Synthesis of 16E-[3-methoxy-4-(2-aminoethoxy)benzylidene]androstene derivatives as potent cytotoxic agents

Article abstract of DOI:10.1016/j.steroids.2008.07.004

The synthesis and cytotoxic studies of a new series of 16E-arylidene androstene derivatives are reported herein. The impact of incorporating bis-tertiary amino functionalities in the steroid skeleton on cytotoxicity has also been observed. The compounds have been evaluated at National cancer Institute, Bethesda, Maryland, USA for their antineoplastic activity against various tumor cell lines. The synthesized 16E-arylidenosteroids exhibited significant cytotoxicity. Bis-tertiary amino steroid 29 possessing a diethylaminoalkoxy functionality was the most promising compound of the series with a total IP and SC score of 20 in in vivo hollow fiber assay and was selected for further detailed in vivo xenograft testing.

Full text of DOI:10.1016/j.steroids.2008.07.004

steroids 7 3 ( 2 0 0 8 ) 1391–1399
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Synthesis of 16E-[3-methoxy-4-(2-aminoethoxy)-
benzylidene]androstene derivatives as potent
cytotoxic agents
Ranju Bansal∗, Sheetal Guleria
University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and cytotoxic studies of a new series of 16E-arylidene androstene derivatives
are reported herein. The impact of incorporating bis-tertiary amino functionalities in the
steroid skeleton on cytotoxicity has also been observed. The compounds have been evalu-
ated at National cancer Institute, Bethesda, Maryland, USA for their antineoplastic activity
against various tumor cell lines. The synthesized 16E-arylidenosteroids exhibited significant
cytotoxicity. Bis-tertiary amino steroid 29 possessing a diethylaminoalkoxy functionality
was the most promising compound of the series with a total IP and SC score of 20 in in vivo
hollow fiber assay and was selected for further detailed in vivo xenograft testing.
© 2008 Elsevier Inc. All rights reserved.
Received 13 June 2008
Received in revised form 3 July 2008
Accepted 8 July 2008
Published on line 23 July 2008
Keywords:
Cytotoxic agents
16-Arylidenosteroids
Antineoplastic activity
Xenograft assays
Bis-tertiary amino steroids
1.
Introduction
steroid nucleus [5]. Investigation of a homologous series of
amino steroids for antitumor activity indicated the essen-
Steroidal alkylating agents have received sporadic attention
as potential antitumor agents for the past few years [1]. The
research efforts have mainly been focused towards reducing
systemic toxicity and improving specificity of cancer ther-
apy using lipophilic steroid nuclei as carriers to deliver the
alkylating agents such as nitrogen and sulphur mustards to a
specific target tissue with ease [2,3]. Further, as expected the
synergistic activity out of these hybrid steroidal structures
was also obtained.
tial requirement of two piperidine substituents. Among these,
2,16-bis(amino) steroid; 2␤,16␤-dipiperidino-5␣-androstane-
3,17-diol dipivalate hydrochloride (1) (DAP) (Fig. 1), showed
significant antitumor activity against transplantable tumors.
The acute toxicity of DAP was similar to that of cyclophos-
phamide and considerably lower than vinblastine [6].
A large number of potent steroidal derivatives with substi-
tution at position 16 have been described in the literature as
potent cytotoxic agents [7–9]. Recently some interesting 16E-
arylidene androstene derivatives (2) (Fig. 1) have also been
reported from our laboratory as strong in vitro inhibitors of
the growth of many types of human tumor cells [10,11]. In
view of the potent cytotoxic activity of bis-tertiary amino
steroids and that of new 16E-arylidenosteroids, we decided
to further explore the anticancer properties of 16-substituted
Interference with neuromuscular transmission has been
reported to be
a side effect of potent antitumor agent
vincristine [4]. Comparison of its molecular structure with
potent neuromuscular blockers indicated drug-receptor sim-
ilarities owing to presence of common structural features
like well-spaced two tertiary amino groups in lipophilic
∗
Corresponding author. Tel.: +91 172 2541142; fax: +91 172 2541142.
E-mail address: ranju29in@yahoo.co.in (R. Bansal).
0039-128X/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2008.07.004

1392
steroids 7 3 ( 2 0 0 8 ) 1391–1399
Fig. 1 – Structures of DAP and 16-benzylidene steroids.
heterosteroidal derivatives by synthesizing new analogues
with suitable structural modifications and also to observe the
impact of incorporating bis-tertiary amino functionalities in
these steroidal products.
2.3.1. 16-[3-Methoxy-4-{2-(piperidin-1-yl)ethoxy}-
benzylidene]-17-oxo-5-androsten-3ˇ-ol (6) (DPJ-RG-1071)
Yield: 97.3%, m.p., 212–214 ◦C. UVmax (MeOH): 242.0 (log ε 3.99)
and 330.6 (log ε 4.31). IR: 3240 (O H), 1711 (C O), 1622 (C C),
1094 (C O). 1H NMR (CDCl3): 0.98 (s, 3H, H-18); 1.07 (s, 3H, H-
19); 2.52 (m, 4H, –N(CH2)2–); 2.83 (t, 2H, –CH2N<); 3.53 (m, 1H,
H-3␣); 3.89 (s, 3H, –OCH3); 4.19 (t, 2H, –OCH2–); 5.40 (d, 1H, H-6);
2.
Experimental
ꢀ
ꢀ
ꢀ
ꢀ
6.93 (d, 1H, J5 ,6 = 8.25 Hz, H-5 ); 7.06 (s, 1H, H-2 ); 7.16 (dd, 1H,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
J2 ,6 = 1.51 Hz, J5 ,6 = 8.37 Hz, H-6 ); 7.38 (s, 1H, H-vinyl). Anal.
calcd for C34H47NO4: C, 76.50; H, 8.87; N, 2.62; found: C, 76.22;
H, 9.07; N, 2.71.
2.1.
General
Melting points were determined on a Veego melting point
apparatus and are uncorrected. UV (wavelengths in nm) were
recorded on Lambda 15 and IR (wavenumbers in cm−1) spec-
tra on PerkinElmer spectrum RX 1, FT-IR spectrophotometer
models using KBr pellets. 1H NMR spectra were recorded
on Brucker AC-300F, 300 MHz using deuterated-chloroform
(CDCl3) or deuterated dimethylsulphoxide (DMSO-d6) con-
taining tetramethylsilane as internal standard (chemical
shifts in ı, ppm). Elemental analyses were carried out
on a PerkinElmer-2400 model CHN analyzer. All solvents
were distilled prior to use according to standard proce-
dures.
