Synthesis of androstene oxime-nitrogen mustard bioconjugates as potent antineoplastic agents

Article abstract of DOI:10.1016/j.steroids.2017.04.005

In the present study, synthesis and antineoplastic activity of phenylacetic acid and benzoic acid nitrogen mustard conjugates of various steroidal oximes are reported for the first time. The conjugation was achieved through a more stable oxime-ester linkage and the resulting newly synthesized conjugates were evaluated in vitro on various human cancer cell lines for cytotoxicity. The extent of their alkylating activity was investigated by the in vitro colorimetric 4-(p-nitrobenzyl)pyridine (NBP) assay. The 17E-steroidal oxime-benzoic acid mustard ester 3β-acetoxy-17E-[p-(N,N-bis(2-chloroethyl)amino)]benzoyloxyimino-androst-5-ene (8) emerged as the most potent conjugate having significant cytotoxicity on most of the NCI 60-cell lines. Outstanding growth inhibition was observed on the IGROV1 ovarian cancer cell line with GI₅₀?=?0.937?μM. In general, the D-ring derived androstene oxime-nitrogen mustard conjugates were found to possess better antineoplastic activity over a variety of cancer cells in comparison to those derived from other rings of the steroid skeleton.

Full text of DOI:10.1016/j.steroids.2017.04.005

Steroids xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of androstene oxime-nitrogen mustard bioconjugates as potent
antineoplastic agents
⁎
Pratap Chandra Acharya, Ranju Bansal
University Institute of Pharmaceutical Sciences, Panjab University, Sector 14, Chandigarh 160 014, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
In the present study, synthesis and antineoplastic activity of phenylacetic acid and benzoic acid nitrogen mustard
conjugates of various steroidal oximes are reported for the first time. The conjugation was achieved through a
more stable oxime-ester linkage and the resulting newly synthesized conjugates were evaluated in vitro on
various human cancer cell lines for cytotoxicity. The extent of their alkylating activity was investigated by the in
vitro colorimetric 4-(p-nitrobenzyl)pyridine (NBP) assay. The 17E-steroidal oxime-benzoic acid mustard ester 3β-
acetoxy-17E-[p-(N,N-bis(2-chloroethyl)amino)]benzoyloxyimino-androst-5-ene (8) emerged as the most potent
conjugate having significant cytotoxicity on most of the NCI 60-cell lines. Outstanding growth inhibition was
observed on the IGROV1 ovarian cancer cell line with GI50 = 0.937 µM. In general, the D-ring derived
androstene oxime-nitrogen mustard conjugates were found to possess better antineoplastic activity over a
variety of cancer cells in comparison to those derived from other rings of the steroid skeleton.
Cytotoxic steroids
Steroid oxime esters
Mustard-steroid conjugates
DNA alkylation
NBP assay
Ovarian cancer
1. Introduction
have their own specific cellular binding site(s) as well [5]. Hormone
receptors present in the cell nucleus might be the main binding site of
Conjugating alkylating agents with steroidal nuclei has been an
the steroidal part [6]. Changing the steroid skeleton or introduction of
heteroatoms in the steroid skeleton have improved the inactive or
already active compounds to strongly active ones. It is also observed
that addition of the steroid moiety confers enhanced activity compared
to the alkylating agent alone [7,8]. In some cases, toxicity remained at
clinically acceptable levels and was significantly lower than the
alkylating agent itself [9].
These facts encouraged us to further explore the steroidal backbone for
attachment with the nitrogen mustards. Earlier, the alkylating agents have
been successfully conjugated to steroid systems through a labile ester or
amide linkage. It is observed that both, the position of attachment of
nitrogen mustard and the basic framework of steroid nucleus, are of equal
importance for the antineoplastic activity. The A- and D-rings-derived aza-
steroids and homo-aza-steroidal lactams (Fig. 2) are preferred platforms
for the improvement in biological activity compared to their B- and C-ring
substituted counterparts [10]. The ideal nitrogen mustard moieties
recognized to conjugate with these steroidal skeletons are chlorambucil,
phenylacetic acid (active metabolite of chlorambucil), benzoic acid and
cinnamic acid mustards (Fig. 2) [6–10]. Benzoic acid mustard and
phenylacetic acid mustards conjugated to C-3 and C-17 positions of A-
and D- ring modified aza-steroids and homo-aza-steroidal lactams have
come out as the best templates to synthesize powerful antineoplastic
agents belonging to this category [5].
attractive approach to fabricate cancer specific cytotoxic agents for
targeting various hormone responsive tumors such as breast, prostate
and endometrium. Conceptualizing that the steroidal part could play
the role of the “biological platform” on which the alkylator could easily
be transported through the cellular barrier to the target site, we
expected several advantages such as reduced systemic toxicity, in-
creased bioavailability, reduced dose, synergistic activity and improved
specificity of cancer therapy following this approach [1]. Conjugates of
nitrogen mustards with various steroidal skeletons like androstane,
estrane, homo-aza-steroids and homo-aza-steroidal lactams have been
synthesized to generate several potent cancer specific cytotoxic agents.
This concept has led to the successful generation of a steroidal
alkylating agent called estramustine phosphate sodium (1) (Fig. 1),
which is being used in the palliative treatment of metastatic and/or
progressive carcinoma of the prostate [2]. However, on further studies
estarmustine was found to be devoid of any hormonal or alkylating
effect, rather it binds to tubulin causing microtubule depolymerization
[
3,4]. This observation suggested that the steroidal alkylating agent
conjugates may have their own mode of action with improved
specificity towards the cancer tissue.
Studies have revealed that the steroidal part is not a mere
biological carrier” as speculated for many years in conjugates, but
“
⁎
Corresponding author.
E-mail address: ranju29in@yahoo.co.in (R. Bansal).
http://dx.doi.org/10.1016/j.steroids.2017.04.005
Received 12 September 2015; Received in revised form 23 March 2017; Accepted 19 April 2017
0
039-128X/ © 2017 Elsevier Inc. All rights reserved.
Please cite this article as: Acharya, P.C., Steroids (2017), http://dx.doi.org/10.1016/j.steroids.2017.04.005

P.C. Acharya, R. Bansal
Steroids xxx (xxxx) xxx–xxx
400 MHz spectrometer (Bruker Corporation, Billerica, USA) using
CDCl
3
as solvents containing tetramethylsilane as the internal standard
(
chemical shifts in δ, ppm). The spin multiplicities are indicated by the
symbols, s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet)
and br (broad). Mass spectra were obtained on an Applied Biosystems
API 2000™ mass spectrometer (AB Sciex Pte. Ltd., Framingham, MA,
USA). Elemental analyses were carried out on a Perkin Elmer-2400
model CHN analyzer (Perkin-Elmer Inc., USA). The purity of the
synthesized conjugates was found to be ≥95% based on the elemental
analysis. 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.
Fig. 1. Structure of estramustine phosphate sodium.
