Design and synthesis of 1,3-biarylsulfanyl derivatives as new anti-breast cancer agents

Article abstract of DOI:10.1016/j.bmc.2011.07.056

A new series of 1,3-biarylsulfanyl derivatives (homodibenzyl core motif) have been designed and synthesized as new estrogen receptor ligands by chopping benzothiophene core of raloxifene to engender seco-raloxifene scaffold. All the synthesized compounds were screened for anti-proliferative, anti-osteoporotic, and anti-implantation activity. Compounds (35, 36) having basic amino anti-estrogenic side chain were exhibiting potential anti-proliferative activity in MCF-7, MDA-MB-231 and ishikawa cell lines. Some of the synthesized compounds having homodibenzyl motif (5, 8, 10) have shown moderate anti-osteoporotic activity.

Full text of DOI:10.1016/j.bmc.2011.07.056

Bioorganic & Medicinal Chemistry 19 (2011) 5409–5419
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of 1,3-biarylsulfanyl derivatives as new anti-breast
cancer agents
Atul Kumar a, , Vishwa Deepak Tripathi a, Promod Kumar a, Lalit Prakash Gupta a, Akanksha a,
⇑
Ritu Trivedi b, Hemant Bid b, V.L. Nayak b, Jawed A. Siddiqui b, Bandana Chakravarti b, Ruchi Saxena b,
Anila Dwivedi b, M.I. Siddiquee c, U. Siddiqui c, Rituraj Konwar b, Naibedya Chattopadhyay b
a Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226 001, India
b Endocrinology Division, CSIR-Central Drug Research Institute, Lucknow 226 001, India
c Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Lucknow 226 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of 1,3-biarylsulfanyl derivatives (homodibenzyl core motif) have been designed and synthe-
sized as new estrogen receptor ligands by chopping benzothiophene core of raloxifene to engender seco-
raloxifene scaffold. All the synthesized compounds were screened for anti-proliferative, anti-osteopo-
rotic, and anti-implantation activity. Compounds (35, 36) having basic amino anti-estrogenic side chain
were exhibiting potential anti-proliferative activity in MCF-7, MDA-MB-231 and ishikawa cell lines. Some
of the synthesized compounds having homodibenzyl motif (5, 8, 10) have shown moderate anti-osteopo-
rotic activity.
Received 16 June 2011
Revised 27 July 2011
Accepted 27 July 2011
Available online 2 August 2011
Keywords:
Anti-breast cancer
SERM
Ó 2011 Elsevier Ltd. All rights reserved.
Anti-implantation
Raloxifene
Osteoporosis
1. Introduction
widely used estrogen antagonist as it shows anti-estrogenic effect9
on estrogen receptor (ER) positive breast cancer cells. Tamoxifen
Breast cancer is the most frequently diagnosed life-threatening
disease in women and more than 1 million women are diagnosed
with breast cancer every year, accounting for 10% of all new can-
cers and 23% of all female cancer cases.1 The disease accounts for
40,000 deaths each year in the United States alone.2 It is estab-
lished from earlier studies that, estrogens are mainly responsible
for initiation and growth of breast cancer.3 Estrogen is important
regulatory hormone which plays vital role in the development
and maintenance of the female reproductive organs, mammary
glands, and other sexual characteristics.4 Recently involvement of
estrogens in the growth and/or function of a number of other tis-
sues, such as in skeleton, cardiovascular system, and central ner-
vous system, has also been well recognized.5 Use of available
chemotherapeutics for the treatment of breast cancer is very lim-
ited because of undesirable side effect. Hence there is critical need
to develop new chemotherapeutic agents for effective treatment of
the breast cancer.6,7
stimulates endometrial proliferation in ovariectomized rats via an
estrogen receptor-dependent mechanism and its resistance limit-
ing the clinical acceptability.10 Raloxifene and arzoxifene, are selec-
tive estrogen receptor modulators and marketed for the prevention
and treatment of osteoporosis. Raloxifene has exhibited better pro-
file at endometrium level than tamoxifen and the difference in pro-
liferative activity in the uterus is the most important difference
between the selective estrogen receptor modulator (SERM) profiles
of tamoxifen and raloxifene.11,12 Raloxifene has also shown to pre-
vent bone loss, reduce serum cholesterol, and provide potent anti-
proliferative effects.13 The search for SERMs that would have an
ideal profile of tissue selectivity for the treatment of breast cancer
and osteoporosis is an active aspect of current pharmaceutical re-
search endeavors.14,15 As a part of our drug discovery effort toward
identifying potential SERMs for the treatment of osteoporosis and
breast cancer, we have synthesized 1,3-biarylsulfanyl derivatives
and evaluated them for theiranti-osteoporotic, anti-implantation
and anti-proliferative activity.
Early drug discovery efforts were focused on the design and
synthesis of non-steroidal estrogen ligands with antagonistic prop-
erties at breast and reproductive tissues.8 Tamoxifen is most
The principal idea for the designing of 1,3-biarylsulfanyl deriv-
atives comes from estradiol, noncyclic structure diethyl stilbestrol
and similar structures (Fig. 1).16,17 It can be argued that the central
bond of diethyl stilbestrol (DES) was important factor for the
correct orientation of ethyl and phenol for binding to estrogen
⇑
Corresponding author.
E-mail address: dratulsax@gmail.com (A. Kumar).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.056

5410
A. Kumar et al. / Bioorg. Med. Chem. 19 (2011) 5409–5419
OH
H3C
OH
OH
OH
H
H
HO
CH3
Dienstrol (III)
HO
HO
HO
Hexestrol (IV)
OH
Estradiol (I)
Diethylstilbestrol (II)
HO
OH
OH
HO
4,4'-(hexane-1,3-diyl)diphenol (VI)
HO
3-ethyl-4-(4-hydroxyphenethyl)phenol (VII)
4,4'-(hexane-1,6-diyl)diphenol (V)
Figure 1. Cyclic versus non-cyclic bioisosteric replacement of estradiol, diethylstilbesterol and similar analogues.
receptor. This observation was further supported by biological
activity, as cis isomer of DES is only 1/14 times active as trans iso-
mer.18 The noncyclic scaffolds such as DES, Dienestrol and hexes-
trol are equipotent as estradiol, however compounds V, VI, VII
did not have significant activity.
1,3-biarylsulfanyl derivatives and keeping the required positioning
of antiestrogenic side chain (Fig. 2).
2. Results and discussion
2.1. Chemistry
Inspired by these finding, we wish to report here design and
synthesis of 1,3-biarylsulfanyl derivatives (seco-raloxifene) a non-
cyclic and nonclassical bioisosteres of known SERMs such as
raloxifene, arzoxifene, trioxifene, ERA-923 and EM-65219 (Fig. 2).
It is interesting to note that all these SERMs have a common pat-
tern that the aryl group bearing anti-estrogen side chain (alkyl-
amino side chain) is three carbons (three atoms) away from aryl
group present in estrogenic subunit. The basic design of 1,3-bia-
rylsulfanyl derivatives is based on combination of two strategies:
(1) noncyclic/seco estradiol retains biological activity; (2) position-
ing of anti-estrogenic side with respect to estrogenic subunits.
These are applied here to generate a new estrogen receptor ligands
by chopping benzothiophene core of raloxifene to build
Substituted chalcones (3a–j) were synthesized by the conden
sation of substituted aldehydes and acetophenones via Claisen–
Schmidt reaction using zirconium chloride as a catalyst. Initially,
the reaction of 1,3-diaryl-2-propenones (3a–j) with mercaptans
in the presence of a catalytic amount of zirconium(IV) chlorides
in anhydrous dichloromethane gave corresponding Michael ad-
ducts (5–26) in high yields at room temperature (Scheme 1). We
have also synthesized compounds 27–32 (Scheme 1.) having alkyl
sulfanyl group by the use of ZrCl4 as a catalyst. Compounds 33–38
were synthesized from para-hydroxy acetophenone in two steps.
p-Hydroxy acetophenone reacted with different chloro alkyl amine
N
O
N
O
O
N
O
O
OH
O
OH
S
HO
OCH3
S
HO
Trioxifene
Raloxifene
Arzoxifene
Antiestrogenic
side chain
Substituents
Substituents
N
O
O
N
OH
O
Aryl Group
Aryl Group
N
OH
HO
Substituents
HO
Core Motif
ERA-923
EM 652
N
O
O
S
Designed compounds
Figure 2. Design of 1,3-biarylsulfanyl derivatives.

A. Kumar et al. / Bioorg. Med. Chem. 19 (2011) 5409–5419
5411
R1
the basis of their molecular conformations and interaction profile
in the protein binding site, the docking study of the compound
35 was carried out as an instructive step that can provide further
insight into the nature of ligand binding of the ER
a. The interac-
O
ZrCl4(25mol%)
R3
tion patterns of compounds 35 suggested that it anchor to the
binding pocket (amino acids Glu353 and Arg394) with dock score
of À27.592 kJ/mol Figure 3.1.
dry DCM or
S
O
solvent free
R2
R1
HS
5-26
R3
R2
3a-j
2.2. Biology
R1
ZrCl4(25mol%)
dry DCM or
solvent free
R3
S
All the compounds synthesized were evaluated for their anti-
proliferative activity in MCF-7, MDA-MB-231 and Ishikawa cell
lines (Tables 1 and 2). Compounds exhibiting promising biological
activity were screened for their relative binding affinity, estroge-
nicity, anti-estrogenicity, anti-osteoporotic and anti-implantation
activity (Table 3). Two compounds (35, 36) with anti-estrogenic
side chains showed significant anti-proliferative activity similar
to that of tamoxifen against MCF-7, MDA-MB-231 cancer cell lines
(Table 2). Compounds 35, 36 have exhibited excellent receptor
binding affinity (RBA) (4.467 and 5.272) and may interact with
estrogen receptor with good affinity. Interestingly, 35 and 36 were
exhibiting low estrogenicity at uterine level (79% and 58%) in com-
parison to tamoxifen (155%) and showed good anti-estrogenicity
(31% and 34%). These compounds have also showed good antioste-
oporotic as well as anti-implantation activity (Table 3).
