Synthesis and biological evaluation of estradiol linked pyrrolo[2,1-c][1,4]benzodiazepine (PBD) conjugates as potential anticancer agents

Article abstract of DOI:10.1016/j.bmc.2011.03.015

A series of new estradiol linked pyrrolo[2,1-c][1,4]benzodiazepine (E ₂-PBD) conjugates (3a-f, 4a-f and 5a-f) with different linker architectures including a triazole moiety have been designed and synthesized. All the 18 compounds have been eva

Full text of DOI:10.1016/j.bmc.2011.03.015

Bioorganic & Medicinal Chemistry 19 (2011) 2565–2581
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of estradiol linked pyrrolo[2,1-c]-
[1,4]benzodiazepine (PBD) conjugates as potential anticancer agents
⇑
Ahmed Kamal , M. Kashi Reddy, M. Janaki Ramaiah, Rajender, J. Surendranadha Reddy, Y. V. V. Srikanth,
⇑
D. Dastagiri, E. Vijaya Bharathi, S. N. C. V. L. Pushpavalli, Pranjal Sarma, Manika Pal-Bhadra
Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 607, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new estradiol linked pyrrolo[2,1-c][1,4]benzodiazepine (E2-PBD) conjugates (3a–f, 4a–f and
5a–f) with different linker architectures including a triazole moiety have been designed and synthesized.
All the 18 compounds have been evaluated for their anticancer activity and it is observed that some of the
compounds particularly 4c–e and 5c,d exhibited significant anticancer activity. The detailed biological
aspects relating to the cell cycle effects and tubulin depolymerization activity have been examined with
a view to understand the mechanism of action of these conjugates. Among all these conjugates, one of the
compound 5c could be considered as the most effective compound particularly against MCF-7 breast can-
cer cell line.
Received 22 January 2011
Accepted 7 March 2011
Available online 12 March 2011
Keywords:
Pyrrolobenzodiazepine
Estradiol
Anticancer agents
Tubulin polymerisation
Apoptosis
Ó 2011 Elsevier Ltd. All rights reserved.
Tunel assay
p53
p21
HDACs
1. Introduction
zine moiety in its linker spacer. Furthermore, piperazine contain-
ing PBDs have also shown significant anticancer activity.13 Many
Pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are
a
family of
1,2,3-triazole derivatives are known to exhibit interesting biologi-
cal activities such as antibiotic,14 tuberculosis,15 and anticancer
activity.16
naturally occurring antitumour antibiotics originated from Strepto-
myces species,1 that exert their cytotoxic activity by binding cova-
lently between C11-position of the PBD and C-2 amino group of the
guanine residues with in the minor groove of DNA.2,3 Recently a
series of C2-fluorinated PBDs have been synthesized and evaluated
for anticancer activity against a number of cancer cell lines,4 in
which fluorine substitution plays an important role for their bio-
logical activity. In the past few years, several hybrid compounds,
in which known anticancer agents tethered to PBD moiety have
been designed, synthesized and evaluated for their biological activ-
ity. C2-Fluorinated PBDs significantly increase the kinetic reactiv-
ity during covalent adduct formation and exhibited considerable
biological activity.5–9 Moreover, a number of piperazine and
1,2,3-triazole molecules have been synthesized as useful chemo-
therapeutic agents for various diseases over the past few years.10,11
Michejda and co-workers12 reported some promising anticancer
class of compounds such as symmetrical bifunctional agents with
remarkable selectivity against colon cancers that possess a pipera-
The naturally occurring PBDs such as anthramycin, chicamycin,
mazethramycin, porothramycin A, sibiromycin, tomaymycin, oxot-
omaymycin, prothracarcin and DC-81 (2), are known as potent
anticancer agents, however they have been excluded from clinical
studies due to problems relating to side effects.17 Hence, there has
been considerable interest in the design and synthesis of conjugate
agents with active moieties of known anticancer agents to enhance
the selectivity as well as anticancer activity. Drug targeting is likely
to improve the selectivity of PBDs and this will allow transporting
the required drug preferably to those sites that need treatment.
When this objectives to be met, not only the efficacy of the treat-
ment can be improved, but also the toxic side effects can be min-
imized to a great extent.
The estrogen receptor is a key biological target that has fasci-
nated considerable interest over the years and it is expressed by
several type of cancers like breast,18 uterus,19 and ovarian.20 The
biological affinity between estradiol and its cognate receptor can
theoretically be used to direct a cytotoxic agent to the target cells.
As a result, over the years various estrogen anticancer hybrids have
been investigated for the treatment of hormone-dependent
cancers.21–28
⇑
Corresponding authors. Tel.: +91 40 27193157; fax: +91 40 27193189.
E-mail addresses: ahmedkamal@iict.res.in (A. Kamal), manika@iict.res.in (M. Pal-
Bhadra).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.015

2566
A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2565–2581
In continuation of our efforts in search of new effective antican-
2. Results and discussion
2.1. Chemistry
cer agents, we have designed and synthesized a series of E2-PBD
conjugates. In the present approach, DC-81 (2), a known anticancer
agent that has been linked to the estradiol (1) through the stable
alkane spacers or a piperazine or 1,2,3-triazole moieties. The pres-
ent study describes the design, synthesis and biological activities of
these new E2-PBD conjugates (3a–f, 4a–f and 5a–f). Representative
estradiol (E2) (1), DC-81 (2) and their conjugates (3a–f, 4a–f and
5a–f) are illustrated in Figure 1.
The synthesis of the estradiol derivatives 6a,b, 9a,b, and 10a,b
was carried out as outlined in Scheme 1. The etherification of
17b-estradiol (1) by employing dibromoalkanes with K2CO3 gave
compounds 6a,b and these compounds were treated with N-Boc
piperazine to obtain compounds 7a,b which upon deprotection
with trifluoroacetic acid provided the intermediates 8a,b. Further,
these compounds on monoalkylation by using dibromoalkanes in
the presence of K2CO3 afford compounds 9a,b. Synthesis of the
azide precursors was carried out by the reaction of halogen deriv-
atives 6a,b with sodium azide to attain the azides 10a,b as illus-
trated in Scheme 1.