OH
H
N
O
2.2. Synthesis of steroidal oxime-nitrogen mustard conjugates
HN
O
The starting nitrogen mustards p-(N,N-bis(2-chloroethyl)amino)
HO
benzoic acid; benzoic acid mustard (2) and p-(N,N-bis-(2-chloroethyl)
amino)phenylacetic acid; phenylacetic acid mustard (3) were synthe-
sized in accordance with the literature procedure [13,14]. The steroidal
H
A-homo-aza-steroidal lactam
D-homo-aza-steroidal lactam
Cl
Cl
oximes, 3β-acetoxy-17E-hydroximino-androst-5-ene (4)
,
17β-acetoxy-
3
E/Z-hydroximino-androst-4-ene (5), 6E-hydroximino-androst-4-ene-
N
N
O
Cl
O
Cl
3,17-dione (7) and 3β-hydroxy-16E-oximino-androst-5-en-17-one (6)
were synthesized as reported, and purified by recrystallization from
methanol to afford sufficiently pure compounds [12,15,16].
HO
HO
Chlorambucil
Cl
Phenylacetic acid mustard
Cl
2.2.1. General method for the preparation of conjugates 8–12
To a mixture of the steroidal oxime (0.60 mmol, 0.207 g for 4, 5 and
N
N
0.19 g for 6) and requisite nitrogen mustard (0.76 mmol, 0.2 g for 2 and
Cl
Cl
0
0
.21 g for 3) in anhydrous dichloromethane (10 mL), DCC (0.2 g,
.97 mmol) and DMAP (0.12 g, 0.98 mmol) were sequentially added.
HO
HO
O
O
The resulting mixture was stirred overnight. The insoluble precipitate
of urea formed was filtered off. The filtrate was evaporated and
acetonitrile (50 mL) was added to precipitate the residual byproduct
(3 times), which was filtered and the filtrate was vacuum evaporated.
The residue obtained was extracted with dichloromethane and sequen-
Benzoic acid mustard
Cinnamic acid mustard
Fig. 2. Structures of the ideal steroid and mustard moieties used in the synthesis of potent
antineoplastic agents.
tially washed with 1 N HCl, H
layer was dried over anhydrous Na
purified either by recrystallization or subjecting to a column of silica
gel (mesh size 80–100) using chloroform as an eluting solvent to yield
the corresponding steroidal nitrogen mustard conjugates 8–12.
2
O, 5% Na
SO
2
CO
3 2
and H O. The organic
Steroidal oximes positioned on the A- and D-rings of androstenes
have structural similarity with the A- and D- ring modified aza-steroids
and homo-aza-steroidal lactams. Moreover, these oximes are known to
have potent cytotoxicity of their own [11,12]. Taking note of these
observations, we envisioned the synthesis of esters of benzoic acid and
phenylacetic acid mustards with oximes present on the A- and D-ring of
androstene and a study of the effect of these modifications on the
antineoplastic properties of the steroid, which we report herein.
2
4
, vacuum evaporated and
2.2.1.1. 3β-Acetoxy-17E-[p-(N,N-bis(2-chloroethyl)amino)]
benzoyloxyimino-androst-5-ene (8). The conjugate 8 was purified by
recrystallization from a mixture of ether and hexane (50:50) to yield
white needle shaped crystals (30%), m.p. 140–142 °C. FT-IRνmax (KBr):
1
2. Experimental section
2956, 1731, 1607, 1520, 1367, 1254, 1179, 1089, 833, 758. H NMR
(
CDCl
4H, -N-(CH
J = 6.88 Hz), 4.59–4.62 (m, 1H, 3α-H), 5.39 (d, 1H, 6-CH,
J = 4.96 Hz), 6.67 (d, 2H, -CH, J = 9 Hz, aromatic), 7.94 (d, 2H,
-CH, J ): δ 16.84 (CH ), 19.35
= 8.88 Hz, aromatic). 13C NMR (CDCl
), 20.47 (CH ), 21.43 (CH), 23.20 (CH ), 27.21 (CH ), 27.70
), 31.33 (CH ), 33.57 (CH ), 36.71 (C), 36.93 (2 X CH ), 38.10
), 40.10 (2 X CH ), 45.03 (C), 49.99 (CH), 53.29 (2 X CH ), 53.99
3
): δ 1.05 (s, 6H, 18 and 19-CH
3
), 2.17 (s, 3H, -OCOCH
CH Cl) J = 6.76 Hz), 3.81 (t, 4H, -N-(CH
3
), 3.66 (t,
2.1. Materials, reagents and instruments
2
2
2
,
2
CH Cl)
2
2
,
Dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopro-
o
pyl)carbodiimide (EDCI), dimethylaminopyridine (DMAP), 4-(p-nitro-
benzyl)pyridine (NBP), deuterated-chloroform (CDCl ) and triethyla-
o
3
3
3
(CH
(CH
(CH
3
2
2
2
2
2
mine were purchased from Sigma-Aldrich Corporation, India. Sodium
sulphate, sodium carbonate, silica gel-G for column chromatography
3
2
2
2
2
(
80–100 mesh size) and solvents were purchased from Merck India Pvt
(CH),), 73.77 (CH), 110.92 (2 X ArCH), 117.93 (C), 121.85 (CH),
Ltd. Solvents were distilled and dried according to the standard
procedures prior to use.
131.76 (2 X ArCH), 140.0 (ArC), 149.76 (ArC), 163.97 (C]O), 170.54
(C]N), 178.82 (C]O), ESI-MS m/z: 589.1 [MH]+
591.1 [MH+2]
+
.
,
Melting points were determined on a precision digital melting point
apparatus (VEEGO instruments corporation, Mumbai, India) and are
uncorrected. UV readings were taken on a Perkin-Elmer LAMBDA-15
double beam spectrophotometer (Perkin-Elmer Inc., USA) supplied with
matched square cuvettes of 1.0 cm light path length. Infrared spectra
Anal. calcd for C32
65.24; H, 7.23; N, 4.80.
2 2 4
H42Cl N O : C, 65.19; H, 7.18; N, 4.75. Found: C,
2.2.1.2. 3β-Acetoxy-17E-[p-(N,N-bis(2-chloroethyl)amino)]
phenylacetoxyimino-androst-5-ene (9). The residue obtained after
workup was purified by column chromatography to yield orange
colored conjugate 9 (46%), m.p. 74–76 °C. FT-IRνmax (KBr): 2942,
−1
(
wavenumbers in cm ) were recorded on Perkin-Elmer RX1 FTIR
spectrophotometer (Perkin-Elmer Inc., USA) model using potassium
1
1
bromide pellets. H NMR spectra were obtained on a Bruker Avance II
1732, 1615, 1520, 1444, 1360, 1244, 1114, 1030, 918, 803. H NMR
2

P.C. Acharya, R. Bansal
Steroids xxx (xxxx) xxx–xxx
(
-
(
CDCl
OCOCH
m, 4H, -N-(CH
J = 5.08 Hz), 6.57 (d, 2H, -CH, J
CH, J
= 8.72 Hz, aromatic). ESI-MS m/z: [MH] 603.5, [MH+2]+
05.5. Anal. calcd for C33 44Cl : C, 65.66; H, 7.35; N, 4.64.
Found: C, 65.70; H, 7.29, N, 4.71.