2.3.2. 16-[3-Methoxy-4-{2-(pyrrolidin-1-yl)ethoxy}-
benzylidene]-17-oxo-5-androsten-
3ˇ-ol (7) (DPJ-RG-1147)
Yield: 92.1%, m.p., 210–212 ◦C. UVmax (MeOH): 246.6 (log ε 3.83)
and 331.0 (log ε 4.21). IR: 3238 (O H), 1710 (C O), 1624 (C C),
1095 (C O). 1H NMR (CDCl3): 0.97 (s, 3H, H-18); 1.07 (s, 3H, H-19);
2.65 (m, 4H, –N(CH2)2–); 2.97 (t, 2H, –CH2N<); 3.50 (m, 1H, H-3␣);
3.88 (s, 3H, –OCH3); 4.20 (t, 2H –OCH2–); 5.39 (d, 1H, H-6); 6.93
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
(d, 1H, J5 ,6 = 8.39 Hz, H-5 ); 7.05 (d, 1H, J2ꢀ,6 = 1.84 Hz, H-2 ); 7.15
ꢀ
ꢀ
ꢀ
ꢀ
(dd, 1H, J2 ,6 = 1.73 Hz, J5 ,6 = 8.39 Hz, H-6 ); 7.38 (s, 1H, H-vinyl).
Anal. calcd for C33H45NO4: C, 76.26; H, 8.73; N, 2.70; found: C,
76.01; H, 9.02; N, 2.55.
2.2.
3–5
General method for the preparation of compounds
2.3.3. 16-[4-(2-Diethylaminoethoxy)-3-
methoxybenzylidene]-17-oxo-5-androsten-
3ˇ-ol (8) (DPJ-RG-1097)
Hydrochloride of requisite dialkylaminoethyl chloride
(6.57 mmol) was added to a stirred and refluxing suspen-
sion 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 stir-
ring until the reaction was completed (monitored by TLC).
The reaction mixture was cooled, filtered and solvent was
removed under reduced pressure to obtain the corresponding
oily residue (3–5), which was used as such for subsequent
reaction.
Yield: 93.4%, m.p., 176–178 ◦C. UVmax (MeOH): 242.8 (log ε 4.02)
and 330.8 (log ε 4.35). IR: 3251 (O H), 1711 (C O), 1622 (C C).
1H NMR (CDCl3): 0.97 (s, 3H, H-18); 1.07 (t, 6H, –N(CH2CH3)2
and s (merged), 3H, H-19); 2.65 (q, 4H, –N(CH2)2); 2.90 (t, 2H,
–CH2N<); 3.51 (m, 1H, H-3␣); 3.89 (s, 3H, –OCH3); 4.13 (t, 2H,
–OCH2–); 5.38 (d, 1H, H-6); 6.93 (d, 1H, J5 ,6 = 8.29 Hz, H-5 );
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
7.05 (d, 1H, J2 ,6 = 1.51 Hz, H-2 ); 7.15 (dd, 1H, J2 ,6 = 1.59 Hz,
ꢀ
ꢀ
ꢀ
J5 ,6 = 8.43 Hz, H-6 ); 7.38 (s, 1H, H-vinyl). Anal. calcd for
C33H47NO4: C, 75.97; H, 9.08; N, 2.68; found: C, 75.99; H, 9.24;
N, 2.71.
2.3.
6–8
General method for the preparation of compounds
2.4.
General Method for the preparation of compounds
9–11
A mixture of dehydroepiandrosterone (2.60 mmol) appropri-
ate aldehyde (oily residues, 3–5) and sodium hydroxide (1 g)
in methanol (20 ml) was stirred at room temperature for 2 h
until the reaction was completed (monitored by TLC). Cold
water was added to the reaction mixture and the precipitate
obtained was filtered, washed with water, dried and crystal-
lized from methanol to yield 6–8.
A mixture of aldol products 6–8 (0.94 mmol), acetic anhydride
(1 ml) and dry pyridine (2 ml) was heated in a stream bath for
2 h. The reaction contents were then poured into cold water
and basified with liquor ammonia. The precipitate obtained
was filtered, washed with water, dried and crystallized from
n-hexane to yield 9–11.

steroids 7 3 ( 2 0 0 8 ) 1391–1399
1393
(m, 3H, H-2ꢀ, H-5ꢀ and H-6ꢀ). Anal. calcd for C34H49NO4: C, 76.22;
H, 9.22; N, 2.61; found: C, 76.35; H, 9.29; N, 2.67.
2.4.1. 16-[3-Methoxy-4-{2-(piperidin-1-yl)ethoxy}-
benzylidene]-17-oxo-5-androsten-3ˇ-yl acetate (9)
(DPJ-RG-1111)
Yield: 70.4%, m.p., 126–128 ◦C. UVmax (MeOH): 242.8 (log ε 3.82)
2.5.2. 16-[3-Methoxy-4-{2-(pyrrolidin-1-yl)ethoxy}-
and 332.0 (log ε 4.36). IR: 1722 (C O), 1617 (C C) 1098 (C O). 1
NMR (CDCl3): 0.97 (s, 3H, H-18); 1.08 (s, 3H, H-19); 2.03 (s, 3H,
–OCOCH3); 2.47–2.52 (m, 4H, –N(CH2)2–); 2.84 (t, 2H, –CH2N<);
H
benzylidene]-5-androstene-3ˇ,17ˇ-diol (13) (DPJ-RG-1217)
Yield: 89.6%, m.p., 113–115 ◦C. UVmax (MeOH): 248.8 (log ε 4.0).
IR: 3368 (O H), 1659 (C C). 1H NMR (CDCl3): 0.72 (s, 3H, H-18);
1.04 (s, 3H, H-19); 2.66 (m, 4H, –N(CH2)2–); 2.96 (t, 2H, –CH2N<);
3.53 (m, 1H, H-3␣); 3.86 (s, 3H, –OCH3); 4.05 (s, 1H, H-17␣); 4.15
(t, 2H, –OCH2–); 5.37 (d, 1H, H-6); 6.44 (d, 1H, H-vinyl); 6.86–6.96
(m, 3H, H-2ꢀ, H-5ꢀ and H-6ꢀ). Anal. calcd for C33H47NO4: C, 75.97;
H, 9.08; N, 2.68; found: C, 75.99; H, 8.91; N, 2.61.
3.89 (s, 3H, –OCH3); 4.18 (t, 2H, –OCH2–); 4.60 (m, 1H, H-3␣);
ꢀ
ꢀ
ꢀ
5.42 (d, 1H, H-6); 6.92 (d, 1H, J5 ,6 = 8.27 Hz, H-5 ); 7.05 (d, 1H,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
J2 ,6 = 1.40 Hz, H-2 ); 7.16 (dd, 1H, J2 ,6 = 1.29 Hz, J5 ,6 = 8.32 Hz,
H-6ꢀ); 7.37 (s, 1H, H-vinyl). Anal. calcd for C36H49NO5: C, 75.09;
H, 8.58; N, 2.43; found: C, 75.10; H, 8.51; N, 2.22.