It is evident from Table 3 that hydroxyl group was essential in
arylsulfanyl moieties (35, 36) for promising activity as well as
receptor binding affinity, whereas corresponding alkyl substitution
were found to be less active. Compounds (35, 36) have been shown
higher RBA value, possibly due to the presence of hydroxyl groups
present at para position of the phenyl ring. Compounds having
anti-estrogenic alkyl amino side chain with piperidine groups were
exhibiting better biological profile in comparison to pyrrolidine
and dimethylamino groups. It is noteworthy that the central
homodibenzylic core (compounds 5, 10) was exhibiting promising
antiosteoporotic activity (43.8% and 39.0%), which is comparable to
H
O
R3
S
R2
27-32
Scheme 1. Synthesis of 1,3-biarylsulfanyl derivatives.
hydrochlorides in the presence of anhydrous K2CO3 to furnish 4a–c
followed by the thia-Michael reaction to give desired products
33–38 (Scheme 2).
The introduction of antiestrogenic side chain in compound 3g
having the free hydroxyl group at para position of the aryl ring
was achieved on reaction with different alkyl mercaptan via thia-
Michael reaction followed by alkylation of hydroxyl group by var-
ious chloro alkyl amine hydrochlorides in the presence of K2CO3
and acetone to furnish compound 39–41 in good yields
(Scheme 3).
All the products were characterized by the 1H,13C NMR and
Mass spectroscopy. Synthesized compounds were screened for
anti-proliferative activity (Tables 1 and 2) antiosteoporotic, recep-
tor binding affinity, estrogenicity, antiestrogenictiy and anti-
implantation activity (Table 3). In order to evaluate the molecular
docking result (Fig. 3) of b-aryl b-mercapto ketone analogues on
R5
O
OH
O
H
O
R5
SH
Cl
R3
dry acetone
R2
ZrCl4 /DCM
O
K2CO3
Reflux
O
R3
O
(4a-c)
S
R2
R5
33-38
R5 = (4a) pyrrolidine
(4b) piperidine
(4c) N,N-dimethyl
Scheme 2. Synthesis of substituted 1,3-biarylsulfanyl derivatives via a multicomponent reaction.
R5
OH
O
O
R5
R3-SH
Cl
O
O
dry acetone
K2CO3, Reflux
R2
ZrCl4(25mol%)
R3
R3
S
OH
S
dry DCM or
3g
solvent free
R2
39-41
R2
Scheme 3. Synthesis of 1,3-biarylsulfanyl derivatives.

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A. Kumar et al. / Bioorg. Med. Chem. 19 (2011) 5409–5419
Table 1
Antiproliferative activity of 1,3-biarylsulfanyl derivatives (5–32)
Entry
R1
R2
R3
Yieldb (%)
Antiproliferative activity
a
a
a
a
MCF-7 IC50
(
lM)
Iska IC50
(l
M)
MDA-MB-231 IC50
(
lM)
HEK-293 IC50 (lM)
5
6
7
8
4-H
4-H
4-H
4-H
4-H
4-H
4-H
4-H
4-Me
4-Cl
4-H
4-Me
4-H
4-Me
4-H
3-Me
4-Me
4-H
4-Cl
4-Cl
4-Cl
4-H
4-H
4-H
4-NO2
4-H
4-Cl
92
96
90
95
94
92
90
88
85
96
94
98
85
90
88
96
78
68
78
90
98
98
88
86
89
70
90
90
24 2.02
NA
NA
20 0.04
NA
NA
26 1.13
17 2.31
16 1.16
NA
NA
22 2.31
NA
57 7.83
NA
NA
—
—
—
—
4-OMe
4-OMe
4-OMe
4-OMe
4-OH
4-OH
4-Me
4-H
4-H
H
4-H
4-OMe
4-H
4-H
4-H
4-H
4-OMe
4-Me
4-H
21 3.21
23 1.33
17 1.74
18 1.49
19 2.37
25 1.34
27 2.03
24 3.14
28 1.07
25 2.35
NA
34 1.32
45 5.02
31 2.35
28 4.33
33 2.34
35 3.07
41 5.14
38 .352
31 2.46
29 2.35
NA
9
4-H
—
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
4-OMe
4-OMe
4-H
4-H
3-Cl
4-OH
4-Cl
4-NO2
4-OMe
4-Cl
38.2
NA
31.4
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
NA
24 1.13
21 1.66
22 0.37
25 2.35
NA
21 4.37
23 3.43
26 2.61
22 2.39
28 2.89
29 2.41
23 1.07
24 1.43
NA
NA
NA
4-OMe
3-NO2
4-H
29 0.37
25 4.37
NA
30 4.65
25 1.17
25 2.09
29 1.98
NA
NA
24 0.16
NA
27 2.05
25 3.34
41 4.02
38 3.74
44 3.74
48 5.67
36 3.12
36 1.87
32 3.36
42 4.31
44 4.25
NA
Napthylc
4-H
3-Cl
3-Cl
4-H
4-H
4-H
4-H
4-OMe
4-H
4-H
4-H
4-H
CH2CH2COOH
CH2CH2COOH
Ethyl
Ethyl
Ethyl
Me
4-OMe
4-OMe
4-OH
4-OMe
4-H
—
—
—
—
NA
NA
25 0.32
NA
NA
NA
—
a
IC50 values for anti-breast cancer activity of ligands using MCF-7, Ishikawa and MDA-MB-231 cell lines, using tamoxifen as a positive control. The results are repre-
sentative of one of three similar experiments each performed in triplicate. Each datum depicts the IC50 for each scaffold. The deviation in each case was less than 5% and NA
represents ligands that were found to be inactive at 50
l
M in MTT assays.
b
Isolated yield.
c
In compound 23 phenyl ring containing R1 is replaced by naphthyl ring.
raloxifene (38%) in anti-resorptive PTH induced resorption of 45Ca
chick bone assay. Compounds 5, 8 and 10 have been showing sig-
nificant RBA (1.25, 2.67, 2.21) hence it appears that compounds
may interact with estrogen receptor.
Compounds (5, 8, 10) were also screened for osteoclastogenesis
in response to RANK (Receptor activator of nuclear factor-kappa B
ligand) and MCSF (macrophage colony-stimulating factor) treat-
ment for 7 days in bone marrow cells (BMCs). These compounds
at 1 lM concentrations, significantly inhibited expression of tar-
trate-resistant acid phosphatise (TRAP), and RANK Fig. 4a and b.
These compounds (5, 8, 10) were showing high estrogenicity
with moderate antiestrogenicity. HEK-293 cell line is derived from
Table 2
Antiproliferative activity alkyl amino substituted 1,3-biarylsulfanyl derivatives (33–41)
R2c
R3
R5
Yieldb (%)
MCF-7 IC50
(
lM)
Iska. IC50
(lM)
MDA-MB-231 IC50
(l
M)
HEK-293 IC50 (lM)
a
a
a
a
Entry
33
34
H
H
H
86
85
>50
>50
20 0.04
NA
27 5.21
28 4.22
NA
NA
N
Me
N
35
3-OH
OH
88
12.2 1.22
17 2.12
19 2.44
65.8
N
N
36
37
38
39
40
H
OH
OH
OH
Et
82
82
85
86
90
9.6 1.24
25 4.37
16.2 1.13
17.3 2.31
16.0 1.16
NA
16.5 1.24
>50
75.2
28.7
27.5
32.8
NA
OH
H
N
28.6 3.02
27.0 2.41
32.8 2.16
27 1.21
38 4.65
40.2 2.64
N
H
N
H
Et
28.5 4.60
N
41
H
Et
80
27 1.43
>50
>50
29.2
10
N
Tamoxifen
12.48 1.23
22.5 0.8
18.9 1.26
a
IC50 values for anti-breast cancer activity of ligands using MCF-7, Ishikawa and MDA-MB-231 cell lines, using tamoxifen as a positive control. The results are repre-
sentative of one of three similar experiments each performed in triplicate. Each datum depicts the IC50 for each scaffold. The deviation in each case was less than 5% and NA
represents ligands that were found to be inactive at 50
l
M in MTT assays.
b
Isolated yield.
c
All the position of R2, at the para position unless otherwise specified.

A. Kumar et al. / Bioorg. Med. Chem. 19 (2011) 5409–5419
5413
Table 3
RBA, estrogencity, antiestrogencity, antiosteoporotic (antiresorptive), and antiimplantation activity of some substituted 1,3-biarylsulfanyl derivatives
Entry
RBAa
Estrogenicity
Antiestrogenicity
(% inhibition in
uterine Wt. gain)c
Anti-resorptive (PTH induced resorption of 45Ca)
Anti-implantation activity
(% increase in
(%) (20 mg/kg)f
T/C ratiod
(%) Inhibitione
uterine weight)b
5
8
10
33
34
35
36
37
38
39
40
41
1.25
2.67
2.21
185
192
187
174
76
16
18
16
12
8
31
34
24
18
4
0.562
0.784
0.610
0.863
0.845
0.812
0.787
0.934
0.902
0.944
0.947
0.963
43.8
21.6
39.0
13.7
15.5
18.8
21.3
6.6
9.8
5.6
5.5
3.7
Inactive
Inactive
Inactive
Inactive
Inactive
100
100
Inactive
60
0.157
0.245
4.467
5.272
5.154
0.670
0.246
0.175
0.191
2.2
79
58
111
101
212
134
152
155
Inactive
Inactive
100
7
9
49
Tamoxifen
Raloxifene
0.620
38
a
Percent (%) of estradiol-17b.
b
Control group of animals received the vehicle alone. The values represent the mean uterine weight and activity expressed as percent increase over that of control used as
basal value.
c
Computed as (E À T) Â 100/E where E and T refer to the mean uterine weights from animals treated with 17
a-ethynylestradiol.
d
e
f
T/C (Treated/Control).