3
): δ 0.91 (s, 3H, 18-CH
), 3.53–3.56 (m, 6H, -N-(CH
CH Cl) ), 4.51 (m, 1H, 3α-H), 5.30 (d, 1H, 6-CH,
= 8.8 Hz, aromatic), 7.12 (d, 2H,
3
), 0.96 (s, 3H, 19-CH
3
), 1.96 (s, 3H,
1342, 1242, 1183, 833, 647. 1H NMR (CDCl
3
): δ 0.98 (s, 3H, 18-CH
CH Cl) , J = 6.68 Hz), 3.73
, J = 6.88 Hz), 4.74 (m, 1H, 3α-H), 5.42 (d, 1H,
= 9.12 Hz, aromatic), 7.86 (d,
3
),
3
2
CH Cl) and -OCOCH
2
2
2
-Ph-), 3.64
1.31 (s, 3H, 19-CH
(t, 4H, -N-(CH CH
3
), 3.58 (t, 4H, -N-(CH
Cl)
2
2
2
2
2
2
2
2
2
o
6-CH, J = 7.96 Hz), 6.59 (d, 2H, -CH, J
2H, -CH, J = 9.2 Hz, aromatic). ESI-MS m/z: 561.6 [MH]
+2] . Anal. calcd for C30 38Cl : C, 64.17; H, 6.82; N, 4.99.
Found: C, 64.22; H, 6.76; N, 5.01.
o
+
+
-
6
o
o
,
563.6 [MH
+
H
2
N
2
O
4
H
2 2 4
N O
2
.2.1.3. 17β-Acetoxy-3E/Z-[p-(N,N-bis(2-chloroethyl)amino)]
2.2.2.2. 3-[p-(N,N-Bis(2-chloroethyl)amino)]phenylacetoxy-16E-
hydroximino-androst-5-en-17-one (16). White solid. Yield: 43%, m.p.
65–67 °C. FT-IRνmax (KBr): 2943, 1726, 1616, 1520, 1455, 1355, 1247,
benzoyloxyimino-androst-4-ene (10). The steroidal conjugate 10 was
synthesized using steroidal oxime 5 [E/Z-isomeric mixture (80:20)].
The solid residue obtained after usual workup was purified by column
chromatography to yield brownish E/Z-isomeric mixture (70:30) of
steroidal oxime ester 10 (49%), m.p. 86–88 °C. FT-IRνmax (KBr): 2935,
1
1156, 1003, 806, 743. H NMR (CDCl
3
): δ 0.97 (s, 3H, 18-CH
-Ph-), 3.57 (t, 4H, -N-(CH
), 4.54 (m, 1H, 3α-H), 5.30 (d, 1H, 6-CH,
J = 5.36 Hz), 6.57 (dd, 2H, -CH, = 6.82 Hz, = 1.98 Hz,
aromatic), 7.09 (dd, 2H, -CH, J = 6.64 Hz, J = 2.08 Hz, aromatic).
ESI-MS m/z: 575.2 [MH]+
577.2 [MH+2]
40Cl : C, 64.69; H, 7.00; N, 4.87. Found: C, 64.74; H, 7.06,
N, 4.91.
3
), 1.23 (s,
3H, 19-CH
3
), 3.42 (s, 2H, -OCOCH
CH Cl)
2
2
CH Cl) ),
2
2
3.64 (t, 4H, -N-(CH
2
2
2
1
730, 1605, 1520, 1443, 1360, 1258, 1178, 1069, 918, 833, 758. 1
H
J
o
J
m
NMR (CDCl
area ratio, 19-CH
CH CH Cl)
J = 6.88 Hz), 4.49–4.55 (m, 1H, 17α-H), 6.01 (s) and 6.41 (s) (1H,
0:30 area ratio, 4-CH), 6.60–6.63 (m, 2H, -CH, aromatic), 7.89–7.93
m, 2H, -CH, aromatic). ESI-MS m/z: 589.2 [MH]+ 591.2 [MH+2]+
: C, 67.34; H, 7.36; N, 2.38. Found: C,
3
): δ 0.76 (s, 3H, 18-CH
3
), 1.0 (s) and 1.02 (s) (3H, 70:30
o
m
+
3
), 1.97 (s, 3H, -OCOCH
3
), 3.60 (t, 4H, -N-
,
. Anal. calcd for
(
2
2
2
,
J = 6.96 Hz),
3.74
(t,
4H, -N-(CH CH Cl)
2
2
2
,
C
31
H
2 2 4
N O
7
(
,
.
2.3. Antineoplastic activity
2 2 4
Anal. calcd for C33H42Cl N O
67.29; H, 7.30; N, 2.42.
2.3.1. In vitro assessment of alkylating activity [17]
2.3.1.1. Preparation of reagents. 4-(p-Nitrobenzyl)pyridine (NBP) reagent:
2
.2.1.4. 17β-Acetoxy-3E-[p-(N,N-bis(2-chloroethyl)amino)]
A 5% w/v solution of NBP was prepared in anhydrous ethyl methyl
ketone.
Triethylamine Reagent: A 50% v/v solution was prepared by taking
analytical grade triethylamine in anhydrous ethyl methyl ketone.
phenylacetoxyimino-androst-4-ene (11). The steroidal oxime ester 11
was synthesized using steroidal oxime [E/Z-isomeric mixture
80:20)]. The solid residue obtained after usual workup was purified
5
(
by column chromatography, which yielded white solid of pure steroidal
oxime ester 11 (48%), m.p. 86–88 °C. FT-IRνmax (KBr): 2934, 1737,
2.3.1.2. Standard curve of p-(N,N-bis(2-choloroethyl)amino)benzoic acid
(2) and p-(N,N-bis-(2-chloroethyl)amino)phenylacetic acid (3). The stock
solutions (1 mM) were prepared by dissolving benzoic acid mustard 2
(5.22 mg) and phenylacetic acid mustard 3 (5.48 mg) in anhydrous
ethyl methyl ketone (20 mL). Five different samples were prepared by
transferring 1, 2, 3, 4 and 5 mL of stock solution to screw capped test
tubes having provision for solvent condensation. To each tube 1 mL of
NBP reagent was added and the solutions were refluxed for 2.5 h. The
solutions were cooled to room temperature and the volume was made
up to 9 mL. To each tube triethylamine reagent (1 mL) was added to
develop the purple color just before taking the absorbance using UV and
the absorbance was measured at 560 nm. The absorbance of a reagent
blank (0 ppm) was determined as above and subtracted from that of the
other samples. Each test was repeated three times. The standard curves
were constructed by plotting the net absorbance against the
concentration of nitrogen mustards using Microsoft Excel program.
1
1
618, 1519, 1446, 1358, 1246, 1106, 1041, 920, 805. H NMR (CDCl
3
):
),
δ 0.75 (s, 3H, 18-CH
.53–3.57 (m, 6H, -N-(CH
CH CH Cl) ), 4.52 (t, 1H, 17α-H), 5.89 (s, 1H, 4-CH), 6.57 (d, 2H, -CH,
= 8.76 Hz, aromatic), 7.13 (d, 2H, -CH, J = 8.76 Hz, aromatic).