2.4.2. 16-(3-Methoxy-4-{2-(pyrrolidin-1-yl)ethoxy}-
benzylidene]-17-oxo-5-androsten-3ˇ-yl acetate (10)
(DPJ-RG-1175)
Yield: 66.6%, m.p., 114–115 ◦C. UVmax (MeOH): 246.8 (log ε
3.74) and 331.0 (log ε 4.15). IR: 1724 (C O), 1625 (C C) 1249
(C( O) O), 1097 (C O). 1H NMR (CDCl3): 0.94 (s, 3H, H-
18); 1.03 (s, 3H, H-19); 2.08 (s, 3H, –OCOCH3); 2.59 (m, 4H,
–N(CH2)2–); 2.90 (t, 2H, –CH2N<); 3.85 (s, 3H, –OCH3); 4.10 (t,
2H –OCH2–); 4.52 (m, 1H, H-3␣); 5.40 (d, 1H, H-6); 6.84 (d, 1H,
2.5.3. 16-[4-(2-Diethylaminoethoxy)-3-
methoxybenzylidene]-5-androstene-3ˇ,17ˇ-diol (14)
(DPJ-RG-1216)
Yield: 79.7%, m.p., 97–99 ◦C. UVmax (MeOH): 264.2 (log ε 4.39).
IR: 3403 (O H), 1595 (C C). 1H NMR (CDCl3): 0.72 (s, 3H, H-
18); 1.07 (t, 6H, –N(CH2CH3)2 and s (merged), 3H, H-19); 2.65
(q, 4H, –N(CH2CH3)2); 2.93 (t, 2H, –CH2N<); 3.54 (m, 1H, H-3␣);
3.86 (s, 3H, –OCH3); 4.05 (s, 1H, H-17␣); 4.10 (t, 2H, –OCH2–); 5.38
ꢀ
ꢀ
(d, 1H, H-6); 6.44 (d, 1H, H-vinyl); 6.87 (d, 1H, J5 ,6 = 8.25 Hz, H-
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
J5 ,6 = 8.43 Hz, H-5 ); 6.95 (d, 1H, J2 ꢀ,6 = 1.35 Hz, H-2 ); 7.06 (dd, 1H,
5ꢀ); 6.91(d, 1H, J2 ,6 = 1.73 Hz, H-2 ); 6.95 (dd, 1H, J2 ,6 = 1.71 Hz,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
J2 ,6 = 1.36 Hz, J5 ,6 = 8.47 Hz, H-6 ); 7.26 (s, 1H, H-vinyl). Anal.
ꢀ
ꢀ
ꢀ
J5 ,6 = 8.35 Hz, H-6 ). Anal. calcd for C33H49NO4: C, 75.68; H, 9.43;
calcd for C35H47NO5: C, 74.83; H, 8.43; N, 2.49; found: C, 74.69;
H, 8.71; N, 2.38.
N, 2.67; found: C, 75.91; H, 9.68; N, 2.56.
2.6.
General procedure for the preparation of
2.4.3. 16-[4-(2-Diethylaminoethoxy)-3-
methoxybenzylidene]-17-oxo-5-androsten-3ˇ-yl
compounds 15–17
acetate (11) (DPJ-RG-1146)
A mixture of diol 12–14 (0.93 mmol), acetic anhydride (1 ml)
and dry pyridine (2 ml) was heated in a steam bath for 2 h.
The contents of the reaction mixture were then poured into
cold water and basified with liquor ammonia. The precipitate
obtained was collected by filtration, washed with water, dried
and crystallized from n-hexane to yield 15–17.
Yield: 66.7%, m.p., 118–120 ◦C. UVmax (MeOH): 242.8 (log ε
4.03) and 330.8 (log ε 4.36). IR: 1731 (C O), 1627 (C C) 1247
(C( O) O), 1095 (C O). 1H NMR (CDCl3): 0.97 (s, 3H, H-18); 1.07
(t, 6H, –N(CH2CH3)2); 1.09 (s, 3H, H-19); 2.03 (s, 3H, –OCOCH3);
2.64 (q, 2H, –N(CH2CH3)2); 2.94 (t, 2H, –CH2N<); 3.88 (s, 3H,
–OCH3); 4.11 (t, 2H, –OCH2–); 4.59 (m, 1H, H-3␣); 5.42 (d, 1H,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
H-6); 6.91 (d, 1H, J5 ,6 = 8.49 Hz, H-5 ); 7.03 (d, 1H, J2 ,6 = 1.78 Hz,
2.6.1. 16-[3-Methoxy-4-{2-(piperidin-1-yl)ethoxy}-
benzylidene]-5-androstene-3ˇ,17ˇ-diol diacetate (15)
(DPJ-RG-1215)
H-2ꢀ); 7.13 (dd, 1H, J2 ,6 = 1.57 Hz, J5 ,6 = 8.47 Hz, H-6 ); 7.39 (s, 1H,
H-vinyl). Anal. calcd for C35H49NO5: C, 74.56; H, 8.76; N, 2.48;
found: C, 74.55; H, 8.92; N, 2.51.
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Yield: 69.2%, m.p., 152–153 ◦C. UVmax (MeOH): 265.8 (log ε 4.25).
IR: 1735 (C O), 1600 (C C), 1237 (C( O) O). 1H NMR (CDCl3):
0.79 (s, 3H, H-18); 1.07 (s, 3H, H-19); 2.03 (s, 3H, OCOCH3-3␤);
2.22 (s, 3H, OCOCH3-17␤); 2.51 (t, 4H, –N(CH2)2–); 2.80 (t, 2H,
–CH2N<); 3.86 (s, 3H, –OCH3); 4.15 (t, 2H, –OCH2–); 4.63 (m, 1H,
H-3␣); 5.36 (s, 1H, H-17␣); 5.40 (d, 1H, H-6); 6.14 (d, 1H, H-vinyl);
6.84–6.93 (m, 3H, H-2ꢀ, H-5ꢀand H-6ꢀ). Anal. calcd for C38H53NO6:
C, 73.63; H, 8.62; N, 2.26; found: C, 73.66; H, 8.96; N, 2.30.
2.5.
12–14
General method for the preparation of compounds
To the stirred suspension of aldol products 6–8 (1.9 mmol)
in methanol (100 ml) at room temperature, sodium borohy-
dride (1.5 g) was added in small fractions over a period of
2 h. The reaction mixture was further stirred for 4 h. Sol-
vent was removed under reduced pressure and ice water
was added to it. The precipitate formed was filtered, washed
with water, dried and crystallized from method to yield
12–14.