Denotes the ability of the test compounds to inhibit PTH induced 45Ca resorption, computed as (C À T) Â 100/C.
All rats pregnant n = 6.
Figure 3. Molecular modeling studies of compound 35 and raloxifene.
normal fetal kidney tissues which of non-cancer origin and hence,
commonly considered as in vitro model for evaluating non-specific
cytotoxicity. Therefore, compounds (35–39, 41) were also tested
for their non-cancer specific cytotoxicity in HEK-293 cell line.
The two most active compounds 35 and 36 were exhibiting low
cytotoxicity than other compounds of the series. All the results
are summarized in Table 3.
exhibited antiproliferative activity comparable to tamoxifen. Thus,
35, 36 cause selective inhibition of ER+ve MCF-7 in comparison to
ERÀve MDA-MB-231 by acting as ER antagonist and this is well
supported by their significant affinity towards ER.
4. Experimental section
4.1. General consideration
3. Conclusions
Unless otherwise specified all the reagents and catalysts were
purchased from Sigma–Aldrich and were used without further
any purification. The common solvents were purchased from
Ranbaxy. Organic solutions were concentrated under reduced pres-
sure on a Büchi rotary evaporator. Chromatographic purification of
products was accomplished using flash chromatography on
230–400 mesh silica gel. Reactions were monitored by thin-layer
chromatography (TLC) on 0.25 mm silica gel plates visualized un-
der UV light, iodine or KMnO4 staining. 1H and 13C NMR spectra
were recorded on a Brucker DRX-200 MHz Spectrometer. Chemical
shifts (d) are given in ppm relative to TMS and coupling constants
(J) in Hertz. IR spectra were recorded on a FTIR spectrophotometer
In present study we have designed and synthesized 1,3-bia-
rylsulfanyl derivatives and evaluated them for their SERM behavior
in various cancer cell lines as well as for their antiosteoporotic
activity. Our finding suggests that b-aryl-b-mercapto ketone ana-
logues (5–32) without antiestrogenic side chain have exhibited
moderate antiproliferative activity in MCF-7, Ishikawa and MDA-
MB-231 cancer cell lines. Whereas, compounds (33–41) with basic
alkyl amino side chain were showing significant antiproliferative
activity. In addition compounds (5, 8, 10) were evaluated for their
antiosteoporotic activity and showed promising activity similar to
that of raloxifene. Most potent compounds 35, 36 of the series

5414
A. Kumar et al. / Bioorg. Med. Chem. 19 (2011) 5409–5419
a
b
Control
Compound 5
1.5
TRAP gene expression
RANK gene expression
1.0
0.5
0.0
*
**
***
10
***
**
Compound 8
Compound 10
***
5
Con
8
Compounds
Figure 4. (a) Compounds (5, 8, 10) were tested for their effects on the induction of TRAP, and RANK, in freshly isolated murine bone marrow cells treated with RANKL and
MCSF for 7 days. QPCR determination of TRAP, and RANK, was made and the data expressed as fold been expressed as the number of animals taken in each group (n = 5). (b)
Representative photomicrograph of TRAP-stained cells that were counted. change over vehicle (control) using quantitative real time PCR. (3.1) Fold change of TRAP positive
cells in control and test compounds Data have.
Shimadzu 8201 PC and are reported in terms of frequency of
absorption (cmÀ1). Mass spectra (ESIMS) were obtained by micro-
mass quattro II instrument.
(t, 1H, J = 7.6, Hz), 7.13–7.54 (m, 13H), 7.85 (d, 2H, J = 7.5 Hz), 13C
NMR (50 MHz, CDCl3) d: 44.1, 48.6, 127.7, 127.9, 128.2, 128.4,
128.8, 129.0, 129.2, 133.1, 133.6, 134.6, 137.1, 141.6, 197.4; MS
(m/z %): 319 [M+H]+; Anal. Calcd for C21H18OS: C, 79.21; H, 5.70.
Found: C, 79.26; H, 5.71.
4.2. Procedure for synthesis of chalcones (3a–j)
Chalcones (3a–j) were synthesized via Clasien–Schmidt con-
densation of substituted benzaldehyde and acetophenones. In dry
DCM substituted benzaldehyde (1.0 mmol), acetophenone
(1.0 mmol) in the presence of zirconium chloride (20 mol %) was
taken. Reaction was stirred at room temperature for 2 h leading
to generation of chalcone. Progress of reaction was monitored by
TLC. After completion of reaction solvent was evaporated under
the reduced pressure and residue was extracted with ethyl acetate
and water. The organic layer was separated and dried over anhy-
drous Na2SO4 and filtered. The filtrate was evaporated under
vacuum on a rotary evaporator.
4.4.2. 1,3-Diphenyl-3-p-tolylsulfanyl-propan-1-one (6)
Yield: 96%; White solid; mp 110–112 °C. IR
m
max: 1601,1664 cm
À1
;
1H NMR (200 MHz, CDCl3) d: 2.29 (s, 3H), 3.56–3.63 (m, 2H),
4.88 (t, 1H, J = 7.0 Hz), 7.01 (d, 2H, J = 7.5 Hz), 7.17–7.54 (m,
10H), 7.85 (d, 2H, J = 7.4 Hz); 13C NMR (50 MHz, CDCl3) d: 21.5,
45.0, 49.0, 127.7, 128.2, 128.4, 128.8, 128.9, 130.0, 130.7, 133.6,
133.8, 137.1, 138.2, 141.7, 197.5; MS (m/z %): 333 [M+H]+; Calcd
for C22H20OS: C, 79.48; H, 6.06. Found: C, 79.51; H, 6.10.
4.4.3. 3-(4-Chloro-phenyl sulfanyl) 1,3-diphenyl-propan-1-one
(7)
Yield: 90%; White solid; mp 90–92 °C. IR
mmax: 688, 1473,
4.3. Representative procedure for the zirconium catalyzed 1, 4
conjugate addition of aryl thiols to 1,3-diaryl-2-propenones
(5–26)
1685 cmÀ1 1H NMR (200 MHz, CDCl3) d: 3.57–3.62 (m, 2H), 4.90
;
(t, 1H, J = 7.0 Hz), 7.14–7.25 (m, 9H), 7.40–7.86 (m, 3H), 7.87–
7.90 (d, 2H, J = 6.9 Hz); 13C NMR (50 MHz, CDCl3) d: 44.9, 48.7,
127.9, 128.1, 128.4, 128.9, 129.0, 129.3, 133.0, 133.7, 134.2,
134.6, 137.1, 141.4, 197.1; MS (m/z %): 353 [M+H]+; Calcd for
To a magnetically stirred mixture of 1,3-diaryl-2-propenone
(1 mmol) with arylmercaptan (1.5 mmol) in dry DCM (1–2 mL),
zirconium chloride (25 mol %) was added. The reaction mixture
was stirred at room temperature for 20–30 min. There was forma-
tion of orange color transition metal complex with evolution of
heat, which gradually converted into the light yellow to ceramic
white after the completion of the reaction. The progress of the
reaction was monitored by TLC. After completion of the reaction,
the reaction mixture was extracted with EtOAc or DCM. The organ-
ic layer was separated and dried over anhydrous Na2SO4 and fil-
tered. The filtrate was evaporated under vacuum on a rotary
evaporator.
C21H17ClOS: C, 71.48; H, 4.86. Found: C, 71.51; H, 4.82.
4.4.4. 3-(4-Methoxyphenyl)-1-phenyl-3-(phenylthio)propan-1-
one (8)
Yield: 95%; White solid; mp 80–82 °C. IR
mmax: 1602, 1665,
3429 cmÀ1 1H NMR (200 MHz, CDCl3) d: 3.52–3.59 (2H, m) 3.83
;
(s, 3H), 4.91 (t, 1H, J = 6.3 Hz), 6.86 (d, 2H, J = 8.8 Hz), 7.34–7.16
(m, 10H), 7.83 (d, 2H, J = 8.8 Hz, Ar-H); 13C NMR (50 MHz, CDCl3)
d: 44.7, 48.7, 55.8, 114.1, 127.7, 127.8, 128.2, 128.8, 129.2, 130.3,
130.7, 133.0, 134.7, 141.7, 164.0, 195.8; MS (m/z %): 349 [M+H]+;
Calcd for C22H20O2S: C, 75.83; H, 5.79. Found: C, 75.85; H, 5.75.
4.4. Spectroscopic and analytical details of b-aryl-b-mercapto
4.4.5. 3-(4-Methoxyphenyl)-1-phenyl-3-(p-tolylthio)propan-1-
ketones (5–26)
one (9)
Yield: 94%; White solid; mp 98–100 °C. IR
mmax: 1600, 1683,
4.4.1. 1,3-Diphenyl-3-phenylsulfanyl-propan-1-one (5)
3420 cmÀ1 1H NMR (200 MHz, CDCl3) d: 2.29 (s, 3H), 3.57–3.51
;
Yield: 92%; White solid; mp 118–120 °C. IR
m
max: 1588, 1677,
(m, 2H), 3.85 (s, 3H), 4.83 (t, 1H, J = 6.4 Hz), 6.86 (d, 2H,
2364 cmÀ1 1H NMR (200 MHz, CDCl3) d: 3.50–3.73 (m, 2H), 4.91
;
J = 8.8 Hz), 7.40–7.00 (m, 9H), 7.83 (d, 2H, J = 8.8 Hz, Ar-H); 13C

A. Kumar et al. / Bioorg. Med. Chem. 19 (2011) 5409–5419
5415
NMR (50 MHz, CDCl3) d: 21.5, 44.5, 49.1, 55.8, 114.1, 127.6, 128.2,
128.7, 128.9, 130.0, 130.2, 130.7, 133.8, 138.1, 141.8, 163.9, 196.0;
MS (m/z %): 363 [M+H]+; Calcd for C23H22O2S: C, 76.21; H, 6.12.