3
), 1.0 (s, 3H, 19-CH
3
), 1.97 (s, 3H, -OCOCH
3
3
(
2
CH Cl) and -OCOCH
2
2
2
-Ph-), 3.64 (t, 4H, -N-
2
2
2
J
o
o
+
+
ESI-MS m/z: 603.6 [MH]
44Cl : C, 65.66; H, 7.35; N, 4.64. Found: C, 65.59; H, 7.31,
N, 4.60.
, 605.6 [MH+2] . Anal. calcd for
C
33
H
2 2 4
N O
2
4
.2.1.5. 6E-[p-(N,N-Bis(2-chloroethyl)amino)]benzoyloxyimino-androst-
-ene-3,17-dione (12). The conjugate 12 was obtained by
recrystallization from methanol as white crystals (23%), m.p.
1
1
68–170 °C. FT-IRνmax (KBr): 2939, 1720, 1606, 1440, 1264, 1171,
1
042, 932. H NMR (CDCl
), 3.68 (t, 4H, -N-(CH
CH Cl) , J = 6.88 Hz), 6.35 (s, 1H, 4-CH), 6.70 (d, 2H, -CH,
= 8.28 Hz, aromatic), 7.95 (d, 2H, -CH, J = 8.08 Hz, aromatic).
3
): δ 0.93 (s, 3H, 18-CH
3
), 1.23 (s, 3H, 19-
CH Cl) , J = 6.84 Hz), 3.83 (t, 4H, -N-
CH
CH
3
2
2
2
(
2
2
2
J
o
o
2.3.1.3. Preparation of test samples. Test samples (0.4 mM) were
prepared by dissolving compounds 8 (5.88 mg), 9 (6.03 mg), 10
(5.88 mg), 11 (6.03 mg), 12 (5.59 mg), 15 (5.61 mg) and 16
(5.75 mg) in anhydrous ethyl methyl ketone (8 mL) in individual
screw capped test tubes. To each tube NBP reagent (1 mL) was added
and solution heated under reflux for 2.5 h. The solutions were cooled to
room temperature and the volume was made up to 9 mL. To each tube
triethylamine reagent (1 mL) was added just before taking the UV
reading to develop the purple color and the absorbance was measured
at 560 nm. The absorbance of a reagent blank (0 ppm.) was determined
as above and subtracted from that of the other samples. Each test was
repeated three times.
+
+
ESI-MS m/z: 559.1 [MH]
36Cl : C, 64.40; H, 6.49; N, 5.01. Found: C, 64.35; H, 6.53;
N, 5.09.
,
561.1 [MH+2] . Anal. calcd for
C
30
H
2 2 4
N O
2
.2.2. General method for the preparation of conjugates 15 and 16
To a solution of EDCI (0.2 g, 1.25 mmol) and DMAP (0.122 g,
mmol) in anhydrous dichloromethane (10 mL), the steroidal oxime
1
(
7, 1 mmol, 0.19 g) and the desired nitrogen mustard (2.1 mmol, 0.5 g
for 2 and 0.55 g for 3) were added and the solution was stirred
overnight. The mixture was extracted with dichloromethane and
sequentially washed with 1 N HCl, H
organic layer was dried over anhydrous Na
and the residue was purified by passing through a column of silica gel
mesh size 80–100) using chloroform as eluting solvent to yield the
pure nitrogen mustard-steroidal oxime conjugates 15 and 16.
2
O, 5% Na
2 3 2
CO and H O. The
2
SO , vacuum evaporated
4
2.3.2. In vitro cancer cell line assay
(
2.3.2.1. 60-Cell line assay (protocol of NCI) [18]. The human tumor cell
lines were grown in RPMI 1640 medium containing 5% fetal bovine
serum and 2 mM l-glutamine. Cells were inoculated into 96 well
microtiter plates in 100 μL at plating densities ranging from 5000 to
40,000 cells/well depending on the doubling time of individual cell
2
.2.2.1. 3-[p-(N,N-Bis(2-chloroethyl)amino)]benzoyloxy-16E-
hydroximino-androst-5-en-17-one (15). White solid. Yield: 34%, m.p.
24–126 °C. FT-IRνmax (KBr): 3327, 2930, 1676, 1622, 1529, 1447,
1
2
lines. The microtiter plates were then incubated at 37 °C, 5% CO , 95%
3

P.C. Acharya, R. Bansal
Steroids xxx (xxxx) xxx–xxx
air and 100% relative humidity for 24 h prior to addition of the
experimental drug conjugates.
The BALB/c mice were induced with intraperitoneal injection of 0.4%
starch and peritoneal macrophages were collected after 48 h. Raw
264.7 cell lines were also maintained and were counted and identified
when attained 60% confluences. About 2 × 105 cells (both the cell
types) were seeded into each well of 96-well tissue culture plates
(Eppendorf) and cultured in RPMI-1640 media supplemented with 10%
FCS. Peritoneal and RAW macrophages were taken in separate 96-well
plate. Cells were treated with different concentrations of the compound
After 24 h, two plates of each cell line were fixed in situ with TCA,
which represent a measurement of the cell population at the time of
drug addition (Tz) for each cell line. Experimental drugs were
solubilized in dimethyl sulfoxide at 400-fold the desired final maximum
test concentration and stored frozen prior to use. At the time of drug
addition, an aliquot of frozen concentrate was thawed and diluted to
twice the desired final maximum test concentration with complete
medium containing 50 μg/ml gentamicin. Additional four, 10-fold or ½
log serial dilutions are made to provide a total of five drug concentra-
tions plus control. Aliquots of 100 μl of these different drug dilutions
were added to the appropriate microtiter wells already containing
8 ranging from 0.7 µM to 10 µM and incubated in presence of 5% CO
2
at 37 °C for 24 h. After that, the cell culture media was replaced with
fresh RPMI (without Phenol Red) containing 1 mg/mL MTT and
incubated at 37 °C for 3 h. The formed formazan crystals were then
dissolved in DMSO by incubation at room temperature for 20 min and
absorbance was measured using an automatic plate reader (Synergy H1
Hybrid) at test wavelength of 550 nm.
1
00 μl of medium, resulting in the required final drug concentrations.