2.6.2. 16-[3-Methoxy-4-{2-(pyrrolidin-1-yl)ethoxy}-
benzylidene]-5-androstene-3ˇ,17ˇ-diol diacetate (16)
(DPJ-RG-1218)
Yield: 68.9%, m.p., 157–158 ◦C. UVmax (MeOH): 250.4 (log ε 4.14).
IR: 1736 (C O), 1601 (C C). 1H NMR (CDCl3): 0.79 (s, 3H, H-18);
1.05 (s, 3H, H-19); 2.03 (s, 3H, OCOCH3-3␤); 2.20 (s, 3H, OCOCH3-
17␤); 2.63 (t, 4H, –N(CH2)2–); 2.95 (t, 2H, –CH2N<); 3.86 (s, 3H,
–OCH3); 4.17 (t, 2H, –OCH2–); 4.61 (m, 1H, H-3␣); 5.36 (s, 1H,
H-17␣); 5.40 (d, 1H, H-6); 6.14 (d, 1H, H-vinyl); 6.85–6.93 (m, 3H,
H-2ꢀ, H-5ꢀand H-6ꢀ). Anal. calcd for C37H51NO6: C, 73. 35; H, 8.49;
N, 2.31; found: C, 73.66; H, 8.29; N, 2.01.
2.5.1. 16-[3-Methoxy-4-{2-(piperidin-1-yl)ethoxy}-
benzylidene]-5-androstene-3ˇ,17ˇ-diol (12) (DPJ-RG-1214)
Yield: 85%, m.p., 174–176 ◦C. UVmax (MeOH): 263.8 (log ε 3.95).
IR: 3363 (O H), 1660 (C C). 1H NMR (CDCl3): 0.71 (s, 3H, H-18);
1.04 (s, 3H, H-19); 2.61 (m, 4H, –N(CH2)2–); 2.80 (t, 2H, –CH2N<);
3.49 (m, 1H, H-3␣); 3.85 (s, 3H, –OCH3); 3.99 (s, 1H, H-17␣); 4.12
(t, 2H, –OCH2–); 5.33 (d, 1H, H-6); 6.39 (d, 1H, H-vinyl); 6.80–6.89

1394
steroids 7 3 ( 2 0 0 8 ) 1391–1399
C33H45NO4: C, 76.26; H, 8.73; N, 2.70; found: C, 76.32; H, 9.01;
2.6.3. 16-[4-(2-Diethylaminoethoxy)-3-
N, 2.68.
methoxybenzylidene]-5-androstene-3ˇ,17ˇ-diol
diacetate (17) (DPJ-RG-1226)
Yield: 69%, m.p., 87–88 ◦C. UVmax (MeOH): 265.6 (log ε 4.14).
IR: 1736 (C O), 1600 (C C), 1238 (C( O) O). 1H NMR (CDCl3):
0.79 (s, 3H, H-18); 1.05 (s, 3H, H-19); 1.12 (t, 6H, –N(CH2CH3)2);
2.03 (s, 3H, OCOCH3-3␤); 2.20 (s, 3H, OCOCH3-17 ␤); 2.72 (t, 4H,
–N(CH2)2–); 3.01 (t, 2H, –CH2N<); 3.85 (s, 3H, –OCH3); 4.15 (t, 2H,
–OCH2–); 4.61 (m, 1H, H-3␣); 5.36 (s, 1H, H-17␣); 5.40 (d, 1H, H-
6); 6.15 (d, 1H, H-vinyl); 6.85–6.93 (m, 3H, H-2ꢀ, H-5ꢀand H-6ꢀ).
Anal. calcd for C37H53NO6: C, 73.11; H, 8.79; N, 2.30; found: C,
73.12; H, 9.01; N, 2.41.
2.8.
General procedure for the preparation of
compounds 24–26
Pyrrolidine (1 ml) was added to the refluxing suspension of
dione 18–20 (1.93 mmol) in methanol (10 ml). The reaction
mixture was further refluxed for 15 min and chilled. The crys-
talline material was filtered, washed with methanol and dried
to afford intermediate enamines 21–23. To the stirred suspen-
sion of the enamines 21–23 so formed (1.71 mmol) in methanol
(100 ml) at room temperature, sodium borohydride (1.5 g) was
added in small amounts over a period of 2 h and stirring
was further continued in vacuum and ice water was added.
The precipitate obtained was filtered, washed with water,
dried and crystallized from methanol to yield corresponding
diaminosteroids 24–26.
2.7.
General method for the preparation of compounds
18–20
The aldol products 6–8 (1.92 mmol) were dissolved in a mixture
of cyclohexanone (10 ml) and dry toluene (150 ml). Azeotropic
distillation was continued at a slow rate while adding a solu-
tion of aluminium isopropoxide (1 g) in dry toluene (15 ml)
dropwise. The reaction mixture was then 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 solid product obtained was filtered, washed with
water, dried and treated with diethyl ether and n-hexane to
yield crystals of 18–20.
2.8.1. 16-[3-Methoxy-4-{2-(piperidin-1-yl)ethoxy}-
benzylidene]-3-pyrrolidino-3,5-androsta dien-17-one (21)
(DPJ-RG-1073)
Yield: 72.7%, m.p., 132–135 ◦C. UVmax (MeOH): 242.0 (log ε 4.12)
and 330.8 (log ε 4.31). IR: 1712 (C O), 1624 (C C), 1025 (C O).
1H NMR (CDCl3): 0.93 (s, 3H, H-18); 1.00 (s, 3H, H-19); 2.37 (brs,
4H, –N(CH2)2–); 2.73 (t, 2H, –CH2N<); 3.07 (brs, 4H, –N(CH2)2-3);
3.80 (s, 3H, –OCH3); 4.10 (t, 2H, –OCH2–); 4.70 (s, 1H, H-4); 5.00
(s, 1H, H-6); 6.70–7.33 (m, 4H, H-2ꢀ, H-5ꢀ, H-6ꢀ and H-vinyl).
2.7.1. 16-[3-Methoxy-4-{2-(piperidin-1-yl)ethoxy}-
benzylidene]-4-androstene-3,17-dione (18) (DPJ-RG-1072)
Yield: 100%, m.p., 88–90 ◦C. UVmax (MeOH): 240.8 (log ε 4.36)
and 331.2 (log ε 4.30). IR: 1713 (C O), 1623 (C C), 1093 (C O).