OH
N
HO
H
N
MeO
Compounds 11a–c were obtained by employing our previously
reported methods.29–32 The etherification of 11a–c with propargyl
bromide in the presence of K2CO3 afforded the terminal alkyne
intermediates 12a–c in good yields. The synthesis of C8-linked
E2-PBD conjugates (3a–f and 4a–f) was carried out from 11a–c
by treating them with estradiol precursors (6a,b and 9a,b) using
K2CO3 to provide the corresponding nitro thioacetals (13a–f and
14a–f) as shown in Scheme 2. Terminal alkynes (12a–c) were re-
acted with azides (10a–c), that underwent the ‘click’ reaction in
the presence of Cu(I) catalyst and sodium ascorbate to produce
the 1,2,3-triazole-containing E2-PBD nitro thioacetals (15a–f) as
shown in Scheme 3. Further, reduction of nitro compounds (13a–
f, 14a–f and 15a–f) by using SnCl2ꢀ2H2O in methanol followed by
deprotection and cyclisation using HgCl2/CaCO3 afford the target
E2-PBD conjugates (3a–f, 4a–f and 5a–f) as shown in Schemes 2
and 3.
O
HO
E2 (1)
DC-81 (2)
HO
N
O
O
H
( )
HO
n
R1
R2
N
O
MeO
3a−f
N
O
n
N
O
N
H
( )
( )
n
HO
R1
R2
N
MeO
4a−f
O
2.2. Evaluation of biological activity
N
N
O
N
( )
N
n
O
2.2.1. Anticancer activity
H
The anticancer activity of these conjugates (3a–f, 4a–f and
5a–f) was carried out in selected human cancer cell lines of breast,
cervix, lung, colon, oral, ovarian and prostate by using Sulforhoda-
R1
R2
N
MeO
5a−f
O
mine B (SRB) method. The compounds exhibiting GI50 6 10ꢁ5
M
Figure 1. Structures of estradiol (E2) (1), DC-81 (2), and E2-PBD conjugates (3a–f,
4a–f and 5a–f).
are considered to be active on the respective cell lines. All these
conjugates have shown good anticancer activity with GI50 values
OH
OH
OH
(ii)
(i)
( )
( )
O
Br
O
Boc
N
N
HO
n
n
6a,b
7a,b
E2 (1)
(iii)
(v)
OH
OH
( )
O
HN
N
n
( )
8a,b
N3
O
n
10a,b
(iv)
OH
( )
( )
Br
O
N
N
n
n
9a: n = 3
9b: n = 5
Scheme 1. Reagents and conditions: (i) dibromoalkanes, K2CO3, DMF, rt, 8ꢁ10 h, 88ꢁ90%; (ii) N-Boc piperazine, K2CO3, dry CH3CN, reflux, 24 h, 78ꢁ82%; (iii) TFA, dry CH2Cl2,
rt, 3 h, 95%; (iv) dibromoalkanes, K2CO3, dry CH3CN, reflux, 24 h, 75ꢁ78%; (v) NaN3, DMF,100 °C, 12 h, 90ꢁ92%.

A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2565–2581
2567
HO
(i)
6a,b
NO2
O
O
O
n
CH(SEt)2
( )
R1
R2
13a−f
N
MeO
(iii), (iv)
HO
O
NO2
HO
CH(SEt)2
R1
R2
N
O
N
MeO
H
( )
n
O
R1
R2
N
MeO
3a−f
11a−c
O
HO
11a: R1 = H; R2 = H
11b: R1 = H; R2 = F
11c: R1 = F; R2 = F
(ii)
9a,b
NO2
N
n
N
O
n
O
CH(SEt)2
( )
( )
R1
R2
N
MeO
14a−f
HO
O
(iii), (iv)
N
N
n
N
O
n
O
H
( )
( )
R1
R2
N
4a−f
MeO
4a: R1 = H; R2 = H; n = 3
4b: R1 = H; R2 = H; n = 5
4c: R1 = H; R2 = F; n = 3
4d: R1 = H; R2 = F; n = 5
4e: R1 = F; R2 = F; n = 3
4f: R1 = F; R2 = F; n = 5
O
Scheme 2. Reagents and conditions: (i) K2CO3, dry CH3COCH3, 60 °C, 10ꢁ12 h, 78ꢁ90%; (ii) K2CO3, dry CH3CN, 80 °C, 30ꢁ48 h, 70ꢁ85%; (iii) SnCl2ꢀ2H2O, CH3OH, reflux,
4ꢁ6 h; (iv) HgCl2, CaCO3, CH3CN–H2O, (4:1), 10–12 h, 60ꢁ70% and 46–55%.
ranging from <0.1 to 2.81
mycin and DC-81 (2) demonstrated the GI50 in the range of 0.10–
7.25 M and 0.10–2.37 M, respectively, as shown in Table 1.
Interestingly all the conjugates have shown significant anticancer
activity against hormone dependent (estrogen receptor positive)
l
M, whereas the positive controls adria-
bution by treating MCF-7 cells at 4 lM concentration for 24 h by
flow cytometry (FACS analysis). The majority of control cells ex-
posed with DMSO showed 14.28% of cells in G2/M phase. E2-PBD
conjugates (4a, 4c, 5c, 5d and 5f) treated cells showed 24.01%,
24.33%, 36.64%, 32.40% and 21.80% of cells in G2/M phase. The left
conjugate partner E2 (1) showed 23.80% of cells in G2/M phase,
whereas the right side conjugate partner, DC-81 (2) has shown
more percent of cells in G1 phase and no effect in the G2/M phase
as shown in Table 2.
l
l
MCF-7 cell line with GI50 values ranging from <0.1 to 0.17 lM,
C2-monofluoro-PBD estradiol conjugates have shown slightly bet-
ter anticancer activity compared to the E2-PBD conjugates particu-
larly in Zr-75-1, A-549, A-2780, HOP-62, GURAV, and PC-3 cell
lines. Further, it is observed that the incorporation of the pipera-
zine as well as triazole moieties in the alkane linker spacer be-
tween the estradiol and PBD subunits has slightly enhanced the
anticancer activity. Whereas PBD conjugates with propyloxy spac-
ers exhibited slightly more activity than the pentyloxy spacers.