Found: C, 76.23; H, 6.17.
d: 44.1, 49.3, 115.9, 117.8, 120.8, 128.1, 128.2, 129.2, 132.4, 134.2,
136.9, 137.6, 141.9, 158.7, 197.8; MS (m/z %): 335 [M+H]+; Calcd
for C21H18O2S: C, 75.42; H, 5.43. Found: C, 75.40; H, 5.39.
4.4.12. 1-(4-Chlorophenyl)-3-(4-chlorophenylsulfanyl)-3-
phenyl-propan-1-one (16)
4.4.6. 1,3-Bis-(4-methoxy-phenyl)-3-phenylsulfanyl-propan-1-
one (10)
Yield: 98%; White solid; mp 84–86 °C. IR
mmax: 688, 1473, 1685,
Yield: 92%; White solid; mp 120–122 °C; IR
m
max: 3435, 1670,
3061 cmÀ1 1H NMR (200 MHz, CDCl3) d: 3.58 (d, 2H, J = 7.1 Hz),
;
1599 cmÀ1 1H NMR (200 MHz, CDCl3) d: 3.49–3.57 (m, 2H), 3.74
;
4.88 (t, 1H, J = 7.1 Hz), 7.16–7.58 (m, 11H), 7.88 (d, 2H, J = 7
8 Hz); 13C NMR (50 MHz, CDCl3) d: 44.8, 48.3, 128.4, 128.9, 129.0,
129.1, 129.4, 129.5, 130.8, 133.9, 134.5, 134.8, 137.5, 140.1,
196.8; MS (m/z %): 387 [M+H]+; Calcd for C21H16Cl2OS: C, 65.12;
H, 4.16. Found: C, 65.10; H, 4.17.
(s, 3H), 3.84 (s, 3H,), 4.89 (t, 1H, J = 6.4 Hz), 6.75 (d, 2H,
J = 8.6 Hz), 6.86 (d, 2H, J = 8.8 Hz), 7.20–7.35 (m, 7H), 7.83 (d, 2H,
J = 8.8 Hz); 13C NMR (50 MHz, CDCl3) d: 44.7, 48.1, 55.6, 55.8,
114.0, 114.1, 127.7, 129.2, 130.2, 130.8, 132.9, 133.6, 134.9,
138.5, 159.0, 163.9, 196.1; MS (m/z %): 379 [M+H]+; Calcd for
C23H22O3S: C, 72.99; H, 5.86. Found: C, 72.96; H, 5.87.
4.4.13. 3-(4-Chlorophenylthio)-1-(4-nitrophenyl)-3-
phenylpropan-1-one (17)
4.4.7. 1,3-Bis-(4-methoxy-phenyl)-3-p-tolyl-sulfanyl-propan-1-
one (11)
Yield: 85%; White solid; mp 94–96 °C. IR
mmax: 688, 1346, 1519,
1681, 3058 cmÀ1 1H NMR (200 MHz, CDCl3) d: 3.65 (d, 2H,
;
Yield: 90%; White solid; mp 130–135 °C. IR
m
max: 1167, 1234,
J = 7.1 Hz), 4.94 (t, 1H, J = 7.1 Hz), 7.15–7.61 (d, 2H, J = 7.8 Hz)
7.88–7.91 (m, 9H), 8.09–8.12 (m, 2H); 13C NMR (50 MHz, CDCl3)
d: 44.3, 48.5, 124.2, 128.2, 128.4, 128.8, 128.9, 129.4, 131.6,
132.2, 134.1, 135.1, 136.6, 149.3, 196.2; MS (m/z %): 398 [M+H]+;
Calcd for C21H16ClNO3S: C, 63.39; H, 4.05; N, 3.52. Found: C,
63.36; H, 4.01; N, 3.54.
1599, 1670, 2364 cmÀ1 1H NMR (200 MHz, CDCl3) d: 2.29 (s,
;
3H), 3.47–3.55 (m, 2H), 3.74 (s, 3H), 3.84 (s, 3H), 4.88 (t, 1H,
J = 6.3 Hz), 6.75 (d, 2H, J = 8.6 Hz), 6.90 (d, 2H, J = 8.8 Hz), 7.05 (d,
2H, J = 8.0 Hz), 7.20–7.25 (m, 4H), 7.87 (d, 2H, J = 8.8 Hz); 13C
NMR (50 MHz, CDCl3) d: 21.5, 44.7, 48.6, 55.6, 55.8, 114.0, 114.1,
127.7, 129.2, 130.0, 130.3, 130.7, 131.0, 133.7, 138.0, 159.0,
163.9, 196.2; MS (m/z %): 393 [M+H]+; Calcd for C24H24O3S: C,
73.44; H, 6.16. Found: C, 73.46; H, 6.13.
4.4.14. 3-(4-Chlorophenylthio)-1-(4-methoxyphenyl)-3-
phenylpropan-1-one (18)
Yield: 90%; White solid; mp 86–90 °C. IR
mmax: 1610, 1674,
4.4.8. 3-(4-Hydroxyphenyl)-1-phenyl-3-(phenylthio)
propan-1-one (12)
2947 cmÀ1 1H NMR (200 MHz, CDCl3) d: 3.53–3.62 (m, 2H), 3.74
;
(s, 3H), 4.93 (t, 1H, J = 8.5 Hz), 6.79 (d, 2H, J = 8.7 Hz), 7.17–7.54
(m, 9H), 7.89 (d, 2H, J = 7.3 Hz); 13C NMR (50 MHz, CDCl3):
d = 45.1, 48.4, 55.6, 114.3, 123.4, 124.1, 124.8, 125.2, 128.5, 129.0,
129.3, 129.4, 133.4, 133.8, 134.1, 137.1, 159.3, 197.3; MS (m/z %):
383 [M+H]+; Calcd for C22H19ClO2S: C, 69.01; H, 5.00. Found: C,
68.98; H, 5.03.
Yield 88%; White solid; mp 103–105 °C; IR
mmax: 1602, 1663,
3420 cmÀ1 1H NMR (200 MHz, CDCl3) d: 3.44–3.67 (m, 2H), 4.89
;
(t, 1H, J = 7.1 Hz), 6.80 (d, 2H, J = 8.7 Hz), 7.16–7.42 (m, 10H),
7.78 (d, 2H, J = 9.4 Hz), 8.12 (s, 1H); 13C NMR (50 MHz, CDCl3) d:
44.6, 48.8, 115.8, 127.7, 127.9, 128.1, 128.8, 129.2, 131.2, 131.5,
133.1, 134.6, 141.5, 161.1, 196.6; MS (m/z %): 335 [M+H]+; Calcd
for C21H18O2S: C, 75.42; H, 5.43. Found: C, 75.45; H, 5.47.
4.4.15. 1-(4-Chlorophenyl)-3-(4-methoxyphenyl)-3-
(phenylthio)propan-1-one (19)
4.4.9. 3-(4-Hydroxyphenyl)-1-phenyl-3-(m-tolylthio)propan-1-
one (13)
Yield: 88%; White solid; mp 84–86 °C. IR mmax: 688, 1473, 1685,
3061 cmÀ1; 1H NMR (200 MHz, CDCl3) d: 3.58 (m, 2H), 3.72 (s, 3H),
4.88 (t, 1H, J = 7.1 Hz), 7.16–7.58 (m, 11H), 7.88 (d, 2H, J = 7 8 Hz);
13C NMR (50 MHz, CDCl3) d: 43.8, 49.3, 55.6, 127.4, 128.6, 129.0,
129.1, 129.2, 129.5, 130.8, 132.9, 134.6, 134.8, 137.5, 142.1,
196.8; MS (m/z %): 383 [M+H]+; Calcd for C22H19ClO2S: C, 69.01,
H, 5.00. Found: C, 68.97; H, 5.02.
Yield: 85%; White solid; mp 98–101 °C. IR
mmax: 1604, 1666,
3422 cmÀ1 1H NMR (200 MHz, CDCl3) d: 2.31 (s, 3H), 3.51–3.57
;
(m, 2H), 4.86 (t, 1H, J = 7.2 Hz), 6.80 (m, 3H), 7.02–7.39 (m, 9H),
7.82 (d, 2H, J = 8.5 Hz); 13C NMR (50 MHz, CDCl3) d: 21.4, 44.6,
48.8, 115.8, 127.7, 127.9, 128.1, 128.8, 129.2, 131.0, 131.4, 133.0,
134.7, 141.5, 161.1, 196.6; MS (m/z %): 349 [M+H]+; Calcd for
C22H20O2S: C, 75.83; H, 5.79. Found: C, 75.88; H, 5.81.
4.4.16. 1-(4-Methoxyphenyl)-3-phenyl-3-(phenylthio)
propan-1-one (20)
4.4.10. 1-(3-Chlorophenyl)-3-p-tolyl-3-(p-tolylthio)propan-1-
one (14)
Yield: 96%; White solid; mp 87–88 °C. IR
mmax: 1580, 1683,
3445 cmÀ1 1H NMR (200 MHz, CDCl3) d: 3.78 (s, 3H), 4.91 (t, 1H,
;
Yield: 96%; White solid; mp 100–104 °C. IR
m
max: 1601, 1674,
J = 8.2 Hz), 3.52–3.59 (m, 2H), 6.78 (d, 2H, J = 8.6 Hz), 7.26–7.54
(m, 10H), 7.85 (d, 2H, J = 7.4 Hz); 13C NMR (50 MHz, CDCl3) d:
44.7, 48.7, 55.8, 114.1, 127.7, 127.8, 128.2, 128.8, 129.4, 130.3,
130.7, 133.0, 134.7, 141.7, 164.0, 195.8; MS (m/z %): 349 [M+H]+;
Calcd for C22H20O2S: C, 75.83; H, 5.79. Found: C, 75.81; H, 5.76.