1H NMR (CDCl3): 1.01 (s, 3H, H-18); 1.25 (s, 3H, H-19); 2.50 (m,
2.8.2. 16-[3-Methoxy-4-{2-(pyrrolidin-1-yl)ethoxy}-
benzylidene]-3-pyrrolidino-3,5-andros-tadien-17-one (22)
(DPJ-RG-1149)
Yield: 63.5%, m.p., 125–127 ◦C. UVmax (MeOH): 241.0 (log ε 3.95)
and 330.8 (log ε 4.10). IR: 1713 (C O), 1622 (C C), 1095 (C O).
1H NMR (CDCl3): 1.00 (s, 3H, H-18); 1.07 (s, 3H, H-19); 2.63
(br, 4H, –N(CH2)2–); 2.96 (t, 2H, –CH2N<); 3.12 (t, 4H, –N(CH2)2-
4H, –N(CH2)2–); 2.84 (t, 2H, –CH2N<); 3.90 (s, 3H, –OCH3); 4.18 (t,
ꢀ
ꢀ
ꢀ
2H, –OCH2–); 5.76 (s, 1H, H-4); 6.93 (d, 1H, J5 ,6 = 8.34 Hz, H-5 );
ꢀ
7.05 (s, 1H, H-2ꢀ); 7.15 (d, 1H, J5 ,6 = 8.66 Hz, H-6 ); 7.39 (s, 1H,
H-vinyl). Anal. calcd for C34H45NO4: C, 76.79; H, 8.53; N, 2.63;
found: C, 76.49; H, 8.72; N, 2.59.
ꢀ
ꢀ
3); 3.90 (s, 3H, –OCH3); 4.18 (t, 2H, –OCH2–); 4.79 (s, 1H, H-4);
ꢀ
ꢀ
ꢀ
5.08 (s, 1H, H-6); 6.92 (d, 1H, J5 ,6 = 8.30 Hz, H-5 ); 7.07 (d, 1H,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2.7.2. 16-[3-Methoxy-4-{2-(pyrrolidin-1-yl)ethoxy}-
J2 6 = 1.20 Hz, H-2 ); 7.15 (d, 1H, J5 ,6 = 8.30 Hz, H-6 ); 7.38 (s, 1H,
benzylidene]-4-androstene-3,17-dione (19) (DPJ-RG-1148)
Yield: 100%, m.p., 104–106 ◦C. UVmax (MeOH): 241.0 (log ε
4.40) and 330.8 (log ε 4.35). IR: 1713 (C O), 1623 (C C), 1095
(C O). 1H NMR (CDCl3): 1.00 (s, 3H, H-18); 1.25 (s, 3H, H-
19); 2.63 (m, 4H, –N(CH2)2–); 2.96 (t, 2H, –CH2N<); 3.89 (s, 3H,
–OCH3); 4.18 (t, 2H, –OCH2–); 5.75 (s, 1H, H-4); 6.92 (d, 1H,
H-vinyl).
2.8.3. 16-[4-(2-Diethylaminoethoxy)-3-
methoxybenzylidene]-3-pyrrolidino-3,5-androsta-
dien-17-one (23) (DPJ-RG-1099)
Yield: 72.6%, m.p., 130–133 ◦C. UVmax (MeOH): 239.4 (log ε 4.08)
and 330.0 (log ε 4.27). IR: 1713 (C O), 1624 (C C), 1026 (C O). 1H
NMR (CDCl3): 0.93 (s, 3H, H-18); 1.03 (t, 6H, –N(CH2CH3)2); 1.13
(s, 3H, H-19); 2.66 (q, 4H, –N(CH2CH3)2); 3.10 (m, 6H, –CH2N<
and –N(CH2)2-3); 3.81 (s, 3H, –OCH3); 4.00 (t, 2H, –OCH2–); 4.57
(s, 1H, H-4); 5.27 (1H, s, H-6); 6.90–7.15 (m, 3H, H-2ꢀ, H-5ꢀ and
H-6ꢀ); 7.50 (s, 1H, H-vinyl).
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ ꢀ
J5 ,6 = 8.32 Hz, H-5 ); 7.04 (d, 1H, J2ꢀ6 = 1.70 Hz, H-2 ); 7.14 (dd, 1H,
ꢀ
ꢀ
ꢀ
ꢀ
J2 ,6 = 1.67 Hz, J5 ,6 = 8.25 Hz, H-6 ); 7.38 (s, 1H, H-vinyl). Anal.
calcd for C33H43NO4: C, 76.56; H, 8.37; N, 2.71; found: C, 76.82;
H, 8.10; N, 3.02.
2.7.3. 16-[4-(2-Diethylaminoethoxy)-3-
methoxybenzylidene]-4-androstene-3,17-dione (20)
(DPJ-RG-1098)
2.8.4. 16-[3-Methoxy-4-{2-(piperidin-1-yl)ethoxy}-
benzylidene]-3ˇ-pyrrolidino-5-androsten-17ˇ-ol (24)
(DPJ-RG-1074)
Yield: 77.3%, m.p., 200–203 ◦C. UVmax (MeOH): 264.8 (log ε 4.44).
IR: 3366 (O H), 1511 (C C). 1H NMR (CDCl3): 0.72 (s, 3H, H-
18); 1.04 (s, 3H, H-19); 2.51 (brs, 4H, –N(CH2)2–); 2.62 (brs, 4H,
–N(CH2)2-3); 2.82 (t, 2H, –CH2N<); 3.87 (s, 3H, –OCH3); 4.05 (s,
1H, H-17␣); 4.15 (t, 2H, –OCH2–); 5.37 (d, 1H, H-6); 6.44 (s, 1H,
Yield: 100%, m.p., 105–108 ◦C. UVmax (MeOH): 240.6 (log ε 4.37)
and 331.0 (log ε 4.31). IR: 1714 (C O), 1623 (C C), 1095 (C O). 1H
NMR (CDCl3): 1.01 (s, 3H, H-18); 1.07 (6H, t, –N(CH2CH3)2); 1.25
(s, 3H, H-19); 2.66 (q, 4H, –N(CH2CH3)2); 2.93 (t, 2H, –CH2N<);
3.89 (s, 3H, –OCH3); 4.14 (t, 2H, –OCH2–); 5.76 (s, 1H, H-4);
ꢀ
ꢀ
ꢀ
ꢀ
6.93 (d, 1H, J5 ,6 = 8.03 Hz, H-5 ); 7.06 (s, 1H, H-2 ); 7.15 (d,
ꢀ
ꢀ
ꢀ
1H, J5 ,6 = 8.82 Hz, H-6 ); 7.39 (s, 1H, H-vinyl). Anal. calcd for

steroids 7 3 ( 2 0 0 8 ) 1391–1399
1395
H-vinyl); 6.85–6.96 (m, 3H, H-2ꢀ, H-5ꢀ and H-6ꢀ). Anal. calcd for
C38H56N2O3: C, 77.50; H, 9.58; N, 4.75; found: C, 77.69; H, 9.71;
N, 4.71.
then removed under reduced pressure to obtain a white solid,
which was crystallized from diethyl ether to afford 27–29.