After the drug addition, the plates were incubated for 48 h at 37 °C, 5%
CO , 95% air, and 100% relative humidity. For adherent cells, the
2
assays were terminated by the addition of cold TCA (trichloroacetic
3
. Results and discussion
acid). Cells were fixed in situ by the gentle addition of 50 μl of cold 50%
(
w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at
3.1. Chemistry
4
°C. The supernatant was discarded, and the plates were washed five
times with tap water and air dried. Sulforhodamine B (SRB) solution
100 μl) at 0.4% (w/v) in 1% acetic acid was added to each well, and
For the preparation of target steroid-nitrogen mustard adducts,
(
synthesis of starting compounds p-(N,N-bis(2-chloroethyl)amino)ben-
zoic acid (benzoic acid mustard) (2) and p-(N,N-bis(2-chloroethyl)
aminophenylacetic acid (phenylacetic acid mustard) (3) was achieved
in a cost effective approach as depicted in Scheme 1 following literature
reports [13,14]. The steroidal oximes 4–7 (Fig. 3) were prepared
according to the reported methods [12,15,16]. In case of testosterone
acetate, oximation using hydroxylamine hydrochloride in pyridine
resulted in the formation of a 80:20 E/Z-isomeric mixture 5. The proton
plates were incubated for 10 min at room temperature. After staining,
unbound dye was removed by washing five times with 1% acetic acid
and the plates were air dried. Bound stain was subsequently solubilized
with 10 mM trizma base, and the absorbance was read on an automated
plate reader at a wavelength of 515 nm. For suspension cells, the
methodology was the same except that the assay was terminated by
fixing settled cells at the bottom of the wells by gently adding 50 μl of
80% TCA (final concentration, 16% TCA). Using the seven absorbance
NMR of E/Z-isomeric mixture showed split singlets of 19-CH at δ 1.08
3
measurements [time zero, (Tz), control growth, (C), and test growth in
the presence of drug at the five concentration levels (Ti)], the
percentage growth was calculated at each of the drug concentrations
levels. Percentage growth inhibition was calculated as:
and 1.12 and of 4-CH at 5.79 and 6.49 ppm (80:20). The stereochem-
istry of the oximino functionality of these derivatives has been assigned
1
E/Z on the basis of H NMR spectroscopy as described in the literature
[
12,16].
The nitrogen mustards 2 and 3 were conjugated with 3β-acetoxy-
7E-hydroximino-androst-5-ene (4), 17β-acetoxy-3E/Z-hydroximino-
[
(Ti−Tz)/(C−Tz)] × 100 for concentrations for whichTi > /=Tz
1
[
(Ti−Tz)/Tz] × 100 for concentrations for which Ti<Tz
androst-4-ene (5), 3β-hydroxy-16E-oximino-androst-5-en-17-one (6)
and 6E-hydroximino-androst-4-en-3,17-dione (7), using coupling
agents DCC and EDCI in the presence of DMAP in anhydrous dichlor-
omethane (Steglich esterification) [20,21] to afford corresponding
steroidal-nitrogen mustard conjugates 8–12, 15 and 16 as shown in
Schemes 2 and 3.
Three dose response parameters were calculated for each experi-
mental agent. Growth inhibition of 50% (GI50) was calculated from
(Ti − Tz)/(C − Tz)] × 100 = 50, which was the drug concentration
[
resulting in a 50% reduction in the net protein increase (as measured by
SRB staining) in control cells during the drug incubation. The drug
concentration resulting in total growth inhibition (TGI) is calculated
from Ti = Tz. The LC50 (concentration of drug resulting in a 50%
reduction in the measured protein at the end of the drug treatment as
compared to that at the beginning) indicating a net loss of cells
following treatment is calculated from [(Ti − Tz)/Tz] × 100 = −50.
Values were calculated for each of these three parameters if the
level of activity is reached; however, if the effect is not reached or is
exceeded, the value for that parameter is expressed as greater or less
than the maximum or minimum concentration tested.
The 17-oximino steroidal esters 8 and 9 exhibited characteristic
carbonyl infrared stretching vibrations of acetyl and oxime ester as a
−1
merged broad peak at 1731 cm . Proton NMR showed singlet for the
2.3.2.2. Selective cell line assay (protocol of ACTREC). In vitro testing on
some selected cell lines was performed using the above described SRB
−
7
assay protocols, each drug being tested at 4 dose levels (1 × 10 M,
−6
−5
−4
1
× 10 M, 1 × 10 M and 1 × 10 M). Appropriate positive
controls were run in each experiment and each experiment was
repeated three times. The dose response curve was plotted by taking
drug concentration verses percentage growth. Various dose response
parameters such as GI50, TGI and LC50 were calculated and reported.
2
.3.3. Determination of cellular toxicity on murine macrophages using MTT
assay [19]
The toxicity of the most active compound 8 was determined on
murine peritoneal macrophages and RAW 264.7 macrophage cell lines.
Scheme 1. Synthesis of benzoic acid mustard (2) and phenylacetic acid mustard (3).
Reagents and reaction conditions: (a) C H OH/H SO , h (b) ethylene chlorohydrin,
2 5 2 4
CaCO3/H O, reflux, 20 h (c) POCl, heating, steam bath (d) concentrated HCl, heating,
2
steam bath, 4 h.
4

P.C. Acharya, R. Bansal
Steroids xxx (xxxx) xxx–xxx
column of silica gel using chloroform as an eluting solvent.
The Steglich esterification reaction of benzoic acid mustard (2) and
E/Z 3-oximino derivative 5 resulted in the formation of conjugate 10 as
a 70:30 isomeric mixture. Repeated attempts to isolate the pure isomers
by crystallization as well as by employing conventional column
chromatography remained unfruitful. The proton NMR showed split
signals (70:30) for 19-CH
and 6.41(s). The singlet for -OCOCH
N-(CH CH Cl) were observed at δ 1.97, 3.60 and 3.74 ppm, respec-
3
at δ 1.0 (s) and 1.02 (s), and 4-CH at 6.01 (s)
3
, triplets for -N-(CH CH Cl) and
2
2
2
-
2
2
2
tively. However in case of esterification of steroid 5 with phenylacetic
acid mustard 3, it was possible to isolate the pure E-isomeric conjugate
1
7β-acetoxy-3E-[p-(N,N-bis(2-chloroethyl)amino)]-phenylacetoxyimi-
no-androst-4-ene (11) using column chromatography. The compound
1 showed singlets of 19-CH and 4-CH at δ 1.0 and 5.89 ppm,
1
3
1
respectively in the H NMR spectra.
Esterification of oxime 6 with mustard 2 using DCC yielded 6E-[p-
(
N,N-bis(2-chloroethyl)amino)]-benzoyloxyimino-androst-4-en-3,17-
dione (12),which was purified by recrystallization from methanol. A
Fig. 3. Structures of various steroidal oximes.
characteristic downfield shift of the 4-CH singlet was observed at δ
+
6
.35 ppm. The molecular ion [MH] peak at 559.1 and a distinct [MH
+
3
β-OCOCH
3
at ∼δ 2 ppm. Two proton triplets of -N-(CH
2
CH
2
Cl)
2
and
+2] peak of isotopic chlorine at 561.1 in the mass spectrum of 12
further confirmed the formation of the steroidal benzoic acid mustard
conjugate. However, repeated efforts to synthesize the conjugate of 6E-
hydroximino-androst-4-ene-3,17-dione (6) and phenylacetic acid mus-
tard (3) remained unsuccessful. Even though progress of the reaction
indicated towards the product formation, but isolation of the desired
compound from the multi-component reaction mixture was highly
challenging. Purification by column chromatography and subsequent
crystallization using several suitable solvents was tried, but the product
could not be isolated.
-
N-(CH CH
2
2
Cl) were observed at ∼δ 3.5 and 3.65 ppm, respectively,
2
whereas, one proton multiplet of 3α-H was found around 4.5 ppm. A
doublet of the 6-CH was seen at ∼δ 5.3 ppm. A characteristically high
+
intensity [MH+2] peak due to the presence of isotopic chlorine in the
mass spectrum provided the confirmatory evidence for the formation of
the desired steroidal oxime esters.