3420 cmÀ1 1H NMR (200 MHz, CDCl3) d: 2.32 (s, 3H), 2.40 (s,
;
3H), 3.57 (d, 2H, J = 7.2 Hz), 4.83 (t, 1H, J = 7.2 Hz), 7.11–7.31 (m,
10H), 7.77 (d, 2H, J = 7.8 Hz); 13C NMR (50 MHz, CDCl3) d: 21.0,
22.0, 44.7, 47.6, 126.4, 126.8, 127.8, 128.2, 128.3, 128.5, 129.7,
130.7, 129.9, 133.9, 144.0, 141.1, 144.6, 196.6; MS (m/z %): 381
[M+H]+; Calcd for C23H21ClOS: C, 75.52; H, 5.56. Found: C, 75.48;
H, 5.57.
4.4.17. 1-(3-Nitrophenyl)-3-phenyl-3-(phenylthio)
propan-1-one (21)
Yield: 78%, White solid; mp 96–98 °C. IR
mmax: 688, 1353, 1549,
4.4.11. 1-(4-Hydroxyphenyl)-3-phenyl-3-(phenylthio)
propan-1-one (15)
1683, 3058 cmÀ1 1H NMR (200 MHz, CDCl3) d: 3.65 (d, 2H,
;
J = 7.1 Hz), 5.00 (t, 1H, J = 7.1 Hz), 7.21–7.60 (m, 11H) 7.91 (d, 2H,
J = 7.1 Hz), 8.09–8.15 (m, 1H); 13C NMR (50 MHz, CDCl3) d: 44.6,
48.2, 123.9, 124.2, 124.7, 125.1, 125.8, 127.6, 128.1, 128.6, 128.9,
129.9, 131.2, 132.0, 134.3, 135.5, 136.2, 149.0, 197.1; MS (m/z %):
364 [M+H]+; Calcd for C21H17O3NS: C, 69.40; H, 4.71; N, 3.85.
Found: C, 69.38; H, 4.68; N, 3.87.
Yield:94%;Whitesolid;mp103–106 °C.IRm ;
max: 1682, 3347 cmÀ1
1H NMR (200 MHz, CDCl3) d: 3.48–3.71 (m, 2H), 4.50–4.55 (m, 1H),
6.68 (t, 1H, J = 7.5 Hz), 6.97 (d, 1H, J = 7.9 Hz), 7.04 (d, 1H,
J = 7.9 Hz), 7.14–7.24 (m, 5H), 7.40–7.45 (m, 2H), 7.50–7.57 (m,
2H), 7.93 (d, 2H, J = 7.2 Hz), 8.14 (s, 1H); 13C NMR (50 MHz, CDCl3)

5416
A. Kumar et al. / Bioorg. Med. Chem. 19 (2011) 5409–5419
4.4.18. 3-(4-Nitro-phenylsulfanyl)-1,3-diphenylpropan-1-one
(22)
4.5.1. 3-(3-Oxo-1,3-diphenylpropylthio)propanoic acid (27)
Yield: 88%; Yellow oil; IR
max: 1598, 1600, 3407 cmÀ1; 1H NMR
m
Yield: 68%, White solid; mp 87–89 °C; IR
m
max: 1679, 3345 cm
(200 MHz, CDCl3) d: 2.52–2.60 (m, 4H), 3.52 (d, 2H, J = 6.9 Hz), 4.59
(t, 1H, J = 6.9 Hz), 7.19–7.59 (m, 8H), 7.88 (d, 2H, J = 7.1 Hz), 11.3
(br s, 1H); 13C NMR (50 MHz, CDCl3) d: 26.4, 34.4, 44.9, 45.6,
127.9, 128.2, 128.5, 129.0, 130.1, 133.7, 137.0, 142.0, 177.3,
197.1; MS (m/z %): 315 [M+H]+; Calcd for C18H18O3S: C, 68.76; H,
5.77. Found: C, 68.72; H, 5.74.
À1
;
1H NMR (200 MHz, CDCl3) d: 3.61–3.66 (m, 2H), 4.91 (t, 1H
J = 7.8 Hz), 7.34–7.37 (m, 4H), 7.42–7.47 (m, 4H), 7.49–7.63 (m,
1H), 7.89–7.91 (m, 2H), 8.03–8.06 (m, 2H), 8.17–8.20 (m, 1H);
13C NMR (50 MHz, CDCl3) d: 45.6, 47.1, 124.4, 124.9, 126.9, 128.2,
128.4, 128.6, 129.2, 129.4, 29.5, 134.1, 140.8, 145.8, 196.7; MS
(m/z %): 364 [M+H]+; Calcd for C21H17NO3S: C, 69.40; H, 4.71; N,
3.85. Found: C, 69.42; H, 4.76; N, 3.87.
4.5.2. 3-(1-(4-Methoxyphenyl)-3-oxo-3-
phenylpropylthio)propanoic acid (28)
Yield: 86%; Yellow semi solid; IR
mmax: 1502, 1599, 1602,
4.4.19. 1-Napthalen-2-yl-3-phenyl-3-phenyl-sulfanyl propan-1-
one (23)
3407 cmÀ1 1H NMR (200 MHz, CDCl3) d: 2.51–2.60 (m, 4H), 3.49
;
(d, 2H, J = 6.9 Hz), 3.85 (s, 3H), 4.58 (t, 1H, J = 6.9 Hz), 6.92 (d, 2H,
J = 8. 8 Hz), 7.26–7.43 (m, 5H), 7.91 (d, 2H, J = 8. 8 Hz) 11.6 (br s,
1H); 13C NMR (50 MHz, CDCl3) d: 26.4, 34.7, 45.1, 45.2, 55.9,
114.1, 127.8, 128.2, 129.0, 130.8, 132.7, 142.2, 164.0, 178.0),
195.7; MS (m/z %): 345 [M+H]+; Calcd for C19H20O4S: C, 66.26; H,
5.85. Found: C, 66.24; H, 5.86.
Yield 78%, White solid; mp 100–101 °C; IR
mmax: 1585, 1687,
3445 cmÀ1 1H NMR (200 MHz, CDCl3) d: 3.76 (d, 2H, J = 6.Hz),
;
5.81 (t, 1H, J = 7.1 Hz), 7.20–7.59 (m, 12H), 7.22 (d, 1H,
J = 8.6 Hz), 7.84–7.92 (m, 3H), 8.32 (d, 1H, J = 9.6 Hz); 13C NMR
(50 MHz, CDCl3) d: 44.3, 48.6, 122.4, 123.2, 123.8, 124.3, 124.6,
125.8, 126.2, 127.1, 127.8, 128.2, 128.7, 129.1, 129.8, 132.6,
132.9, 134.6, 136.3, 138.9, 196.4; MS (m/z %): 369 [M+H]+; Calcd
for C25H20OS: C, 81.49; H, 5.47. Found: C, 81.45; H, 5.48.
4.6. Typical experimental procedure for the zirconium catalyzed
1, 4 conjugate addition of alkyl thiols to 1,3-diaryl-2-
propenones (29–32)
4.4.20. 3-(4-Chlorophenylthio)-3-(4-methoxyphenyl)-1-
phenylpropan-1-one (24)
In a typical experiment 1,3-diaryl-2-propenone (1 mmol), zirco-
nium chloride (25 mol %) in dry DCM (2–3 mL) were mixed to-
gether and then alkyl thiol (2 mmol) was added drop wise with
help of syringe. The solution stirred at room temperature under
an air atmosphere for appropriate time of period. After completion
of the reaction (monitored by TLC), the reaction mixture was
worked up by simple extraction with ethyl acetate and water.
The organic layer was separated and dried over anhydrous Na2SO4
and filtered. The filtrate was evaporated under vacuum on a rotary
evaporator to afford the desired product in high purity. The further
purification was performed by crystallization.
Yield: 90%, White solid; mp 100–109 °C. IR mmax: 1601, 1674,
3420 cm-1; 1H NMR (200 MHz, CDCl3) d: 3.51–3.56 (m, 2H), 3.85
(s, 3H), 4.91 (t, 1H, J = 7.0 Hz), 6.91 (d, 2H, J = 7.7 Hz), 7.15–7.28
(m, 9H), 7.86 (d, 2H, J = 7.7 Hz); 13C NMR (50 MHz, CDCl3) d:
45.0, 48.6, 55.4, 113.9, 128.1, 129.2, 129.0, 129.6, 133.3, 133.9,
132.6, 134.0, 134.2, 137.8, 159.6, 197.4; MS (m/z %): 383 [M+H]+;
Calcd for C22H19ClO2S: C, 69.01; H, 5.00. Found: C, 68.98; H, 5.02.