2.9.1. 16-[3-Methoxy-4-{2-(piperidin-1-yl)ethoxy}-
benzylidene]-3ˇ-pyrrolidino-5-androsten-17ˇ-yl acetate (27)
(DPJ-RG-1087)
2.8.5. 16-[3-Methoxy-4-{2-(pyrrolidin-1-yl)ethoxy}-
benzylidene]-3ˇ-pyrrolidino-5-androsten-
17ˇ-ol (25) (DPJ-RG-1122)
Yield: 84%, m.p., 110–113 ◦C. UVmax (MeOH): 265.2 (log ε 4.33).
IR: 1720 (C O), 1514 (C C). 1H NMR (CDCl3): 0.79 (s, 3H, H-18);
1.02 (s, 3H, H-19); 2.20 (s, 3H, –OCOCH3); 2.50 (m, 4H, –N(CH2)2–);
2.58 (brs, 4H, –N(CH2)2-3); 2.80 (t, 2H, –CH2N<); 3.87 (s, 3H,
–OCH3); 4.14 (t, 2H, –OCH2–); 5.36 (s, 2H, H-17␣ and H-6); 6.14 (s,
1H, H-vinyl); 6.84–6.94 (m, 3H, H-2ꢀ, H-5ꢀ and H-6ꢀ). Anal. calcd
for C40H58N2O4: C, 76.14; H, 9.26; N, 4.44; found: C, 76.49; H,
9.66; N, 4.49.
Yield: 69.5%, m.p., 181–183 ◦C. UVmax (MeOH): 265.0 (log ε 4.32).
IR: 3351 (O H), 1512 (C C). 1H NMR (CDCl3): 0.71 (s, 3H, H-
18); 1.03 (s, 3H, H-19); 2.61 (m, 8H, 2× –N(CH2)2–); 2.94 (t, 2H,
–CH2N<); 3.86 (s, 3H, –OCH3); 4.03 (s, 1H, H-17␣); 4.15 (t, 2H,
–OCH2–); 5.35 (d, 1H, H-6); 6.44 (s, 1H, H-vinyl); 6.86–6.96 (m,
3H, H-2ꢀ, H-5ꢀ and H-6ꢀ). Anal. calcd for C37H54N2O3: C, 77.31;
H, 9.47; N, 4.87; found: C, 77.39; H, 9.09; N, 5.01.
2.8.6. 16-[4-(2-Diethylaminoethoxy)-3-
methoxybenzylidene]-3ˇ-pyrrolidino-5-androsten-17ˇ-ol
(26) (DPJ-RG-1100)
2.9.2. 16-[3-Methoxy-4-{2-(pyrrolidin-1-yl)ethoxy}-
benzylidene]-3ˇ-pyrrolidino-5-androsten-17ˇ-yl acetate (28)
(DPJ-RG-1170)
Yield: 70.8%, m.p., 97–98 ◦C. UVmax (MeOH): 265.8 (log ε 4.28). IR:
1735 (C O), 1513 (C C). 1H NMR (CDCl3): 0.76 (s, 3H, H-18); 1.01
(s, 3H, H-19); 2.21 (s, 3H, –OCOCH3); 2.62 (m, 8H, 2× –N(CH2)2–);
2.95 (t, 2H, –CH2N<); 3.87 (s, 3H, –OCH3); 4.15 (t, 2H, –OCH2–);
5.36 (m, 2H, H-17␣ and H-6); 6.14 (s, 1H, H-vinyl); 6.74–6.80 (m,
3H, H-2ꢀ, H-5ꢀ and H-6ꢀ). Anal. calcd for C39H56N2O4: C, 75.93;
H, 9.15; N, 4.54; found: C, 75.81; H, 9.22; N, 4.49.
Yield: 67.5%, m.p., 179–181 ◦C. UVmax (MeOH): 264.8 (log ε 4.32);
IR: 3375 (O H), 1512 (C C). 1H NMR (CDCl3): 0.71 (s, 3H, H-18);
1.07 (t, 6H, –N(CH2CH3)2) and s (merged), 3H, H-19); 2.64 (m,
8H, –N(CH2CH3)2 and –N(CH2)2–); 2.90 (t, 2H, –CH2N<); 3.85 (s,
3H, –OCH3); 4.02 (s, 1H, H-17␣); 4.08 (t, 2H, –OCH2–); 5.34 (d, 1H,
H-6), 6.43 (d, 1H, H-vinyl); 6.83–6.94 (m, 3H, H-2ꢀ, H-5ꢀand H-6ꢀ).
Anal. calcd for C37H56N2O3: C, 77.04; H, 9.79; N, 4.86; found: C,
77.41; H, 10.01; N, 4.92.
2.9.3. 16-[4-(2-Diethylaminoethoxy)-3-
2.9.
General procedure for the preparation of
methoxybenzylidene]-3ˇ-pyrrolidino-5-androsten-17ˇ-yl
acetate (29) (DPJ-RG-1110)
compounds 27–29
Yield: 72.8%, m.p., 110–111 ◦C. UVmax (MeOH): 266.0 (log ε 4.38).
IR: 1726 (C O), 1513 (C C). 1H NMR (CDCl3): 0.80 (s, 3H, H-
18); 1.04 (s, 3H, H-19); 1.07 (t, 6H, –N(CH2CH3)2); 2.21 (s, 3H,
–OCOCH3); 2.64 (m, 8H, –N(CH2CH3)2 and –N(CH2)2-3); 2.92 (t,
2H, –CH2N<); 3.86 (s, 3H, –OCH3); 4.09 (t, 2H, –OCH2–); 5.36 (s,
2H, H-17␣ and H-6); 6.14 (d, 1H, H-vinyl); 6.86–6.93 (m, 3H, H-2ꢀ,
H-5ꢀand H-6ꢀ). Anal. calcd for C39H58N2O4: C, 75.68; H, 9.45; N,
4.53; found: C, 75.69; H, 9.54; N, 4.61.
A mixture containing respective diol 24–26 (0.85 mmol), acetic
anhydride (1 ml) and dry pyridine (0.5 ml) was heated in a
steam bath for 1.5 h. The reaction contents were poured in
a mixture of crushed ice and dichloromethane. The resul-
tant turbid solution was basified with liquor ammonia and
extracted with dichloromethane (3 × 50 ml). The combined
organic extract was washed with water and dried. Solvent was
Scheme 1 – Reagents and reaction conditions: (a) MeOH, KOH, RT; (b) (CH3CO)O/dry pyridine, steam bath, 2H.