In case of 17-oxime based steroidal nitrogen mustard, 3β-acetoxy-
7E-[p-(N,N-bis(2-chloroethyl)amino)]phenylacetoxyiminoandrost-5-
1
ene (9), purification of the product was achieved by passing through a
Scheme 2. Synthesis of steroidal nitrogen mustard conjugates 8–12. Reagents and reaction conditions: (a) DCC, DMAP, anhy. DCM, overnight stirring, rt.
5

P.C. Acharya, R. Bansal
Steroids xxx (xxxx) xxx–xxx
Scheme 3. Synthesis of steroidal nitrogen mustard conjugates 15–16. Reagents and reaction conditions: (a) DCC, DMAP, anhy. DCM, overnight stirring, rt (b) EDCI, DMAP, anhy. DCM,
overnight stirring, rt.
Table 1
Table 2
Absorbance values of benzoic acid mustard (2) and phenylacetic acid mustard (3) at
various concentrations.
Absorbance values and percentage alkylation of steroidal test compounds (0.4 mM) in
comparison to the standard mustards.
Conc. (mM)
Absorbance
Comp. No.
Absorbance
Percentage alkylation
Compound 2 (Benzoic acid
mustard)
Compound 3 (Phenylacetic acid
mustard)
2
3
8
9
10
11
12
15
0.098
0.207
0.031
0.078
0.023
0.107
0.017
0.040
0.163
100
100
31.63
37.68
23.46
51.69
17.34
40.84
78.74
0
0
0
0
0
.1
.2
.3
.4
.5
0.006
0.035
0.069
0.095
0.131
0.0580
0.113
0.225
0.295
0.3685
1
6
The virtual insolubility of the steroidal oxime 7 in dichloromethane
oxime 7 in dichloromethane was greatly enhanced after the addition of
the coupling agent EDCI. This observation allowed us to esterify the
steroidal oxime 7 with the benzoic acid mustard 2 and phenylacetic
acid mustard 3 using EDCI and DMAP in dichloromethane. However,
the workup resulted in the isolation of compounds 3-[p-(N,N-bis(2-
chloroethyl)amino)]benzoyloxy-16E-hydroximino-androst-5-en-17-one
or chloroform made it difficult to synthesize its benzoic acid mustard
ester using DCC. Therefore, esterification of 3β-hydroxy-16E-oximino-
androst-5-en-17-one (7) with benzoic acid mustard (2) was tried in
presence of DMAP using DCC as coupling agent and anhydrous
dimethylformamide (DMF) as solvent. Although, product formation
was observed in the TLC, but isolation and purification could not be
accomplished even with extreme care. An alternate route was adopted
to synthesize the target molecules 13 and 14 using EDCI as coupling
agent (Scheme 3). It was observed that the solubility of the steroidal
(
15) and 3-[p-(N,N-bis(2-chloroethyl)amino)]phenylacetoxy-16E-hy-
droximino-androst-5-en-17-one (16) instead of the target steroidal
derivatives 13 and 14, respectively. Anticipating that the esterification
would occur at both the 3-hydroxy and 16-hydroximino positions of 7,
Fig. 4. Calibration curves of benzoic acid mustard (2) and phenylacetic acid mustard (3).
6

P.C. Acharya, R. Bansal
Steroids xxx (xxxx) xxx–xxx
Table 3
Table 3 (continued)
Percentage growth of cancer cells treated with compounds 8, 10 and 12 at 10 µM.
Cell lines
Percentage growth
Cell lines
Percentage growth
8
10
12
8
10
12
MDA-MB-468
Mean% growth
79.88
44.05
67.16
64.08
–
Leukemia
HL-60 (TB)
CCRF-CEM
K-562
MOLT-4
RPMI-8226
SR
73.96
17.49
–
5.00
3.85
26.57
−24.87
33.74
58.48
–
57.97
34.80
51.11
61.47
14.82
18.25
57.68
70.78
−7.87
the reaction was performed with two equivalents of the nitrogen
mustard acids 2 and 3. The ester, however, was formed only at 3-
hydroxy position leading to formation of conjugates 15 and 16, even
after using higher molar equivalents of nitrogen mustard and employ-
ing longer reaction time. Steric hindrance at C16 and greater affinity of
C -OH for esterification might be some of the possible reasons. The final
3
purification of the conjugates 15 and 16 was achieved by subjecting
them to a column of silica gel and eluting with chloroform.
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
2.05
23.06
−60.83
–
76.07
100.62
45.83
21.03
27.60
56.56
–
57.71
36.99
92.66
–
64.42
78.36
78.81
60.61
65.91
78.57
92.54
93.95
69.66
91.66
−4.94
79.21
The carbonyl infrared stretching bands of the ester and C17 keto
−
1
groups of steroid 15 were found merged as a broad peak at 1676 cm
Proton NMR showed triplets for -N-(CH CH Cl) and-N-(CH CH Cl)
δ 3.58 and 3.73 ppm, respectively, indicating the intact nitrogen
mustard moiety. The singlet of 3α-H resonated downfield at
4.74 ppm indicating the esterification at C position of the steroid.
.
2
2
2
2
2
2
at
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
δ
69.72
80.27
9.02
55.17
32.55
47.59
84.08
83.69
98.58
54.58
71.56
46.84
73.56
83.44
86.84
89.01
54.70
61.51
69.46
59.83
107.44
3
The doublet of 6-CH was observed at δ 5.42, while the doublets for the
aromatic protons were present at 6.59 and 7.86 ppm. The careful
analysis of the 1H NMR spectrum helped in identification of the
structure, which suggested that the esterification took place only at
3
C –OH group. The structure of 15 was further confirmed by the mass
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
+
spectrometry, which showed an [MH] and a characteristic high
5.40
63.46
51.50
37.67
22.49
−27.25
62.85
81.53
75.58
62.95
70.48
59.29
46.53
78.52
85.18
73.10
62.50
70.18
intensity [MH+2]+ peak due to the presence of isotopic chlorine at
5
61.6 and 563.6, respectively.
The 1H NMR spectrum of conjugate 16 showed a singlet for
-
(
OCOCH
2
-
at δ3.42 and triplets for -N-(CH
2
CH
2
Cl)
2
and-N-
CH CH Cl) at 3.57 and 3.64 ppm, respectively. A downfield shift
2 2 2
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
was seen for 3α-H, which appeared as a singlet at δ4.54 ppm. A doublet
5.24
15.51
76.29
89.57
78.27
103.99
107.51
67.46
105.41
72.52
82.99
113.75
92.09
83.90
120.66
96.65
89.71
110.28
79.99
92.19
59.38
47.08
99.20
81.82
73.19
97.17
68.38
of 6-CH was observed at δ 5.30, while the doublets for the aromatic
+
protons were observed at 6.57 and 7.09 ppm. A molecular ion [MH]
peak at 575.2 and [MH+2]+ peak of approximately 66% intensity of
the molecular ion peak at 577.2 were also observed confirming the
formation of the conjugate 16.