Among all the conjugates prepared, compounds 4c–e and 5c,d
exhibited significant anticancer activity.
2.2.3. Effect of PBD conjugates on tubulin polymerization
Generally, the inhibition of tubulin polymerization is associated
with an arrest in cell cycle progression at the G2/M phase transi-
tion by interrupting mitotic spindle formation33 and chromosome
segregation.34 From previous studies, it is known that molecules
that alter the microtubule polymerization, cause mitotic arrest
which ultimately leads to apoptosis. Therefore, these E2-PBD con-
jugates (5c and 5d) were evaluated for the inhibitory effects on
MTT assay was also carried out to identify the cytotoxic effect of
some of these E2-PBD conjugates (4a, 4c, 5c, 5d and 5f) in MCF-7
cells at 4 lM. Further, the cytotoxicity of compounds E2 (1), and
tubulin polymerization at 4 lM for 24 h by taking Nocodazole as
a positive control DC-81 (2) was also examined to substantiate
the cytotoxic effect of these E2-PBD conjugates and it is interesting
to observe that these conjugates have shown higher cytotoxicity
than both the controls (1 and 2) as shown in Figure 2.
the standard. While E2 (1) and DC-81 (2), were also used as positive
controls. After 24 h 5c and 5d cells were fixed and stained with
cy3-conjugated b-tubulin antibody and DAPI was used for in situ
observation of microtubule network and nuclei fluorescence
microscopy. It was observed that the disruption of microtubule
network (b-tubulin) was effected significantly in conjugates 5c
than 5d as shown in Figure 3.
2.2.2. Effect of PBD conjugates on cell cycle
To investigate the mechanism underlying the antiproliferative
effect of these E2-PBD conjugates, we analyzed the cell cycle distri-

2568
A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2565–2581
NO2
O
HO
CH(SEt)2
(i)
(ii)
R1
R2
11a−c
N
MeO
O
N
12a−c
N
N
O
( )
NO2
O
12a: R1 = H; R2 = H
12b: R1 = H; R2 = F
12c: R1 = F; R2 = F
n
CH(SEt)2
R1
R2
15a−f
HO
N
MeO
O
(iii), (iv)
N
N
N
O
( )
N
O
n
H
R1
R2
5a−f
5a: R1 = H; R2 = H; n = 3
5b: R1 = H; R2 = H; n = 5
5c: R1 = H; R2 = F; n = 3
5d: R1 = H; R2 = F; n = 5
5e: R1 = F; R2 = F; n = 3
5f: R1 = F; R2 = F; n = 5
N
MeO
O
Scheme 3. Reagents and conditions: (i) propargyl bromide, K2CO3, dry CH3COCH3, 60 °C, 10ꢁ12 h, 92ꢁ95%; (ii) 10a,b; CuSO4ꢀ5H2O (1 mol %), Na2S2O4, (5 mol %), t-BuOH/H2O
(1:1), rt, 8ꢁ10 h, 80ꢁ90%; (iii) SnCl2ꢀ2H2O, CH3OH, reflux, 6 h; (iv) HgCl2, CaCO3, CH3CN–H2O (4:1), 12 h, 55ꢁ65%.
Table 1
GI50 valuesa (in
lM) for E2-PBD conjugates (3aꢁf, 4aꢁf and 5aꢁf) in selected human cancer cell lines
Compound
Zr-75-1c
A-549b
A-2780d
HOP-62b
KBe
SiHah
Guravb
MCF7c
Colo205f
DWDe
PC3g
3a
3b
3c
3d
3e
3f
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
1.19
1.19
0.14
0.17
0.18
0.19
0.12
0.17
<0.1
<0.1
0.12
0.12
0.19
0.11
<0.1
<0.1
0.17
0.18
1.79
2.37
2.10
2.20
0.18
0.18
2.81
2.81
0.12
0.16
<0.1
0.11
0.12
0.13
0.18
0.19
<0.1
<0.1
0.17
0.17
7.25
0.16
1.19
1.19
0.17
0.19
2.10
2.12
1.30
1.70
<0.1
0.11
0.11
0.16
0.17
0.17
<0.1
<0.1
1.27
1.80
0.16
0.14
2.23
2.24
0.18
0.19
2.11
2.11
1.14
1.17
0.11
0.13
0.15
0.17
1.18
1.69
<0.1
0.11
0.18
0.19
0.14
0.15
2.11
2.12
2.18
2.12
2.31
2.81
2.81
0.13
0.17
0.15
0.16
0.16
0.19
1.14
1.17
<0.1
0.11
1.12
1.21
0.17
0.17
2.05
2.04
0.19
0.18
2.18
2.19
0.14
0.19
0.11
0.13
0.13
0.15
0.17
0.18
<0.1
0.11
0.17
0.19
0.17
0.16
0.17
0.17
0.14
0.15
0.17
0.17
0.11
0.14
<0.1
0.13
<0.1
0.14
0.12
0.14
<0.1
<0.1
0.11
0.11
0.17
0.17
0.16
0.19
0.16
0.17
0.10
0.20
0.13
1.16
0.13
0.14
0.14
0.17
0.17
0.17
0.11
0.12
0.14
0.16
0.14
0.11
0.17
0.18
1.70
1.90
2.04
2.17
0.12
0.17
0.10
0.15
0.10
1.32
0.16
0.17
<0.1
0.12
0.17
0.18
0.10
1.49
2.08
2.19
0.19
0.19
2.40
2.42
0.14
0.19
<0.1
0.17
0.13
0.19
0.19
0.19
<0.1
<0.1
0.14
0.17
1.81
0.20
2.13
2.16
2.17
0.15
0.14
0.19
0.19
0.12
0.14
0.17
0.81
0.31
0.80
<0.1
<0.1
0.19
0.19
0.17
0.17
5e
5f
ADRi
DC-81 (2)
a
50% growth inhibition and the values are mean of three determinations.
b
c
d
e
f
Lung cancer.
Breast cancer.
Ovarian cancer.
Oral cancer.
Colon cancer.
Prostate cancer.
Cervix cancer.
