3), 1.12 (s, 3H, 19-CH3), 2.04 (s, 6H,
2 -OCOCH3), 2.30 (s, 6H, -N(CH3)2), 2.62 (t, 2H, -CH2N), 4.13 (m, 2H,
-OCH2-), 4.64 (m, 2H, 3a-H and 17a-H), 6.48 (s, 1H, 6-CH). Anal.
calcd. for C27H42N2O5: C, 68.32; H, 8.92; N, 5.90. Found: C, 68.57;
H, 8.61; N, 5.82.
7-[O-{2-(Pyrrolidin-1-yl)ethyl}oximino]-5-androstene-
7-[O-{2-(Piperidin-1-yl)ethyl}oximino]-5-androstene-
3ꢀ,17ꢀ-diol diacetate 5e
3ꢀ,17ꢀ-diol 6a
Yield: 56.45%, m.p.: 123–1258C; IR mmax (KBr) cm–1: 2949.3, 2790.2,
Yield: 95.24%, m.p.: 123–1258C; IR mmax (KBr) cm–1: 3291.1,
2936.3, 1638.0, 1455.6, 1349.4, 1315.5, 1189.0, 1129.8, 1054.0,
948.7, 915.1, 839.0; 1H-NMR (CDCl3): 0.78 (s, 3H, 18-CH3), 1.12 (s,
3H, 19-CH3), 2.45 (brs, 4H, -N-(CH2)2-, piperidine), 2.63 (t, 2H,
-CH2N), 3.62 (m, 2H, 3a-H and 17a-H), 4.16 (m, 2H, -OCH2-), 6.43 (s,
1H, 6-CH). Anal. calcd. for C26H42N2O3: C, 72.51; H, 9.83; N, 6.51.
Found: C, 72.82; H, 9.88; N, 6.58.
1
1735.1, 1632.8, 1455.6, 1373.2, 1245.0, 1032.2, 887.7, 841.9; H-
NMR (CDCl3): 0.82 (s, 3H, 18-CH3), 1.14 (s, 3H, 19-CH3), 2.04 (s, 3H,
3b-OCOCH3), 2.05 (s, 3H, 17b-OCOCH3), 2.54 (m, 4H, -N-(CH2)2-, pyr-
rolidine), 2.76 (t, 2H, -CH2N), 4.17 (m, 2H, -OCH2-), 4.63 (m, 2H, 3a-
H and 17a-H), 6.48 (s, 1H, 6-CH). Anal. calcd. for C29H44N2O5: C,
69.57; H, 8.86; N, 5.60. Found: C, 69.49; H, 8.67; N, 5.88.
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7-[O-{2-(Morpholin-4-yl)ethyl}oximino]-5-androstene-
7-[O-{3-(Imidazol-1-yl)propyl}oximino]-5-androstene-
3ꢀ,17ꢀ-diol 6b
3ꢀ,17ꢀ-diol 6h
Yield: 83.33%, m.p.: 117–1198C; IR mmax (KBr) cm–1: 3379.7, 2939.4,
2867.7, 1635.9, 1456.6, 1350.8, 1305.2, 1112.7, 1058.2, 955.9,
910.8, 838.8; 1H-NMR (CDCl3): 0.78 (s, 3H, 18-CH3), 1.12 (s, 3H, 19-
CH3), 2.53 (t, 4H, -N-(CH2)2-, morpholine), 2.67 (t, 2H, -CH2N), 3.62
(m,2H, 3a-H and 17a-H), 3.73 (m, 4H, -O-(CH2)2-, morpholine), 4.18
(m, 2H, -OCH2-), 6.41 (s, 1H, 6-CH). Anal. calcd. for C25H40N2O4: C,
69.41; H, 9.32; N, 6.48. Found: C, 69.49; H, 9.86; N, 6.66.
Yield: 23.45%, m.p.: 205–2068C; IR mmax (KBr) cm–1: 3301.5, 3107.3,
2940.0, 2871.2, 1631.3, 1511.8, 1456.6, 1377.7, 1231.1, 1187.9,
1081.6, 1059.7, 1025.6, 952.0, 921.8, 889.0, 823.3; 1H-NMR
(CDCl3): 0.78 (s, 3H, 18-CH3), 1.13 (s, 3H, 19-CH3), 2.37 (m, 2H,
-OCH2CH2CH2N), 3.65 (m, 2H, 3a-H and 17a-H), 4.03 (m, 4H,
-OCH2CH2CH2N), 6.39 (s, 1H, 6-CH), 6.95 (brs, 1H, 5-CH, imidazole),
7.07 (brs, 1H, 4-CH, imidazole), 7.49 (brs, 1H, 2-CH, imidazole).
Anal. calcd. for C25H37N3O3: C, 70.23; H, 8.72; N, 9.83. Found: C,
70.19; H, 8.94; N, 9.76.