4.4.21. 1-(3-Chlorophenyl)-3-(phenylthio)-3-p-tolylpropan-1-
one (25)
Yield: 98%, White solid; mp 106–108 °C. IR
mmax: 1601, 1674,
4.6.1. 3-(Ethylthio)-3-(4-methoxyphenyl)-1-phenylpropan-1-
one (29)
3420 cmÀ1 1H NMR (200 MHz, CDCl3) d: 2.39 (s, 3H), 3.54–3.52
;
(m, 2H), 4.89 (t, 1H, J = 7.0 Hz), 7.16–7.31 (m, 11H), 7.80 (d, 2H,
J = 7.7 Hz); 13C NMR (50 MHz, CDCl3) d: 22.8, 44.5, 47.3, 126.4,
126.6, 127.6, 128.0, 128.2, 128.7, 129.0, 129.2, 130.7, 133.7,
141.1, 143.8, 144.0, 196.1; MS (m/z %): 367 [M+H]+; Calcd for
Yield: 89%; White solid; mp 90–92 °C. IR
mmax: 1600, 1680,
3428 cmÀ1 1H NMR (200 MHz, CDCl3) d: 1.15 (t, 3H, J = 7.3 Hz),
;
2.28–2.40 (m, 2H), 3.45 (d, 2H, J = 7.0 Hz), 3.82 (s, 3H), 4.54 (1H,
J = 7.0 Hz), 6.86 (d, 2H, J = 8.8 Hz), 7.19–7.32 (m, 5H), 7.87 (d, 2H,
J = 8.8 Hz); 13C NMR (50 MHz, CDCl3) d: 14.7, 25.8, 44.6, 45.3,
55.8, 114.1, 127.5, 128.2, 128.8, 129.2, 133.0, 134.7, 141.7, 164.0,
195.0; MS (m/z %): 301 [M+H]+; Calcd for C18H20O2S: C, 71.96; H,
6.71. Found: C, 71.92; H, 6.68.
C22H19ClOS: C, 72.02; H, 5.22. Found: C, 72.00; H, 5.19.
4.4.22. 1-(3-Chlorophenyl)-3-phenyl-3-(phenylthio)propan-1-
one (26)
Yield: 98%; White solid; mp 106–108 °C; IR
mmax: 1601, 1674,
3420 cmÀ1 1H NMR (200 MHz, CDCl3) d: 3.54–3.52 (m, 2H), 4.89
;
4.6.2. 3-(Ethylthio)-3-(4-hydroxyphenyl)-1-phenylpropan-1-
one (30)
(t, 1H, J = 7.0 Hz), 7.16–7.31 (m, 12H), 7.80 (d, 2H, J = 7.7 Hz); 13C
NMR (50 MHz, CDCl3) d: 44.5, 47.3, 126.4, 126.6, 127.6, 128.0,
128.2, 128.7, 129.0, 129.2, 130.7, 133.7, 141.1, 143.8, 144.0,
196.1; MS (m/z %): 353 [M+H]+; Calcd for C21H17ClOS: C, 71.48;
H, 4.86. Found: C, 71.46; H, 4.85.
Yield: 70%; White semi solid; IR m ;
max: 1605, 1673, 3422 cmÀ1
1H NMR (200 MHz, CDCl3) d: 1.10 (t, 3H, J = 7.3 Hz), 2.20–2.41
(m, 2H), 3.46 (d, 2H, J = 7.1 Hz), 4.51 (t, 1H, J = 7.1 Hz), 6.84 (d,
2H, J = 7.7 Hz), 7.15–7.81 (m, 5H), 7.85 (d, 2H, J = 9.8 Hz), 8.12 (s,
1H); 13C NMR (50 MHz, CDCl3) d: 14.7, 25.8, 44.8, 45.3, 116.0,
126.3, 127.6, 128.1, 129.7, 131.3, 142.4, 161.7, 197.3; MS (m/z %):
287 [M+H]+. Calcd for C17H18O2S: C, 71.30; H, 6.34. Found: C,
71.34; H, 6.38.
4.5. Typical experimental procedure for the zirconium catalyzed
1, 4 conjugate addition of mercapto carboxylic acid to
1,3-diaryl-2-propenones (27–28)
4.6.3. 3-Ethylsulfanyl-1,3-bis-(4-methoxy-phenyl)-propan-1-
one (31)
To a magnetically stirred mixture of 1,3-diaryl-2-propenone
(1 mmol), zirconium chloride (25 mol %) and mercapto carboxylic
acid (2.5 mmol) were added at room temperature in solvent free
condition. The reaction was stirred for 6–8 h. The precipitate was
filtered and washed with water and hexane. The solid or oily resi-
due was crystallized by ethyl acetate and hexane to afford the pure
products in high yield.
Yield: 90%; White solid; mp 159–161 °C. IR
mmax: 1669, 1680,
3430 cmÀ1 1H NMR (200 MHz, CDCl3) d: 1.12 (t, 3H, J = 7.3 Hz),
;
2.28–2.38 (m, 2H), 3.43 (d, 2H, J = 7.1 Hz), 3.77 (s, 3H), 3.85 (s,
3H), 4.51 (t, 1H, J = 7.0 Hz), 6.80–6.88 (m, 4H), 7.30 (d, 2H,
J = 8.6 Hz), 7.87 (d, 2H, J = 8.8 Hz); 13C NMR (50 MHz, CDCl3) d:
14.7, 25.7, 44.0, 45.5, 55.6, 55.8, 110.0, 114.1, 114.2, 129.2, 130.3,

A. Kumar et al. / Bioorg. Med. Chem. 19 (2011) 5409–5419
5417
130.8, 134.6, 158.9, 163.9, 196.0; MS (m/z %): 331 [M+H]+; Calcd for
19H22O3S: C, 69.06; H, 6.71. Found: C, 69.10; H, 6.67.
129.2, 129.5, 130.8, 133.0, 134.8, 140.8, 163.3, 195.8; MS (m/z %):
478 [M+H]+; Calcd for C28H31NO4S: C, 70.41; H, 6.54; N, 2.93.
Found: C, 70.39; H, 6.52; N, 2.95.
C
4.6.4. 1,3-Diphenyl-3-(p-tolylthio)propan-1-one (32)
Yield: 90%; White solid; mp 155–157 °C IR max: 693, 750,1594,
1677 cmÀ1 1H NMR (200 MHz, CDCl3) d: 2.39 (s, 3H), 3.51 (d, 2H,
J = 7.0 Hz), 4.55(t, 1H, J = 7.0 Hz), 7.16–7.33 (m, 7H), 7.39–7.54
(m, 5H), 7.89 (d, 2H, J = 7.5 Hz); 13C NMR (50 MHz, CDCl3) d:
14.7, 25.8, 44.4, 45.7, 127.6, 128.2, 128.5, 128.9, 129.0, 133.6,
137.2, 142.6, 197.3; MS (m/z %): 333 [M+H]+; Calcd for C22H20OS:
C, 79.48; H, 6.06. Found: C, 79.45; H, 6.01.
4.7.4. 3-(4-Hydroxyphenylthio)-3-phenyl-1-(4-(2-(piperidin-1-
yl)ethoxy)phenyl)propan-1-one (36)
m
;
Yield: 82%; White solid; mp 122–123 °C. IR
mmax: 689, 1582,
1680, 3336 cmÀ1 1H NMR (200 MHz, CDCl3) d: 2.46–2.70 (m,
;
6H), 2.86 (t, 4H, J = 6.0 Hz), 4.89 (t, 2H, J = 6.0 Hz), 3.53–3.59 (2H,
m), 4.34 (t, 2H, J = 6.1 Hz), 4.96 (t, 1H, J = 7.0 Hz), 6.98 (d, 2H,
J = 8.4 Hz), 7.18–7.28 (m, 6H), 7.31 (d, 2H, J = 8.4 Hz), 7.38–7.41
(m, 4H); 13C NMR (50 MHz, CDCl3) d: 21.7, 23.9, 30.1, 32.6, 44.7,
48.7, 53.8, 54.6, 55.2, 56.0, 67.7, 114.7, 127.3, 127.6, 127.9, 128.2,
128.8, 129.2, 129.5, 130.8, 133.0, 134.8, 141.0, 163.2, 197.8; MS
(m/z %): 462 [M+H]+; Calcd for C28H31NO3S: C, 72.85; H, 6.77; N,
3.03. Found: C, 72.84; H, 6.74; N, 3.05.
4.7. Spectroscopic and analytical details of basic alkyl amino
side chain analogues b-aryl-b-mercapto ketones (33–41)
In 100 mL of round bottom flask, compound (4c) (1 mmol),
substituted aldehyde (1 mmol) and arylmercaptan (1.5 mmol)
was taken in dry DCM, zirconium chloride (25 mol %) was added
as a catalyst. Reaction mixture was stirred at room temperature
60 min. There is a formation of colored complex with evolution
of heat, which gradually converts light yellow to ceramic white
at the completion of the reaction. The progress of the reaction
was monitored by checking TLC. After completion of the reaction,
the reaction mixture was extracted with EtOAc or DCM. The organ-
ic layer was separated and dried over anhydrous Na2SO4 and fil-
tered. The filtrate was evaporated under vacuum on a rotary
evaporator. The crude solid was obtained which was further
purified by simple crystallization to obtain product in good yield.
4.7.5. 3-(4-Hydroxyphenyl)-3-(4-hydroxyphenylthio)-1-(4-(2-
(pyrrolidin-1-yl)ethoxy)phenyl)propan-1-one (37)
Yield: 82%; Yellow solid; mp: 132–135 °C. IR
mmax: 688, 1679,
2782, 2932, 2952 cmÀ1 1H NMR (200 MHz, CDCl3) d: 1.59–1.6
;
(m, 4H), 2.25–2.32 (m, 4H), 2.78 (t, 2H, J = 6.0 Hz), 3.18–3.21(m,
2H), 4.08 (t, 2H, J = 6.0 Hz), 4.54 (t, 1H, J = 7.1 Hz), 6.87 (d, 2H,
J = 8.2 Hz), 7.03–7.11 (m, 2H), 7.14–7.19 (m, 8H), 7.78 (d, 2H,
J = 8.2 Hz); 13C NMR (50 MHz, CDCl3) d: 24.9, 44.5, 47.7, 52.5,
54.0, 72.9, 114.4, 124.9, 126.3, 126.8, 127.9, 128.5, 128.9, 129.6,
130.3, 135.6, 142.7, 163.2, 195.8; MS (m/z %): 464 [M+H]+; Calcd
for C27H29NO4S: C, 69.95; H, 6.31; N, 3.02. Found C, 69.93; H,
6.29; N, 3.05.