Adriamycin.
g
h
i
2.2.4. Effect of PBD conjugates on the expression of Cdk1
Further, to understand the molecular events involved in G2/M
phase arrest, the effect on the expression level of CDKs particularly
Cdk1,35,36 was also investigated in MCF-7 cells that were treated
with these conjugates. Accordingly, Western blot analysis was car-
ried out and it was observed that there was a reduction of Cdk1 le-
vel as shown in Figure 4. The reduction of Cdk1 protein level is
more in case of 5c compared to compounds E2 (1) and DC-81 (2).
which ultimately leads to apoptosis. Hence, it was considered of
interest to investigate the apoptosis mediated by these conjugates
employing Tunel assay wherein E2 (1) and DC-81 (2) are used as
positive controls. Results of the Tunel assay reveals that the Apo-
alert DNA fragmentation is more prominent in case of 5c and 5d
compared to the controls as shown in Figure 5a.
Further, we have focused our attention on the molecules that
are involved in apoptosis. Studies of Basu and co-workers have re-
ported Bax as an important signaling molecule involved in apopto-
sis and its activity was dependent on p53 status.37 Moreover,
caspases-3 and -7 are also known to cause cleavage of substrates
and manifest the apoptotic process.38 Hence it was considered of
2.2.5. Effect of PBD conjugates on apoptosis
From previous studies, it was established that PBD conjugates
that alter the microtubule polymerization, cause mitotic arrest

A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2565–2581
2569
(p65) proteins for 5c treated cells thus suggesting the inhibitory
role of this compound as shown in Figure 7.
3. Conclusions
In conclusion, new classes of E2-PBD conjugates were synthe-
sized and these exhibit promising anticancer activity against sev-
eral cancer cell lines particularly for the estrogen positive breast
cancer (MCF-7) cells. Further, from the MTT proliferation assay it
was observed that the conjugates 5c and 5d are more effective as
antiproliferative agents than the naturally occurring DC-81 (2) in
MCF-7 cells at 4 lM concentration. Flow cytometric data of these
conjugates showed increased cells in G2/M phase, which is sugges-
tive of G2/M cell cycle arrest. Moreover, compound 5c showed the
disruption of microtubules as well as fragmentation of the nuclei
and interestingly, this compound was also identified as an effective
CDK1 inhibitor. Further, it was observed from the results of the de-
tailed biological assays that the up regulation of p53, p21, BAX and
concomitant down regulation of procaspase-7, NF-kB and HDACs
when treated with compounds 5c and 5d, it was more pronounced
in case of 5c. These studies suggest that compound 5c has a poten-
tial to the taken up for detailed preclinical investigations, particu-
larly in the treatment of breast cancer.
Figure 2. Effect of E2-PBD conjugates (4a, 4c, 5c, 5d and 5f) on cell viability (in vitro
cytotoxicity). MCF-7 cells were treated with 4 V concentration of PBD conjugates
as indicated for 24 h in 96-well plates seeded with 10,000 cells per well. O.D
readings were taken at 570 nm wave length to measure the percentage of cell
viability after treatment with the respective compounds. E2 (1) and DC-81 (2) were
used as the positive controls. Control: Control cells treated with DMSO.
l
Table 2
Cell cycle distribution of MCF-7 cell line at 4
DC-81 (2) and E2-PBD conjugates (4a, 4c, 5c, 5d and 5f)
lM concentration of compounds E2 (1),
4. Experimental section
4.1. Chemistry
Compound
G0
G1
S
G2/M
Control (C)
E2 (1)
DC-81 (2)
4a
4c
5c
5d
5f
1.03 0.05
0.83 0.13
8.50 0.50
2.84 0.19
0.87 0.06
2.00 0.05
0.82 0.09
0.74 0.02
61.9.0 1.00
50.00 1.05
73.66 1.52
47.90 0.79
50.53 1.03
30.76 1.78
42.50 1.06
55.00 1.60
22.10 1.00
25.26 1.40
13.00 1.00
25.20 0.61
24.01 0.53
30.59 1.10
25.80 0.58
22.30 0.77
14.28 0.72
23.80 2.60
04.83 0.28
24.01 0.23
24.33 0.67
36.64 1.57
32.40 1.69
21.80 1.68
All chemicals were purchased from Aldrich (Sigma–Aldrich, St.
Louis, MO, USA), Lancaster (Alfa Aesar, Johnson Matthey Company,
Ward Hill, MA, USA) or Spectrochem Pvt. Ltd (Mumbai, India) and
were used without further purification. Reactions were monitored
by TLC, performed on silica gel glass plates containing 60 GF-254,
and visualizat ion on TLC was achieved by UV light or iodine indi-
cator. Column chromatography was performed with Merck 60–120
mesh silica gel. 1H NMR and 13C NMR spectra were recorded on
Gemini Varian-VXR-unity (200 MHz) or Bruker UXNMR/XWIN-
NMR (300 MHz) instruments. Chemical shifts are reported in parts
per million (d in ppm) relative to the peak for tetramethylsilane
(TMS) as an internal standard, coupling constants are reported in
hertz (Hz). Elemental analysis was 0.4% of the theoretical values.
ESI spectra were recorded on Micro mass, Quattro LC using ESI+
software with capillary voltage 3.98 kV and ESI mode positive ion
trap detector. Melting points were determined with an Electro
thermal melting point apparatus, and are uncorrected.
interest to investigate the levels of Bax and procaspase-7. It was
observed from the results that there was up regulation of Bax as
well as cleavage of procaspase-7 indicating apoptosis in case of
5c and 5d as shown in Figure 5b.