1396
steroids 7 3 ( 2 0 0 8 ) 1391–1399
Scheme 2 – Reagents and reaction conditions: (a) NaBH4, MeOH, RT; (b) (CH3CO)O/dry pyridine, steam bath, 2H; (c)
Al(t-BuO)3, cyclohexanone, reflux, 5H; (d) pyrrolidine/MeOH; (e) NaBH4; (f) (CH3CO)O/dry pyridine.
bridged proton of the reduced products appeared at an upfield
3.
Results and discussion
position (ı ∼ 6.4 ppm) as compared to their corresponding par-
ent aldol compounds indicating the reduction of 17-keto to
17-hydroxy functionality. Similarly acetylation of 12–14 with
acetic anhydride and dry pyridine in a steam bath afforded the
corresponding 3␤,17␤-diacetoxy compounds 15–17, respec-
tively. The –CH– of benzylidene in these diacetoxy compounds
was found further little upfield (␦ ∼ 6.14 ppm) as compared to
their 3␤,17␤-diol counterparts.
In view of the literature reports that 3,17-diketo steroids
with higher degree of unsaturation in A-ring exhibit potent
aromatase inhibitory activity and significant cytotoxicity,
Oppenauer oxidation [15] of 6–8 was carried out to afford
␣,␤-unsaturated-3-keto steroids 18–20, respectively. A singlet
for the 4-CH proton at about ı 5.76 ppm was observed in
the 1H NMR spectra of all the Oppenauer products. Further
treatment of these 3,17-diketo derivatives with pyrrolidine in
refluxing methanol yielded the corresponding unstable dien-
amines 21–23, which on immediate reduction with sodium
borohydride in methanol at room temperature afforded their
corresponding 3␤,16-bis-tertiary amino steroids 24–26. In the
process the 17-keto got reduced to 17-hydroxy functionality
as indicated by an upfield shift (ı 6.44 ppm) for the vinylic pro-
ton of 16-arylidene. The configuration at positions 3 and 17
was assigned ␤ in accordance with earlier reports [16]. Further
3.1.
Chemistry
The synthesis of various 16E-arylidenoandrostene derivatives
have been carried out as depicted in Schemes 1 and 2. Aldol
condensation [12] of dehydroepiandrosterone with substi-
tuted aldehydes 3–5 at room temperature in alkaline medium
afforded 16E-benzylidene steroids 6–8, respectively. The struc-
tures of the compounds were established using various
spectral, elemental and X-ray crystallographic studies. The
methine-bridged proton at C16 appeared at ı 7.38 ppm in
the 1H NMR spectra of all these aldol products. The con-
figuration at C16 with respect to the carbonyl at C17 has
been assigned E on the basis of earlier reports from our
laboratory as well as the X-ray crystallographic studies of
some of the synthesized compounds [10,11,13,14]. These aldol
products were further subjected to various structural mod-
ifications to afford newer derivatives of medicinal interest.
Acetylation of 6–8 using acetic anhydride and dry pyridine
in a steam bath to afford 3␤-acetoxy derivatives 9–11 and
reduction with sodium borohydride in methanol affording
3␤,17␤-diol products 12–14 were carried out using a simple
method [1]. The proton resonance signals for the methine-

steroids 7 3 ( 2 0 0 8 ) 1391–1399
1397
Table 1 – Growth percentage at 10−4 molar concentration in the 3-cell line in vitro screening
S. no.
Compound
NSC no.
Growth percentage
Breast (MCF-7)
Non-small cell lung (NCI-H460)
CNS (SF-268)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
6 (DPJ-RG-1071)
7 (DPJ-RG-1147)
8 (DPJ-RG-1097)*
9 (DPJ-RG-1111)*
10 (DPJ-RG-1175)*
11 (DPJ-RG-1146)*
12 (DPJ-RG-1214)*
13 (DPJ-RG-1217)*
14 (DPJ-RG-1216)*
15 (DPJ-RG-1215)*
16 (DPJ-RG-1218)*
17 (DPJ-RG-1226)*
18 (DPJ-RG-1072)*
19 (DPJ-RG-1148)*
20 (DPJ-RG-1098)*
24 (DPJ-RG-1074)
25 (DPJ-RG-1122)
26 (DPJ-RG-1100)*
27 (DPJ-RG-1087)*
28 (DPJ-RG-1170)*
29 (DPJ-RG-1110)*
727078
727088
727083
727082
728323
727087
730472
730476
730474
730473
730477
730475
727079
727089
727084
727080
727090
727085
727081
728322
727086
98
103
0
0
0
0
0
0
8
56
0
4
0
0
36
97
80
57
14
0
125
102
0
0
0
0
0
0
1
6
0
1
0
0
5
111
73
23
3
112
122
3
0
0
0
0
1
4
101
2
85
0
0
39
117
103
74
24
0
0
0
0
0
∗
Compounds active in 3-cell line assay.
acetylation of 24–26 using acetic anhydride and dry pyridine
in a steam bath and careful processing of reaction mixture in
ice, afforded 17␤-acetoxy derivatives 27–29, respectively.
The 3-cell line actives, which reduced the growth of any one
of the cell lines to approximately 32% or less, were passed
on for evaluation in the full panel of 60-cell lines over a
minimum of 5-log dose range at ten fold dilutions, the high-
est being 10−4 M. A 48 h continuous drug exposure protocol
was used, and a sulforhodamine B (SRB) protein assay was
used to estimate the cell viability or growth [17,18]. Mean
log dose response parameters such as GI50 (drug concentra-
tion resulting in a 50% reduction in the net protein increase),
TGI (drug concentration of total growth inhibition) and LC50
(concentration of drug resulting in a 50% reduction in the
measured protein at the end of the drug treatment as com-
pared to that at the beginning) are summarized in Table 2.
Two standard drugs, meaning that their activities against
3.2.
Biological activity
The compounds 6–20 and 24–29, selected by National Cancer
Institute, Bethesda, Maryland, USA on the basis of degree of
novelty of the structure and computer modeling techniques
for evaluation of antineoplastic activity, were first assayed
using one dose (10−4 M) primary anticancer in vitro assay
against tumor in the 3-cell line panel consisting of MCF-7
(breast), NCI-H460 (lung) and SF-268 (CNS) [17,18]. The results
in the 3-cell line in vitro screening are shown in Table 1.