In general, with respect to the physical properties of nitrogen
mustard-steroid conjugates, it was observed, that the melting points
of all the phenylacetic acid mustard-steroidal adducts 9, 11 and 16
were significantly lower than their corresponding benzoic acid mustard
analogues 8, 10 and 15. Also, the phenylacetic acid mustards were
obtained in much higher (∼ two times) yields in comparison to the
benzoic acid mustard counterparts.
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
11.60
53.55
–
81.71
56.40
44.02
102.40
−59.06
59.67
72.01
101.5
54.54
80.29
60.92
–
80.91
76.40
56.65
98.32
−13.19
114.39
3.2. In vitro antineoplastic activity
Renal Cancer
7
86-0
47.60
56.96
46.39
50.19
44.11
47.30
79.76
−11.90
87.03
63.28
58.14
73.87
67.95
72.57
92.64
−32.01
62.42
69.27
91.11
70.92
73.48
77.74
120.80
56.16
3.2.1. In vitro assessment of alkylating activity
A498
A simple and suitable technique to determine the activity of
alkylating agents is the colorimetric NBP assay method. The nucleo-
phile NBP acts as a trap for alkylating agents with nucleophilic
characteristics similar to those of DNA bases [17,22].
The synthesized steroidal alkylating agent conjugates were sub-
jected to the NBP assay to determine their possible DNA alkylation
activity and the extent of alkylation was compared to standard
alkylating agents following a method developed by Epstein et al.
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate Cancer
PC-3
DU-145
47.77
78.66
77.06
79.56
52.31
103.48
[
17]. The duration of reaction time was optimized for test and standard
Breast Cancer
MCF7
MDA-MB-31/ATCC
HS 578T
BT-549
T-47D
samples for best results.
38.98
40.47
27.02
51.42
17.71
99.34
78.45
26.32
109.67
43.79
67.31
50.68
56.00
67.03
50.83
The absorbance values obtained for different concentrations of
standard compounds 2 and 3 are shown in the Table 1, and their
calibration curves are presented in Fig. 4. The alkylating activity was
measured in terms of percentage of alkylation (Table 2) compared to
the corresponding standard nitrogen mustard, which is assigned to have
7

P.C. Acharya, R. Bansal
Steroids xxx (xxxx) xxx–xxx
Table 4
100% alkylating activity at the same concentration.
Dose response parameters such as GI50, TGI and LC50 of compound 8 in 60-cell line assay.
The steroidal oxime esters of nitrogen mustards developed purple
color in the NBP assay indicating the possibility of DNA alkylation as
one of the possible modes of action of these derivatives. In general, the
phenylacetic acid mustard conjugates produced better alkylating activ-
ity than their benzoic acid mustard counterparts.
Cell lines
GI50 (µM)
TGI (µM)
LC50 (µM)
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
1.25
7.01
3.90
4.91
5.68
1.40
–
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
5.98
3.2.2. In vitro cancer cell line assay
Out of the newly synthesized steroidal mustard derivatives 8–12, 15
and 16 were submitted to NCI for anticancer screening, and only
compounds 8, 10 and 12 were selected for the in vitro antineoplastic
screening on 60-cell lines. The results of the single dose screen are
summarized in Table 3.
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
1.73
4.22
2.77
13.8
20.6
4.21
> 100
3.13
4.21
–
> 100
> 100
91.7
> 100
> 100
> 100
> 100
> 100
> 100
> 100
9.52
Benzoic acid mustard conjugated 17E-oximino androstane 8 pro-
duced excellent growth inhibition with mean percentage growth of
44.05% against 60-cell lines at 10 µM. The steroidal oxime ester
conjugate displayed most prolific activity against the leukemia cell
lines with mean percentage growth of 15.85% at 10 µM. The excellent
growth inhibition pattern produced by the steroidal derivative 8
qualified the compound for further evaluation against the 60 cell line
panel at five concentration levels, where it displayed significant
cytotoxicity on most of the 60-cell lines. The compound showed
prominent antileukemic activity with GI50 values of 1.25, 7.01, 3.90,
4.91, 5.68 and 1.40 µM against CCRF-CEM, HL-60, K-562, MOLT-4,
RPMI-8226 and SR leukemia cell lines, respectively. Significant growth
inhibition was also observed on the melanoma cell lines. Outstanding
growth inhibition was also observed on the ovarian cancer cell line
IGROV1 with GI50 = 0.937 µM. The oxime ester also effectively
inhibited growth of other ovarian cell lines such as OVCAR-3,
OVCAR-4, OVCAR-8 and NCI/ADR-RES with GI50 values of 6.72,
> 100
> 100
> 100
> 100
> 100
68.6
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
> 100
30.8
1.88
8.69
4.55
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
6.94
> 100
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
2.55
3.31
6.89
3.09
3.82
1.33
7.71
33.7
> 100
96.3
35.9
8.27
> 100
> 100
> 100
> 100
> 100
> 100
7.06, 4.02 and 5.07 µM, respectively. The same pattern of growth
inhibition was also observed in case of the panel of CNS cancer, renal
cancer, breast cancer, non-small cell lung cancer and colon cancer cell
lines with GI50 values ranging from 1.33 to 6.89, 1.66 to 4.98, 2.26 to
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
2.72
42.8
6.65
6.41
31.7
> 100
19.4
> 100
6.63
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
20.3, 1.73 to 20.6 and 1.88 to 8.69 µM, respectively. Dose response
parameters (GI50, TGI and LC50) of the steroidal derivative 8 in the 60-
cell line assay are given in table 4 and the dose response growth curves
against various cell lines with GI50 < 5 µM are depicted in Fig. 5.
The excellent growth inhibition pattern produced by the steroid-
nitrogen mustard conjugate 8 suggests the importance of C17 position
for conjugating with nitrogen mustard. Probably, the well placed
nitrogen mustard at the far end of the steroidal oxime helps in greater
interaction of the cellular target and the alkylating moiety to produce
the enhanced cytotoxicity.
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
0.937
6.72
7.60
53.0
4.02
–
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
The benzoic acid mustard conjugates 10 and 12 of 3E-oximino and
6
E-oximino steroids, respectively, produced reasonable cytotoxicity on
leukemia cell lines with mean percentage growth of 31.23% and
2.76%, respectively, at 10 µM. Corresponding mean percentage
5.07
> 100
5
Renal Cancer
growth of ovary cell lines were found to be 41.21% and 58.28%.