2.2.6. Effect of PBD conjugates on tumor suppressor proteins
(p53, p21) and modulators of chromation (HDACs)
Moreover, activation of tumor suppressor genes such as p53
and p21 are considered an important in the regulation of apoptotic
pathway induced by various stimuli.39,40 In order to understand
the effect of these E2-PBD conjugates on p53 and p21 dependent
apoptotic pathway, MCF-7 cells were treated with conjugates 5c
4.2. General procedures
and 5d at 4 lM concentration for 24 h and Western blot analysis
4.2.1. 3-(3-Bromopropyloxy)-17-hydroxy-1,3,5-estratrien (6a)
To a solution of compound 17b estradiol 1 (E2, 272 mg, 1 mmol)
in dry DMF (1 mL), anhydrous K2CO3 (414 mg, 3 mmol) and 1,3-
dibromopropane (0.3 mL, 3 mmol) were added and the reaction
was stirred at rt over 8 h. After completion of the reaction, ice-cold
water (15 mL), added to the reaction mixture. The solution was ex-
tracted into ethyl acetate (3 ꢂ 30 mL), and the combined organic
layers were washed with brine (2 ꢂ 30 mL), dried over anhydrous
Na2SO4, and solvent was evaporated under reduced pressure to
get the crude product. This was further purified by column chro-
matography using ethyl acetate–hexane (15%) to afford the com-
pound 6a as a white solid. Yield 346 mg, 88%; mp 76–78 °C; 1H
NMR (CDCl3, 300 MHz): d 7.17 (d, 1H, J = 8.3 Hz, ArH), 6.68 (dd,
1H, J = 2.3, 8.3 Hz, ArH), 6.61 (s, 1H, ArH), 5.32 (br s, 1H, E2 –OH),
4.06 (t, 2H, J = 6.2 Hz, –JCY2–, 3.71 (t, 1H, J = 8.3 Hz, E2 –CH–JY),
3.58 (t, 2H, J = 6.8 Hz, –CH2Br), 2.80–2.86 (m, 2H, E2 –CH 2–),
was carried out. In these studies up regulation of p53 and p21 pro-
tein levels is observed particularly in 5c apart from 5d as shown in
Figure 6a and b.
Recent studies have revealed that HDAC inhibitors,41 cause up
regulation of p21 protein level in estrogen positive breast cancer
cells such as MCF-7.42–45 Moreover, HDAC inhibitors have the po-
tential to inhibit the inducible as well as constitutive NF-jB activa-
tion to cause an apoptotic response. Since an increase was
observed in the protein levels specific to antitumor genes such as
p53, p21 we became further interested to examine the effect of
these compounds for the inhibition Histone deacetylases (HDAC)
being the main modulators of chromatin. Thus the estrogen posi-
tive MCF-7 cells were treated with compounds for 24 h and Wes-
tern blot analysis was carried out. It was observed from these
results that there is down regulation of HDAC-1, 3 and NF-kB

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A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2565–2581
Figure 3. Effect of PBD conjugates on tubulin polymerization. MCF-7 cells were exposed to E2 (1), DC-81 (2) and 5c and 5d at 4 lM concentration for 24 h. b-Tubulin antibody
was used in this immunofluorescence studies. Nocadozole was used as a positive control.
3.73 (t, 1H, J = 8.2 Hz, E2 –CH–JY), 3.43 (t, 2H, J = 7.2 Hz, –OCH2–),
2.80–2.90 (m, 2H, E2 –CH2–), 2.06–2.35 (m, 3H, E2 3ꢂ –CH–), 1.84–
1.99 (m, 4H, 2ꢂ –CH2–), 1.75–1.83 (m, 2H, –CH2–), 1.15–1.74 (m,
10H, E2 5ꢂ –CH2–), 0.78 (s, 3H, E2 –CY3); MS (ESI): m/z 422 (M+1)+.
4.2.3. 3-{3-Piperazine tert-butyl-1-carboxylate)-propyloxy}-17-
hydroxy-1,3,5-estra trien (7a)
Figure 4. Effect of PBD conjugates on Cdk1 level. MCF-7 cells were treated with E2
(1), DC-81 (2), 5c, and 5d at 4 V concentration for 24 h. The cell lysates were
l
To a solution of compound 6a (393 mg, 1 mmol) in dry acetoni-
trile (20 mL), anhydrous K2CO3 (552 mg, 4 mmol) and tert-butyl-1-
piperazinecarboxylate (374 mg, 2 mmol) were added and the
mixture was refluxed for 24 h. After completion of the reaction,
potassium carbonate was removed by suction filtration and the
solvent was evaporated under reduced pressure to get the crude
product. This was further purified by column chromatography
using ethyl acetate–hexane (60%) to afford the compound 7a as a
white solid. Yield 388 mg, 78%; mp 118–120 °C; 1H NMR (CDCl3,
300 MHz): d 7.18 (d, 1H, J = 8.2 Hz, ArH), 6.69 (dd, 1H, J = 2.2,
8.2 Hz, ArH), 6.63 (s, 1H, ArH), 5.34 (br s, 1H, E2 –OH), 3.97 (t,
2H, J = 6.6 Hz, –OCH2–), 3.73 (t, 1H, J = 8.1 Hz, E2 –CH–JY), 3.38–
3.45 (m, 8H, –N(–CH2–CH2–)2N–), 2.76–2.90 (m, 2H, E2 –CH2–),
2.56 (t, 2H, J = 8.0 Hz, –CH2N–), 1.80–2.37 (m, 5H, E2 3ꢂ –CH–,
–CH2–), 1.12–1.78 (m, 19H, tert-butyl 3ꢂ –CH3, E2 5ꢂ –CH 2–),
0.78 (s, 3H, E2 –CY3); MS (ESI): m/z 499 (M+H)+.
collected and expression level of Cdk1 were determined by Western blot analysis.
b-Actin was used as a loading control.
2.24–2.36 (m, 5H, E2 3ꢂ –CH–, –CH2–), 1.12–2.00 (m, 10H, E2 5ꢂ –
CH2–), 0.77 (s, 3H, E2 –CY3); MS (ESI): m/z 394 (M+1)+.
4.2.2. 3-(5-Bromo pentyloxy)-17-hydroxy-1,3,5-estratrien (6b)
The compound 6b was prepared following the method de-
scribed for the preparation of the compound 6a, employing 1 (E2,
272 mg, 1 mmol) and 1,5-dibromopentane (0.3 mL, 3 mmol), and
the crude product was purified by column chromatography using
ethyl acetate–hexane (17%) to afford the compound 6b as a white
solid. Yield 380 mg, 90%; mp 82–84 °C; 1H NMR (CDCl3, 300 MHz):
d 7.19 (d, 1H, J = 9.3 Hz, ArH), 6.69 (dd, 1H, J = 2.1, 9.3 Hz, ArH), 6.61
(s, 1H, ArH), 5.33 (br s, 1H, E2 –OH), 3.94 (t, 2H, J = 6.2 Hz, –OCH2–),

A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2565–2581
2571
Figure 5a. Effect of PBD conjugates on apoptosis. MCF-7 cells were treated with E2 (1), DC-81 (2), 5c and 5d at 4
l
M concentration for 24 h. The cells were subjected to Tunel
staining, green colour stained cells represents Tunel positive cells. Here lack of staining in control (untreated) cells, represents that the cells are actively proliferating, without
apoptotic cell death.