Table 2 – Mean log dose response parameters such as GI50, TGI and LC50 of the 60-cell line assay
S. no.
Compound
Mean log10 GI50 (molar)
Mean log10 TGI (molar)
Mean log10 LC50 (molar)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
8 (DPJ-RG-1097)
9 (DPJ-RG-1111)
10 (DPJ-RG-1175)
11 (DPJ-RG-1146)
12 (DPJ-RG-1214)
13 (DPJ-RG-1217)
14 (DPJ-RG-1216)
15 (DPJ-RG-1215)
16 (DPJ-RG-1218)
17 (DPJ-RG-1226)
18 (DPJ-RG-1072)
19 (DPJ-RG-1148)
20 (DPJ-RG-1098)
26 (DPJ-RG-1100)
27 (DPJ-RG-1087)
28 (DPJ-RG-1170)
29 (DPJ-RG-1110)
−5.59
−5.48
−5.39
−5.30
−6.23
−5.01
−5.18
−4.78
−5.05
−5.05
−5.42
−5.68
−5.55
−5.30
−5.87
−5.55
−5.85
−4.43
−4.95
−4.91
−4.74
−5.67
−4.59
−4.63
−4.47
−4.64
−4.61
−4.81
−5.17
−4.94
−4.69
−5.36
−5.13
−5.34
−4.00
−4.48
−4.50
−4.30
−5.09
−4.35
−4.39
−4.35
−4.38
−4.36
−4.34
−4.56
−4.34
−4.18
−4.87
−4.75
−4.81

1398
steroids 7 3 ( 2 0 0 8 ) 1391–1399
Table 3 – Data summarizing the preliminary in vivo hollow fiber assay reports
S. no.
Compound
NSC no.
IP score
SC score
Total score
Cell kill
1
2
27 (DPJ-RG-1087)
29 (DPJ-RG-1110)
727081
727086
8
12
0
8
8
20
No
No
the cell lines are well documented, were tested against each
cell line: NSC 19893 (5-Fluorouracil) and NSC 123127 (Adri-
amycin).
significant cytotoxicity. As expected and in accordance with
our previous reports, introduction of hydroxyl group in steroid
skeleton leads to reduction in cytotoxicity as observed for
compounds 6 and 7 (–OH is present at 17 position) and 24
and 25 (–OH at 3 position). These compounds were not able to
reduce the growth of any one of the cell lines to the required
percentage in 3-cell line assay and did not qualify for evalu-
ation in full panel of 60-cell line. In contrast, monoacetoxy
steroids 9–11 (acetoxy at 3-posiion), 27–29 (acetoxy at 17-
posiion) and 15–17 (acetoxy at 3- and 17-position) exhibit
potent cytotoxicity. Similarly diketo compounds 18–20 with
higher degree of oxidation in ring A exhibited more activity.
Of all the three series of steroidal derivatives, the ones substi-
tuted with diethylamino moiety like 8, 11, 14, 17, 20, 23, 26
and 29 appear to be the most promising one. This observation
is further supported by the fact that out of hydroxy substi-
tuted compounds 6–8 and 24–26, only 8 and 26 showed good
cytotoxicity, which may be attributed to presence of diethy-
lamino functionality in side chain. It can be said that open
ring analogues are better cytotoxic agents than their cyclic
counterparts.
Out of all 60-cell line actives, only bis-tertiary amino
steroids 27 and 29 qualified for in vivo hollow fiber assay. Two
well-placed tertiary amino groups further enhance the cyto-
toxic potential of the compounds, which is also the aim of the
current study. The most promising compound 29 possessing
diethylamino group with a total IP and SC score of 20 in in
vivo hollow fiber assay was selected for further detailed in vivo
xenograft testing. The test agent suppressed tumor growth
but unfortunately failed to meet the established antitumor
activity criteria.
Out of all the compounds screened for 60-cell line assay,
27 and 29 qualified for preliminary in vivo testing using hollow
fiber assay [19] and have shown interesting IP and SC scores. In
general, a compound is selected for in vivo studies if its mean
log10 GI50 ≤ −6 in 60-cell line assay. The total pattern of activ-
ity of the compounds is also taken into consideration by the
Biological Evaluation Committee for Cancer Drugs to select
the compound for further in vivo evaluation. These deriva-
tives lie outside the category of adequately studied class of
antitumor agents and are among the small percentage, which
have been selected for testing in the in vivo hollow fiber assays.
Data summarizing the in vivo hollow fiber assay performed by
the Developmental Therapeutic Program on the compounds
is given in Table 3. A standard panel of 12 tumor cell lines
was used for the preliminary in vivo hollow fiber screen-
ing of the in vitro actives. These include NCI-H23, NCI-H522,
MDA-MB-231, MDA-MB-435, SW-620, COLO 205, LOX, UACC-62,
OVCAR-3, OVCAR-5, U251 and SF-295. A total of three differ-
ent tumor lines are prepared for each experiment so that each
mouse receives three intraperitoneal (IP) implants (one for
each tumor line) and three subcutaneous (SC) implants (one of
each tumor line). Each compound is assessed in a total of four
experiments (3-cell lines/experiment × 4 experiments = 12 cell
lines). The test compound was administered into athymic
nude mice implanted with twelve selected human tumor
cell lines encased in hollow fibers. After 6–8 days, the fibers
were collected, cells were removed and growth inhibition
was measured using MTT assay. The percent net growth
for each cell line in each treatment group was calculated
and compared to the percent net growth in the vehicle
treated controls. Taxol was the positive control used in the
hollow fiber assay. A 50% or greater reduction in percent
net growth in the treated samples compared to the vehicle
control samples is considered a positive result. Each posi-
tive result is given a score of 2 and all of the scores are
totaled for a given compound. The maximum possible score
for an agent is 96 (12 cell lines × 2 sites × 2 dose levels × 2
(score)).
In conclusion, 16-arylidenosteroids represents a novel class
of cytotoxic agents. Further research efforts should be directed
towards rational structural modifications of such compounds
to produce better antineoplastics.
Acknowledgements
The financial support provided by the CSIR, New Delhi,
India for this project is gratefully acknowledged. The authors
express appreciation to National Cancer Institute, Bethesda,
Maryland, USA for evaluation of compounds for antineoplastic
activity, and Cipla Ltd., Bombay, India for the generous supply
of steroids.
Compounds giving promising results in these assays are
referred to Biological Evaluation Committee for cancer drugs
to select for further in vivo evaluation using xenograft models.
A compound is referred for xenograft testing if it has a com-
bined IP + SC score of 20 or greater, a SC score of 8 or greater, or
produces cell kill of any cell line at either dose level evaluated.
The total pattern of activity of the compounds is also taken
into consideration for further in vivo evaluation. Biological
Evaluation Committee for Cancer Drugs selected compound
29 for further detailed in vivo xenograft testing.
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