4.7.1. 1-(4-(2-(Dimethylamino)ethoxy)phenyl)-3-phenyl-3-
(phenylthio)propan-1-one (33)
4.7.6. 3-(4-Hydroxyphenylthio)-3-phenyl-1-(4-(2-(pyrrolidin-1-
yl)ethoxy)phenyl)propan-1-one (38)
Yield: 86%; Creamish solid; mp 119–120 °C. IR
mmax: 1679,
Yield 85%; Yellow oil; IR
m
max: 687, 1678, 2784, 2942, 2950 cm
3345 cmÀ1 1H NMR (200 MHz, CDCl3) d: 2.28 (s, 6H), 2.79 (t, 2H,
;
À1
;
1H NMR (200 MHz, CDCl3) d: 1.62–1.64 (m, 4H), 2.28–2.32
J = 5.7 Hz), 3.22–3.27 (m, 2H), 4.10 (t, 2H, J = 5.7 Hz), 4.39 (t, 1H,
J = 7.1 Hz, SCH), 6.85–7.02 (m, 3H), 7.08–7.16 (m, 5H), 7.18–7.23
(m, 4H), 7.80 (d, 2H, J = 8.4 Hz); 13C NMR (50 MHz, CDCl3) d:
41.8, 42.2, 47.9, 58.9, 71.9, 114.3, 124.9, 126.6, 126.9, 127.9,
128.2, 128.7, 129.1, 129.7, 135.8, 141.0, 163.8, 196.7; MS (m/z %):
406 [M+H]+; Calcd for C25H27NO2S: C, 74.04; H, 6.71; N, 3.45.
Found: C, 74.08; H, 6.70; N, 3.48.
(m, 4H), 2.82 (t, 2H, J = 5.8 Hz), 3.20–3.26 (m, 2H), 4.10 (t, 2H,
J = 5.8 Hz), 4.56 (t, 1H, J = 7.2 Hz), 5.27 (s, 1H), 6.87–6.98 (m, 5H),
7.07–7.19 (m, 6H), 7.79 (d, 2H, J = 8.5 Hz); 13C NMR (50 MHz,
CDCl3) d: 21.8, 24.9, 43.5, 48.2, 54.0, 72.9, 114.4, 124.8, 126.2,
126.8, 127.9, 128.4, 128.7, 129.3, 130.1, 137.6, 141.9, 163.0,
196.0; MS (m/z %): 448 [M+H]+; Calcd for C27H29NO3S: C, 72.45;
H, 6.53; N, 3.13. Found: C, 72.48; H, 6.58; N, 3.10.
4.7.2. 1-(4-(2-(Dimethylamino)ethoxy)phenyl)-3-phenyl-3-(p-
tolylthio)propan-1-one (34)
4.7.7. 1-(4-(2-(Dimethylamino)ethoxy)phenyl)-3-(ethylthio)-3-
phenylpropan-1-one (39)
Yield: 85%; Yellow solid; mp 127–129 °C. IR mmax
: 1679,
Yield: 86%; yellow semi solid; IR
mmax: 688, 1582, 1679,
3345 cmÀ1 1H NMR (200 MHz, CDCl3) d: 2.26 (s, 6H), 2.29 (s, 3H)
;
3345 cmÀ1 1H NMR (200 MHz, CDCl3) d: 1.24 (t, 3H, J = 7.0 Hz),
;
2.81 (t, 2H, J = 5.7 Hz), 3.21–3.28 (m, 2H), 4.12 (t, 2H, J = 5.7 Hz)
4.41(t, 1H, J = 7.1 Hz), 6.82–6.86 (3H, m, Ar-H), 6.97–6.99 (2H, m,
Ar-H), 7.04–7.12 (4H, m, Ar-H), 7.23–7.28 (2H, m, Ar-H), 7.78
(2H, d, J = 8.6 Hz, Ar-H); 13C NMR (50 MHz, CDCl3) d: 41.8, 42.2,
47.9, 50.2, 58.9, 71.9, 114.3, 124.9, 126.6, 126.9, 127.9, 128.2,
128.7, 129.1, 129.7, 135.8, 141.0, 163.8, 196.7; MS (m/z %): 420
[M+H]+; Calcd for C26H29NO2S: C, 74.43; H, 6.97; N, 3.34. Found:
C, 74.40; H, 6.92; N, 3.36.
2.30 (s, 6H), 2.48–2.51 (m, 2H), 2.72–2.79 (m, 2H), 3.21–3.28 (m,
2H), 4.12 (t, 2H, J = 5.8 Hz), 4.49 (t, 1H, J = 7.1 Hz), 6.84 (d, 2H,
J = 8.6 Hz), 7.08–7.21 (m, 5H), 7.81 (d, 2H, J = 8.4 Hz); 13C NMR
(50 MHz, CDCl3) d: 21.2, 23.2, 24.8, 41.9, 47.9, 58.2, 71.8, 114.4,
126.6, 128.4, 128.9, 129.0, 129.9, 138.4, 163.2, 196.1; MS (m/z %):
358 [M+H]+; Calcd for C21H27NO2S: C, 70.55; H, 7.61; N, 3.92.
Found: C, 70.53; H, 7.58; N, 3.95.
4.7.3. 3-(3-Hydroxyphenyl)-3-(4-hydroxyphenylthio)-1-(4-(2-
(piperidin-1-yl)ethoxy)phenyl)propan-1-one (35)
4.7.8. 3-(Ethylthio)-3-phenyl-1-(4-(2-(piperidin-1-
yl)ethoxy)phenyl)propan-1-one (40)
Yield: 88%; Yellow solid; mp 137–139 °C. IR mmax
:
1679,
Yield: 90%; Yellow solid; mp 142–145 °C. IR mmax
: 1689,
3345 cmÀ1 1H NMR (200 MHz, CDCl3) d: 2.44–2.68 (6H, m, CH2),
;
3354 cmÀ1 1H NMR (200 MHz, CDCl3) d: 1.16 (t, 3H, J = 7.3 Hz),
;
2.81 (t, 4H, J = 5.5 Hz), 2.99 (t, 2H, J = 5.5 Hz), 3.43–3.49 (m, 2H),
4.04 (t, 2H, J = 6.1 Hz), 4.86 (t, 1H, J = 7.0 Hz), 6.78 (d, 2H,
J = 8.4 Hz), 7.10–7.22 (m, 6 H), 7.38–7.41 (m, 4H), 7.51 (d, 2H,
J = 8.4 Hz); 13C NMR (50 MHz, CDCl3) d: 23.9, 30.1, 32.6, 44.7,
54.5, 55.1, 67.7, 114.7, 126.3, 127.6, 127.7, 127.9, 128.2, 128.8,
2.41–2.20 (m, 2H), 2.41–2.44 (m, 6H), 2.68 (t, 4H, J = 5.8 Hz), 2.99
(t, 2H, J = 5.8 Hz), 3.45 (d, 2H, J = 7.1 Hz), 4.04 (t, 2H, J = 6.1 Hz),
4.57 (t, 1H, J = 7.1 Hz), 6.89 (d, 2H, J = 8.4 Hz), 7.10–7.22 (m, 5H),
7.31–7.34 (d, 2H, J = 8.8 Hz); 13C NMR (50 MHz, CDCl3) d: 14.7,
24.5, 25.8, 26.3, 26.7, 44.5, 54.5, 58.1, 66.6, 114.6, 127.5, 128.2,

5418
A. Kumar et al. / Bioorg. Med. Chem. 19 (2011) 5409–5419
128.9, 130.8, 130.9, 142.7, 163.2, 195.8; MS (m/z %): 398 [M+H]+;
Calcd for C24H31NO2S: C, 72.50; H, 7.86; N, 3.52. Found: C, 72.44;
H, 7.83; N, 3.47.
concentration relative to that of estradiol gave the affinity of the
test compound to estrogen receptor relative to estradiol. This when
multiplied with 100 gave the percentage value designated as RBA.
4.7.9. 3-(Ethylthio)-3-phenyl-1-(4-(2-(pyrrolidin-1-
yl)ethoxy)phenyl)propan-1-one (41)
4.11. Cell culture conditions
Yield: 80%; Yellow solid; mp 122–125 °C. IR
m
max: 688, 1549,
Primary cultures of mouse osteoclast precursor cells in the form
of bone marrow-derived monocytes/macrophages (BMMs) were
isolated as described by others. Briefly, bone marrow cells from
the long bones of 4 to 6-week-old Balb/c mice were collected
and plated overnight in a MEM (Life Technologies, Rockville, MD,
1682, 2428, 2745 cmÀ1 1H NMR (200 MHz, CDCl3) d: 1.26 (t, 3H,
;
J = 7.2 Hz), 1.60–1.66 (m, 4H), 2.31–2.36 (m, 4H), 2.46–2.49 (m,
2H), 2.99 (t, 2H, J = 5.8 Hz), 3.25–3.35 (m, 2H), 4.09 (t, 2H,
J = 5.8 Hz), 4.59 (t, 1H, J = 7.0 Hz), 6.85 (d, 2H, J = 8.5 Hz), 7.08–
7.12 (m, 3H), 7.21 (m, 2H),7.78 (d, 2H, J = 8.5 Hz); 13C NMR
(50 MHz, CDCl3) d: 18.7, 23.2, 26.3, 40.5, 48.0, 51.9, 54.2, 72.0,
114.3, 127.0, 128.4, 128.9, 129.1, 129.8 140.1, 163.2, 196.2; MS
(m/z %): 384 [M+H]+. Calcd for C23H29NO2S: C, 72.02; H, 7.62; N,
3.65. Found: C, 71.98; H, 7.58; N, 3.61.
USA) supplemented with 10% heat-inactivated FBS, 2 mM
L-gluta-
mine, 100 g/ml penicillin, 100 g/ml streptomycin, and 10 ng/
l
l
ml macrophage-colony stimulating factor (M-CSF). The next day,
The nonadherent cells were collected and after centrifugation, cul-
tured on 48-well tissue culture plates in a-MEM with heat-inacti-
vated fetal bovine serum (10%), RANKL (50 ng/ml), and M-CSF
4.8. Cells and cell culture condition
(10 ng/ml). Cells were exposed to test agents at 1 lM concentra-
tion culture media and indicated supplement(s) were changed
MCF-7 (Estrogen receptor +ve breast cancer cell line), MDA-MB-
231 (estrogen receptor Àve breast cancer cell lines), Ishikawa
(estrogen receptor +ve endometrial cancer cell line) and HEK-293
(human embryonic kidney epithelial cell line) cells were obtained
from ATCC, USA. Cells were cultured at 37 °C with 5% CO2 in DMEM
medium with 10% fetal bovine serum (FBS), 100 U/ml penicillin,
100 U/ml streptomycin, except in growth inhibition assays, where
the FBS supplement was reduced to 2%.
every 2 d for a total of 7 d.