Figure 5b. Effect of PBD conjugates on the expression of Bax and Procaspase-7
proteins. MCF-7 cells were treated with E2 (1), DC-81 (2), 5c, and 5d at 4
Figure 6a. Effect of PBD conjugates on the expression of p53 and p21 protein levels.
MCF-7 cells were treated with E2 (1), DC-81 (2), 5c and 5d at 4 M concentration for
l
V
l
concentration for 24 h. The cell lysates were collected and expression levels of Bax
and procaspase-7 proteins were determined by Western blot analysis. b-Actin was
used as a loading control.
24 h. The cell lysates have been collected and the levels of p53, p21 were
determined by Western blot analysis using specific antibodies. b-Actin was used as
loading control.

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A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2565–2581
4.2.6. 3-(5-Piperazinopentyloxy)-17-hydroxy-1,3,5-estratrien
(8b)
The compound 8b was prepared following the method de-
scribed for the preparation of the compound 8a, employing 7b
(526 mg, 1 mmol) and tri fluoroaceticacid (0.8 mL, 10 mmol),
Without further purification this compound was used next step,
to afford the compound 8b as a light yellow oil. Yield 401 mg, 95%.
4.2.7. 3-{3-(4-(3-Bromopropyl)-piperazin-1-yl)-propyloxy}-17-
hydroxy-1,3,5-estra trien (9a)
To a solution of compound 8a (398 mg, 1 mmol) in dry acetoni-
trile (20 mL), anhydrous K2CO3 (553 mg, 4 mmol) and 1,3-dibro-
mopropane (0.3 mL, 3 mmol) were added and the mixture was
refluxed for 24 h. After completion of the reaction, potassium car-
bonate was removed by filtration and the solvent was evaporated
under reduced pressure to get the crude product. This was further
purified by column chromatography using ethyl acetate–hexane
(62%) to afford the compound 9a as a light yellow oil. Yield
388 mg, 75%; 1H NMR (CDCl3, 300 MHz): d 7.19 (d, 1H, J = 8.2 Hz,
ArH), 6.71 (dd, 1H, J = 2.2, 8.2 Hz, ArH), 6.61 (s, 1H, ArH), 5.34 (br
s, 1H, E2 –OH), 3.94–4.12 (m, 2H, –OCH2–), 3.72 (t, 1H, J = 8.1 Hz,
E2 –CH–JY), 3.56 (t, 2H, J = 5.6 Hz, –CH2Br), 3.36–3.44 (m, 8H, –
N(–CH2–CH2–)2N–), 2.77–2.89 (m, 2H, E2 –CH 2–), 2.40–2.66 (m,
4H, 2ꢂ –CH2N–), 1.73–2.36 (m, 7H, 2ꢂ –CH 2–, E2 3ꢂ –CH–),
1.13–1.71 (m, 10H, E2 5ꢂ –CH 2–), 0.77 (s, 3H, E2 –CY3); MS
(ESI): m/z 520 (M+1)+.
Figure 6b. Effect of PBD conjugates on p21 promoter (ꢁ208/+8) activity. MCF-7
cells were transfected with 1.5 lg of p21 promoter fused with luciferase reporter
gene and after 24 h, the cells were subjected to compound treatments E2 (1), DC-81
(2), and 5c at 4 M concentration for 24 h. The cell lysates have been collected and
l
observed for luciferase activity which is the indicator for p21 promoter activity after
compound treatment. Here C: Control (untreated).
4.2.8. 3-{5-(4-(5-Bromopentyl)-piperazin-1-yl)-pentyloxy}-17-
hydroxy-1,3,5-estra trien (9b)
The compound 9b was prepared following the method de-
scribed for the preparation of the compound 9a, employing com-
pound 8b (426 mg, 1 mmol) and 1,5-dibromopentane (0.3 mL,
3 mmol), and the crude product was purified by column chroma-
tography using ethyl acetate–hexane (64%) to afford the compound
9b as a light yellow oil. Yield 447 mg, 78%; 1H NMR (CDCl3,
200 MHz): d 7.19 (d, 1H, J = 8.3 Hz, ArH), 6.71 (dd, 1H, J = 2.3,
8.3 Hz, ArH), 6.60 (s, 1H, ArH), 5.34 (br s, 1H, E2 –OH), 3.90–4.00
(t, 2H, 2ꢂ –OCH2–), 3.73 (t, 1H, J = 8.3 Hz, E2 –CH–JY), 3.57 (t,
2H, J = 5.9 Hz, –CH2Br), 3.40–3.47 (m, 8H, –N(–CH2–CH2–)2N–),
2.76–2.90 (m, 2H, E2 –CH 2–), 2.45–2.66 (m, 4H, 2ꢂ –CH2N–),
2.38–2.41 (m, 3H, E2 3ꢂ –CH–), 1.80–2.30 (m, 12H, 6ꢂ –CH2 –),
1.12–1.78 (m, 10H, E2 5ꢂ –CH 2–), 0.78 (s, 3H, E2 –CY3); MS
(ESI); m/z 576 (M+1)+.
Figure 7. Effect of PBD conjugates on expression of the NF-kB (p65), Histone
deacetylases proteins. MCF-7 cells were treated with E2 (1), DC-81 (2), 5c and 5d at
4 lM concentration for 24 h. Western blot analysis was carried out with antibodies
against p65 and HDAC1, 3.