The steroidal benzoic acid mustard conjugates 10 and 15 and the
phenylacetic acid mustard conjugates 9, 11 and 16 were further
screened on four selected cell lines namely K-562 (leukemia), HL60
7
86-0
3.54
2.58
4.98
3.80
1.80
4.81
2.88
1.66
> 100
> 100
> 100
> 100
62.2
> 100
54.1
4.31
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
(
leukemia), OVCAR-3 (ovary cancer) and A-2780 (ovary cancer) at
ACTREC, for antineoplastic activity. The cell lines were chosen on the
basis of the activity data obtained from the NCI for the steroidal
conjugates 8, 10 and 12. The dose response curves are shown in Figs. 6
and 7. The calculated dose response parameters such as GI50, TGI and
LC50 for these compounds are summarized in Table 5. The phenylacetic
acid mustard derived C17-substituted steroidal oxime ester 9 exhibited
significant growth inhibition on the four selected cell lines K-562,
HL60, OVCAR-3 and A2780 with GI50 value of 13.6, 36.0, 48.7 and
UO-31
Prostate Cancer
PC-3
DU-145
7.60
79.5
> 100
> 100
> 100
> 100
Breast Cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
7.59
4.46
2.26
6.45
4.92
20.3
> 100
> 100
8.39
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
19.1 µM, respectively. Although, it displayed a lower GI50 value than
the benzoic acid mustard counterpart 8, but the growth pattern
observed from the growth curves looks quite similar. The results, yet
again, highlighted the significance of substitution of the nitrogen
mustard at C17 position.
MDA-MB-468
8

P.C. Acharya, R. Bansal
Steroids xxx (xxxx) xxx–xxx
Fig. 5. Dose response curves of compound 8 against various cancer cell lines with GI50 < 5 μM.
Fig. 6. Dose response curves of compounds 9–11, 15 and 16 against human leukemia cell lines K-562 and HL60.
The C
3
-substituted 3-oximinotestosterone acetate and benzoic acid
bond in the C3-substituted derivatives does not affect the antineoplastic
activity. The nitrogen mustard conjugation to the B-ring oxime also
results in less active 6E-steroidal benzoic acid mustard derivative 12.
Considering both the results of in vitro cytotoxicity and alkylating
activity some conclusions can be drawn regarding the mode of action
and antineoplastic activity of all the newly synthesized heterosteroidal
derivatives.
mustard conjugate 10 exhibited moderate to good cytotoxicity against
the four selected cell lines K-562, HL60, OVCAR-3 and A2780 with GI50
value of 12.4, 31.2, 51.1 and 17.9 µM, respectively, while the pheny-
lacetic acid mustard steroidal analogue 11 showed moderate activity
with GI50 value of 40.1, 61.2, 72.5 and 35.5 µM, respectively.
The C
3
-substituted phenyl acetic acid mustard conjugate 16 of the
1
6E-oxime steroid displayed a moderate degree of cytotoxicity with
The C17-substituted steroidal mustard derivatives 8 and 9 exhibited
the most prominent in vitro antineoplastic activity, but with a little
lower degree of alkylation of 31.63 and 51.69%, respectively. The
greater stability offered by the oxime ester linkage than the normal
ester linkage is probably the cause for the lower degree of alkylation in
these compounds. Furthermore, it is also expected that hydrolysis of the
oxime ester linkage occurs in the favorable biological condition (i.e.
enzymatic hydrolysis) in these conjugates to exert the high antineo-
plastic activity.
GI50 of 28.2, 51.1, 31.1 and 15.0 µM on K-562, HL60, OVCAR-3 and
A2780 cell lines, respectively. The corresponding benzoic acid mustard
conjugate 15 of the 16E-oxime steroid proved to be ineffective on these
cell lines as the GI50 values exceeds 100 µM.
In general, nitrogen mustards esterified to D-ring steroidal oxime,
especially the C17-androstene oxime, seems to be a preferred module for
the cytotoxicity in comparison to the C
decreased cytotoxicity in the case of both types of C
nitrogen mustard conjugates, 15 and 16 having a C -C double bond
along with a free oxime function at C16 and steroidal derivatives 10 and
1 having a C -C double bond, suggests that the position of the double
3
or C
6
oxime esters. The
3
-substituted
5
6
Although conjugating nitrogen mustards to 3β-hydroxy-16E-oximi-
no-androst-5-en-17-one in derivatives 15 and 16 produced maximum
alkylation 40.84 and 78.74%, respectively, but a reduction in in vitro
1
4
5
9

P.C. Acharya, R. Bansal
Steroids xxx (xxxx) xxx–xxx
Fig. 7. Dose response curves of compounds 9–11, 15 and 16 against human ovary cancer cell lines OVCAR-3 and A278.
Table 5
Dose response parameters such as GI50, TGI and LC50 (µM) of compounds 9–11, 15 and 16 against various human cancer cell lines.
Cell lines
Compound
9
K-562
GI50
HL60
GI50
OVCAR-3
GI50
A2780
GI50
TGI
LC50
TGI
LC50
TGI
LC50
TGI
LC50
13.6
12.4
40.1
> 100
28.2
66.4
89.9
> 100
> 100
89.9
> 100
> 100
> 100
> 100
> 100
36.0
31.2
61.2
> 100
51.1
78.1
73.5
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
48.7
55.1
72.5
> 100
31.1
> 100
> 100
> 100
> 100
60.1
> 100
> 100
> 100
> 100
89.1
19.1
17.9
35.3
58.8
15.0
62.7
52.2
> 100
> 100
61.6
> 100
86.5
> 100
> 100
> 100
10
11
15
16
Table 6
Toxicity study results of compound 8 against murine peritoneal macrophage and RAW
64.7 macrophage.
1
20
00
2
1
Cell lines
Murine peritoneal macrophage
RAW 264.7 macrophage
8
6
4
2
0
0
0
0
0
Conc. (µM)
% Toxicity
% Viability
% Toxicity
% Viability
%
toxicity
0
0
0
0
0
0
0
0
0
0
0
0
0
1
.7
.8
.9
.93
1
2
3
4
5
6
7
8
9
8.54
15.82
3.79
91.46
84.18
96.19
87.03
79.75
78.49
76.90
84.50
67.41
74.06
60.76
79.12
81.58
76.56
8.87
8.61
7.96
14.03
2.40
3.39
8.61
7.97
12.53
17.59
19.01
18.80
20.22
8.87
91.13
91.39
92.04
85.97
97.60
96.61
91.39
92.03
87.47
82.41
80.99
81.20
79.78
91.13
% viability
12.97
20.25
21.51
23.10
15.50
32.59
25.94
39.24
20.88
18.42
23.44
Fig. 8a. Toxicity of compound 8 against murine peritoneal macrophage.
1
20
00
0
1
8
6
4
0
0
0
antineoplastic activity was observed. The nitrogen mustards are con-
jugated to the steroid nucleus at C via the simple ester linkage in these
derivatives owing to its high susceptibility for hydrolysis into the
individual components, which could be the possible justification for the
higher alkylating activity and lower in vitro antineoplastic activity.
3
%
toxicity
% viability
20
0
3.3. Cellular toxicity against murine macrophages
The cytotoxicity of the most potent steroidal conjugate 8 was also
studied on the host cell in order to determine its toxicity on the normal
cells. For this purpose, the murine peritoneal macrophages and RAW
Fig. 8b. Toxicity of compound 8 against RAW 264.7 macrophage.
264.7 macrophage cell lines were treated with the sub GI50 doses of
1
0

P.C. Acharya, R. Bansal
Steroids xxx (xxxx) xxx–xxx
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[
[
[
(
Table 6, Fig. 8b). These results indicate that the compound 8 has very
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