4.16. Anti-osteoporotic activity (anti-bone resorption assay)
The in vitro assay of anti-resorptive activity using 45Ca pre-
labeled rat fetal bone was done according the literature method.
Three month old Sprague–Dawley female rats (180–220 g) main-
tained at standard conditions (22 1 °C) with alternate 12 h
light/dark periods and free access to pellet diet and tap water were
used throughout the study. Females were mated to males of pro-
4.9. MTT assay
ven fertility. 250 lCi/200 lL of 45CaCl2 was administered subcuta-
The antiproliferative activities of the compounds were
determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) reduction assay. 1 Â 104 cells/well (MCF-7,
neously to each rat on day 18 of pregnancy and labeled femur and
radio-ulna bones were isolated 48 h thereafter under sterile condi-
tions. Bones were cultured in 200 lL of the BGJb medium supple-
mented with antibiotic, antifungal and buffer (pH 7.3) for 24 h.
MDA-MB-231, Ishikawa, and HEK-293) were seeded in 100
l
l
DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with
10% FBS in each well of 96-well microculture plates and incubated
for 24 h at 37 °C in a CO2 incubator. Compounds, diluted to the
desired concentrations in culture medium. After 48 h of incubation,
The bones were washed twice with PBS and transferred to BJGb
medium containing PTH (0.4
the presence or absence of test compound (100
(0.1% ethanol/DMSO) in 200 L of BGJb, medium. Contralateral fe-
lM) and these cultured for 96 h in
lM) or the vehicle
l
media were removed and to each well 10
added and the plates were further incubated for 4 h. Supernatant
from each well was carefully removed, formazon crystals were
l
l MTT (5 mg/mL) was
mur of each fetus served as corresponding control. On termination
of the culture, bones were transferred to 0.1 N HCl for 24 h. Radio-
activity due to 45Ca in the spent medium collected at 48 and 96 h
of culture and the HCl extracts at 96 h of culture were quantified by
liquid scintillation spectrophotometer. Bone resorbing activity was
expressed as percentage of released 45Ca and the effect of test
compounds as percent of corresponding control or T/C ratio. All
the synthesized compounds were screened for in vitro bone
resorption inhibitory activity in 21 days old fetal rat by measuring
the inhibition of PTH (Parahyriod hormone) induced bone resorp-
tion inhibitory activity.
dissolved in 100
was recorded.
ll of DMSO and absorbance at 540 nm wavelength
4.10. Estrogen receptor binding affinity assay
The RBA of the compounds for the estrogen receptor was deter-
mined by competition assay, employing radio labeled estradiol
(3H-E2) as the reference compound. The test ligands and (3H-E2)
were incubated (4 °C) with cytosol estrogen receptors obtained
from immature 20 to 21-day-old rat uteri. Aliquots of the uterine
4.17. Osteoclastogenesis
cytosol (200 lL concentrated 1 uterus per ml) prepared in TEA buf-
fer (10 mM Tris, 1.5 mM EDTA, and 0.02% sodium azide, pH 7.4)
were incubated in triplicate with a fixed concentration of radio la-
beled estradiol with or without various concentrations of the com-
Primary cultures of mouse osteoclast precursor cells in the form
of bone marrow-derived monocytes/macrophages (BMMs) were
isolated as described by others. Briefly, bone marrow cells from
the long bones of 4 to 6-week-old Balb/c mice were collected and
plated overnight in a MEM (Life Technologies, Rockville, MD, USA)
petitor substance dissolved in 60 lL of the TEA buffer containing
DMF as co-solvent (final concentration of DMF in the incubation
medium never exceeded 5%) for 18 h at 4 °C. At the end of this per-
iod, dextran-coated charcoal (DCC) (5% Norit 0.5% dextran) suspen-
supplemented with 10% heat-inactivated FBS 2 mM
2 mM -glutamine, 100 g/ml penicillin, 100 g/ml streptomycin,
and 10 g/ml macrophage-colony stimulating factor (M-CSF). The
L-glutamine,
L
l
l
sion in 100
l
L of TEA buffer was added into each tube, which was
l
briefly vortexed and allowed to stand for 15 min. DCC was precip-
itated by centrifugation (800g 10 min) and the supernatants
counted for radioactivity in 10 ml of a dioxane-based scintillation
fluid. RBA of the text compound was computed from a graph plot-
ted between percent bound radioactivity versus log concentration
of the test substance. At 50% inhibition, log of the competitor
next day, The nonadherent cells were collected and after centrifu-
gation, cultured on 48-well tissue culture plates in a-MEM with
heat-inactivated fetal bovine serum (10%), RANKL (50 ng/ml), and
M-CSF (10 ng/ml). Cells were exposed to test agents at 1 lg/ml con-
centration culture media and indicated supplement(s) were chan-
ged every 2 d for a total of 7 d. Compounds (5, 8 and 10) were

A. Kumar et al. / Bioorg. Med. Chem. 19 (2011) 5409–5419
5419
tested for their effects on the induction of TRAP and RANK in freshly
4.20. Anti-implantation activity
isolated murine bone marrow cells treated with RANKL and MCSF
for 7 days. QPCR determination of TRAP and RANK levels was made
and the data expressed as fold change over vehicle (control) using
quantitative real time PCR (Fig. 4a). Total RNA was extracted from
the cultured cells using Trizol (Invitrogen). cDNA was synthesized
Anti-implantation activity of the compounds was studied in
sperm positive adult (180–220 g) Sprague–Dawley female albino
rats mated to coeval males of proven fertility. The compounds
were administered orally as a suspension in gum acacia to colony
bread adult mated female rats on days 1–5 post-coitum using five
to seven animals in each group. The animals were examined by
laprotomy on day 10 of pregnancy for the number and status of
implantations and corpora lutea. The results were scored as posi-
tive only if implantations were totally absent in both the uterine
horns of each animal.
from 2 lg total RNA with the reverse script reverse transcription
kit (FERMENTAS). SYBR green chemistry was used to perform quan-
titative determinations of the mRNAs for TRAP, RANK, and house-
keeping gene, cyclophilin A, following an optimized protocol.
Light cycler (Roche) was used. The design of sense and antisense oli-
gonucleotide primers was based on published cDNA sequences
using primer express software (version 2.0.0, Applied Biosystems).
At 1
lM concentrations, Compounds (5, 8 and 10) significantly
Acknowledgments
inhibited expression of TRAP and RANK induced by RANKL and
MCSF (Fig. 4b).
All the authors would like to acknowledge generous funding by
Ministry of Health and Family Welfare, New Delhi. Funding by DST,
New Delhi (to RT) is acknowledged. Fellowship grants from the
Council of Scientific and Industrial Research, Government of India
(V.D.T., P.K., L.P.G., B.C.) and University Grant Commission (JAS)
are acknowledged. This manuscript bears CDRI Communication
No. 7787. Authors also acknowledge SAIF-CDRI for providing all
analytical and spectral data.
4.18. Estrogen agonistic activity
Twenty one day old immature female Spraugue–Dawley rats
were bilaterally ovariectomized under light ether anesthesia and
after post-operative rest for 7 days were randomized into different
treatment groups. Each rat received the compound of the invention
once daily for 3 consecutive days on days 28–30 of age by oral route.
A separate group of animals received only the vehicle for similar
duration served as control. At autopsy 24 h after the last treatment
on day 31 of age, vaginal smear of each rat was taken and uterus
was carefully excised, gently blotted, weighed. Premature opening
of vagina, cornification of vaginal epithelium and increase in uter-
ine fresh weight were taken as parameters for evaluation of estro-
gen agonistic activity in comparison to rats of vehicle control
group. The objective was to evaluate estrogen agonistic effect of
the compounds on the uterus and vagina. Compounds exhibiting
no or negligible estrogen agonistic activity in this assay were iden-
tified for further development as antiimplantation agents.
References and notes
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4.19. Estrogen antagonistic activity
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Twenty-one-day-old immature female Sprague–Dawley rats
were bilaterally ovariectomized under light ether anesthesia and
after post-operative rest for 7 days were randomized into different
treatment groups. Each rat received the compound of the invention
and 0.02 mg kgÀ1 dose of ethynyl estradiol in 10% ethanol-distilled
water once daily for 3 consecutive days on days 28–30 of age by
oral route. A separate group of animals receiving only ethynylest-
radiol (0.02 mg/kg) in 10% ethanol distilled water for similar dura-
tion were used for comparison. At autopsy on day 31 of age vaginal
smear of each rat was taken and uterus was carefully excised,
gently blotted, weighed and fixed for histology. Inhibition in ethy-
nylestradiol induced cornification of vaginal epithelium and in-
crease in uterine fresh weight were taken as parameters for
evaluation of estrogen antagonistic effect of the compounds.
Compounds exhibiting potent estrogen antagonistic activity in this
assay were identified for further development as antiimplantation
agents.
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