4.2.4. 3-{5-Piperazine tert-butyl-1-carboxylate)-pentyloxy}-17-
hydroxy-1,3,5-estra trien (7b)
The compound 7b was prepared following the method
described for the preparation of the compound 7a, employing
6b (421 mg, 1 mmol) and tert-butyl-1-piperazinecarboxylate
(374 mg, 2 mmol), and the crude product was purified by column
chromatography using ethyl acetate–hexane (64%) to afford the
compound 7b as a white solid. Yield 431 mg, 82%; mp 148–
150 °C; 1H NMR (CDCl3, 200 MHz): d 7.19 (d, 1H, J = 8.3 Hz, ArH),
6.70 (dd, 1H, J = 2.3, 8.3 Hz, ArH), 6.61 (s, 1H, ArH), 5.33 (br s, 1H,
E2 –OH), 3.98 (t, 2H, J = 7.1 Hz, –OCH2–), 3.72 (t, 1H, J = 8.3 Hz, E2
–CH–JY), 3.39–3.46 (m, 8H, –N(–CH2–CH2–)2N–), 2.77–2.90 (m,
2H, E2 –CH2–), 2.59 (t, 2H, J = 8.1 Hz, –CH2N–), 2.22–2.51 (m, 3H,
E2 3ꢂ –CH–), 1.79–2.20 (m, 6H, 3ꢂ –CH2–), 1.14–1.76 (m, 19H,
tert-butyl 3 –CH3, E2 5ꢂ –CH2–), 0.78 (s, 3H, E2 –CY3); MS (ESI):
m/z 527 (M+H)+.
4.2.9. 3-(3-Azidopropyloxy)-17-hydroxy-1,3,5-estratrien (10a)
Azidation reaction, compound 6a (393 mg, 1 mmol) in dry DMF
(3 mL), and NaN3 (265 mg, 4 mmol) was added and the mixture
was heated at 60–70 °C for 6–8 h, in this transformation bromo
substituted with azide, Later this reaction mixture was poured into
ice-cold water and extracted with ethyl acetate (3 ꢂ 40 mL), the
organic layer was washed with brine (2 ꢂ 20 mL), and dried over
anhydrous Na2SO4, solvent was evaporated under reduced pres-
sure to get the crude product. This was further purified by column
chromatography using ethyl acetate–hexane (18%) to afford the
compound 10a as a white solid. Yield 324 mg, 90%; mp 60–62 °C;
1H NMR (CDCl3, 200 MHz): d 7.19 (d, 1H, J = 8.3 Hz, ArH), 6.69
(dd, 1H, J = 2.3, 8.3 Hz, ArH), 6.63 (s, 1H, ArH), 5.34 (br s, 1H,
E2 –OH), 4.00 (t, 2H, J = 5.4 Hz, –JCY2–), 3.72 (t, 1H, J = 8.5 Hz, E2
–CH–JY), 3.41 (t, 2H, J = 6.7 Hz, –CH2N3), 2.77–2.89 (m, 2H, E2
–CH2–), 2.25–2.38 (m, 5H, –CH 2–, E2 3ꢂ –CH–), 1.14–2.10 (m,
10H, E2 5ꢂ –CH2–), 0.78 (s, 3H, E2 –CY3); MS (ESI): m/z 356 (M+H)+.
4.2.5. 3-(3-Piperazinopropyloxy)-17-hydroxy-1,3,5-estratrien
(8a)
The compound 7a (498 mg, 1 mmol) in dry dichloromethane
(25 mL), at 0 °C, and trifluoroacetic acid (0.8 mL, 10 mmol) were
added slowly to this reaction mixture and the mixture was stirred
at room temperature for 2 h. After completion of the reaction, the
reaction mixture was concentrated and the resulting compound
was suspended in NaHCO3 solution (30 mL), the solution was ex-
tracted into dichloromethane (3 ꢂ 40 mL), and the combined or-
ganic layer were washed with brine (2 ꢂ 30 mL), dried over
anhydrous Na2SO4, and solvent was evaporated under reduced
pressure to get the product. Without further purification this com-
pound was used next step, to afford the compound 8a as a light
yellow oil. Yield 380 mg, 95%.
4.2.10. 3-(5-Azidopentyloxy)-17-hydroxy-1,3,5-estratrien (10b)
The compound 10b was prepared following the method de-
scribed for the preparation of the compound 10a, employing 6b
(421 mg, 1 mmol) and NaN3 (265 mg, 4 mmol), and the crude

A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2565–2581
2573
product was purified by column chromatography using ethyl ace-
4.2.14. (2S)-N-[4-{3-(17-Hydroxy-1,3,5-estratrien-3-yl)-oxy}-
tate–hexane (20%) to afford the compound 10b as a white solid.
Yield 352 mg, 92%; mp 69–71 °C; 1H NMR (CDCl3, 200 MHz): d
7.18 (d, 1H, J = 9.2 Hz, ArH), 6.70 (dd, 1H, J = 3.0, 7.9 Hz, ArH),
6.62 (s, 1H, ArH), 5.32 (br s, 1H, E2 –OH), 3.96 (t, 2H, J = 7.1 Hz, –
OCH2–), 3.71 (t, 1H, J = 8.2 Hz, E2 –CH–JY), 3.42 (t, 2H, J = 7.2 Hz,
–CH2N3), 2.79–2.90 (m, 2H, E2 –CH2–), 2.06–2.36 (m, 3H, E2 3ꢂ –
CH–), 1.83–1.96 (m, 4H, 2ꢂ –CH2–), 1.74–1.80 (m, 2H, –CH2–),
1.14–1.72 (m, 10H, E2 5ꢂ –CH 2–), 0.78 (s, 3H, E2 –CY3); MS
(ESI): m/z 384 (M+H)+.
propyloxy)-5-methoxy-2-nitrobenzoyl]-pyrrolidine-2-
carboxaldehyde diethylthioacetal (13a)
To a solution of (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]-
pyrrolidine-2-carboxaldehyde diethylthioacetal 11a (400 mg,
1 mmol) in acetone (20 mL) anhydrous K2CO3 (552 mg, 4 mmol)
and the compound 6a (393 mg, 1 mmol) were added. The reaction
mixture was heated to reflux for 10 h. After completion of the
reaction as indicated by TLC, potassium carbonate was removed
by suction filtration and the solvent was removed under vacuum.