7-[O-(2-Diethylaminoethyl)oximino]-5-androstene-
3ꢀ,17ꢀ-diol 6c
Preparation of aromatase
Yield: 84.06%, m.p.: 176–1788C; IR mmax (KBr) cm–1: 3320.1,
2933.0, 2871.8, 2807.9, 1641.1, 1446.8, 1383.3, 1352.0, 1292.1,
1251.2, 1131.4, 1051.6, 947.9, 914.0, 891.6, 838.5; 1H-NMR
(CDCl3): 0.78 (s, 3H, 18-CH3), 1.04 (t, 6H, -N(CH2CH3)2), 1.12 (s, 3H,
19-CH3), 2.60 (q, 4H, -N(CH2CH3)2), 2.75 (t, 2H, -CH2N), 3.63 (m, 2H,
3a-H and 17a-H), 4.10 (m, 2H, -OCH2-), 6.44 (s, 1H, 6-CH). Anal.
calcd. for C25H42N2O3: C, 71.73; H, 10.11; N, 6.69. Found: C, 71.82;
H, 10.45; N, 6.72.
The enzyme was obtained from the microsomal fraction of
freshly delivered human term placental tissue according to the
procedure of Thompson and Siiteri [16]. The isolated micro-
somes were suspended in the minimum volume of phosphate
buffer (0.05 M, pH 7.4) and stored at –308C as described. No loss
of activity was observed within four months.
Inhibition of aromatase in vitro
This assay was performed similar to the described methods [17,
18] monitoring the enzyme activity by measuring the 3H2O
formed from [1b, 2b-3H] testosterone during aromatization. Each
incubation tube contained 0.225 lCi of [1b, 2b-3H] testosterone,
5 lM unlabeled testosterone, 2 mM NADPH, 20 mM glucose-6-
phosphate, 1 EU glucose-6-phosphate dehydrogenase, and inhib-
itor (0–250 lM) 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 pre-incubated for 5 min at
308C in a shaking water bath. Microsomal protein (0.5 mg) was
added to start the reaction. The total volume of each incubation
was 0.5 mL. The reaction was terminated by withdrawing 100 lL
aliquots at 0, 7, 14, and 21 min and pipetting them into 200 lL
of a cold 1 mM HgCl2 solution. After addition of 200 lL of an
aqueous dextran-coated charcoal (DCC) suspension (2%), the
vials were shaken for 20 min and centrifuged at 15006g 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 spectrom-
eter (Pharmacia LKB, Freiburg, Germany).
7-[O-(2-Dimethylaminoethyl)oximino]-5-androstene-
3ꢀ,17ꢀ-diol 6d
Yield: 72.92%, m.p.: 179–1808C; IR mmax (KBr) cm–1: 3372.8, 2943.0,
2872.8, 1633.5, 1464.5, 1350.1, 1263.9, 1055.5, 963.7, 914.8,
1
839.0; H-NMR (CDCl3): 0.76 (s, 3H, 18-CH3), 1.12 (s, 3H, 19-CH3),
2.29 (s, 6H, -N (CH3)2), 2.60 (t, 2H, -CH2N), 3.59 (m, 2H, 3a-H and
17a-H), 4.10 (m, 2H, -OCH2-), 6.41 (s, 1H, 6-CH). Anal. calcd. for
C23H38N2O3: C, 70.73; H, 9.81; N, 7.17. Found: C, 70.85; H, 9.99; N,
7.20.
7-[O-{2-(Pyrrolidin-1-yl)ethyl}oximino]-5-androstene-
3ꢀ,17ꢀ-diol 6e
Yield: 84.13%, m.p.: 167–1698C; IR mmax (KBr) cm–1: 3218.9,
1
2940.3, 1639.3, 1463.1, 1353.3, 1057.6, 912.6; H-NMR (CDCl3):
0.78 (s, 3H, 18-CH3), 1.12 (s, 3H, 19-CH3), 2.57 (brs, 4H, -N-(CH2)2-,
pyrrolidine), 2.76 (t, 2H, -CH2N), 3.63 (m, 2H, 3a-H and 17a-H),
4.17 (m, 2H, -OCH2-), 6.45 ppm (s, 1H, 6-CH). Anal. calcd. for
C25H40N2O3: C, 72.07; H, 9.68; N, 6.73. Found: C, 72.22; H, 9.65; N,
6.78.
The authors are thankful to University Grants Commission, India for
providing financial assistance and National Cancer Institute,
Bethesda, Maryland, USA for screening the compounds for antineoplas-
tic activity. The generous supply of steroids by Cipla Ltd., India is grate-
fully acknowledged.
7-[O-(3-Chloropropyl)oximino]-5-androstene-3ꢀ,17ꢀ-diol
6f
Yield: 78.8%, m.p.: 186–1878C; FTIR mmax (KBr) cm–1: 3324.9,
2936.9, 1637.1, 1453.7, 1350.1, 1129.5, 1053.5, 949.1, 914.5,
1
839.1; H-NMR (CDCl3): 0.78 (s, 3H, 18-CH3), 1.12 (s, 3H, 19-CH3),
2.12 (m, 2H, -OCH2CH2CH2Cl), 3.63 (m, 4H, -CH2Cl, 3a-H and 17a-
H), 4.15 (m, 2H, -OCH2-), 6.43 (s, 1H, 6-CH). Anal. calcd. for
C22H34NO3Cl: C, 66.73; H, 8.66; N, 3.54. Found: C, 66.92; H, 8.58;
N, 3.62.
The authors have declared no conflict of interest.
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