The crude product was purified by column chromatography using
ethyl acetate–hexane (60%) to afford the pure compound 13a as a
light yellow solid. Yield 613 mg, 86%; mp 90–92 °C; 1H NMR
(CDCl3, 300 MHz): d 7.72 (s, 1H, ArH), 7.20 (d, 1H, J = 9.4 Hz,
ArH), 6.81 (s, 1H, ArH), 6.72 (dd, 1H, J = 3.1, 9.4 Hz, ArH), 6.65 (s,
1H, ArH), 5.35 (br s, 1H, E2 –OH), 4.88 (d, 1H, J = 3.9 Hz, –CHS2),
4.66–4.76 (m, 1H, pro –NCH–), 4.29 (t, 2H, J = 6.2 Hz, –OCH2–),
4.15 (t, 2H, J = 5.5 Hz, –OCH2–), 3.93 (s, 3H, –OCH3), 3.72 (t, 1H,
J = 8.6 Hz, E2 –CY–OH), 3.18–3.29 (m, 2H, pro –NCH2–), 2.69–2.90
(m, 6H, 2ꢂ –SCH2–, E2 –CH2–), 2.14–2.41 (m, 3H, E2 3ꢂ –CY–),
1.53–2.10 (m, 6H, pro 2ꢂ –CY2–, –CY2–), 1.22–1.48 (m, 16H,
E2 5ꢂ –CY2–, –CH32), 0.78 (s, 3H, E2 –CH3); MS (ESI): m/z 713
(M+H)+.
4.2.11. (2S)-N-[5-Methoxy-2-nitrobenzoyl-4-(2-propynyloxy]-
pyrrolidine-2-carboxaldehyde diethylthioacetal (12a)
To a solution of (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]
pyrrolidine-2-carbox aldehyde diethylthioacetal 11a (400 mg,
1 mmol) in dry acetone (10 mL), anhydrous K2CO3 (552 mg,
4 mmol) and the propargyl bromide (302 mg, 2 mmol) were added
to the reaction mixture. The reaction mixture was heated to reflux
for 4–6 h. After completion of the reaction as indicated by TLC,
potassium carbonate was removed by suction filtration and the
solvent was removed under vacuum. The crude product thus ob-
tained was purified by column chromatography using ethyl ace-
tate–hexane (35%) to afford the pure compound 12a as a light
yellow solid. Yield 395 mg, 90%; mp 84–86 °C; 1H NMR (CDCl3,
500 MHz): d 7.88 (s, 1H, ArH), 6.87 (s, 1H, ArH), 4.88 (d, 2H,
J = 3.9 Hz, –OCH2–), 4.86 (d, 1H, J = 2.3 Hz, –CHS2), 4.68–4.76 (m,
1H, pro –NCH–), 3.97 (s, 3H, –OCH3), 3.18–3.35 (m, 2H, pro –
NCH2–), 2.67–2.89 (m, 4H, 2ꢂ –SCH2–), 2.50–2.62 (m, 1H, „CH),
1.93–2.32 (m, 4H, pro 2ꢂ –CH2–), 1.20–1.41 (m, 6H, –CH32); MS
(ESI): m/z 461 (M+Na)+.
4.2.15. (2S)-N-[4-{3-(17-Hydroxy-1,3,5-estratrien-3-yl)-oxy}-
pentyloxy)-5-methoxy-2-nitrobenzoyl]-pyrrolidine-2-
carboxaldehyde diethylthioacetal (13b)
This compound was prepared according to the method de-
scribed for compound 13a, employing compound 11a (400 mg,
1 mmol) and compound 6b (423 mg, 1 mmol). The crude product
was purified by column chromatography using ethyl acetate–
hexane (65%) to afford the compound 13b as a light yellow solid.
Yield 667 mg, 90%; mp 86–88 °C; 1H NMR (CDCl3, 300 MHz): d
7.67 (s, 1H, ArH), 7.19 (d, 1H, J = 8.3 Hz, ArH), 6.82 (s, 1H, ArH),
6.69 (dd, 1H, J = 2.9, 8.3 Hz, ArH), 6.63 (s, 1H, ArH), 5.34 (br s,
1H, E2 –OH), 4.88 (d, 1H, J = 3.8 Hz, –CHS2), 4.68–4.76 (m, 1H,
pro –NCH–), 4.12 (t, 2H, J = 5.3 Hz, –OCH2–), 3.97 (t, 2H,
J = 6.8 Hz, –OCH2–), 3.94 (s, 3H, –OCH3), 3.73 (t, 1H, J = 8.3 Hz,
E2 –CY–OH), 3.19–3.33 (m, 2H, pro –NCH2–), 2.64–2.89 (m,
6H, 2ꢂ –SCH2–, E2 –CH2–), 2.03–2.40 (m, 7H, E2 3ꢂ –CY–, 2ꢂ
–CH2–), 1.78–2.01 (m, 6H, pro 2ꢂ –CH2–, –CH2–), 1.25–1.76 (m,
16H, E2 5ꢂ –CY2–, –CH32), 0.78 (s, 3H, E2 –CH3); MS (ESI): m/z
741 (M+H)+.
4.2.12. (2S,4R)-N-[5-Methoxy-2-nitrobenzoyl-4-(2-propynyl-
oxy]-4-fluoropyrrolidine-2-carboxaldehyde diethylthioacetal
(12b)
This compound was prepared according to the method
described for compound 12a, employing compound (2S,4R)-N-[4-
hydroxy-5-methoxy-2-nitrobenzoyl]-4-fluoropyrroli dine-2-carbox-
aldehyde diethylthioacetal 11b (418 mg, 1 mmol) and propargyl
bromide (305 mg, 2 mmol). The crude product was purified by
column chromatography using ethyl acetate–hexane (38%) to af-
ford the compound 12b as a light yellow solid. Yield 374 mg,
82%; mp 93–95 °C; 1H NMR (CDCl3, 200 MHz): d 7.86 (s, 1H,
ArH), 6.92 (s, 1H, ArH), 4.86 (d, 2H, J = 3.3 Hz, –OCH2–),4.72–4.83
(m, 1H, pro –NCH–), 4.62 (d, 2H, J = 2.2 Hz, –CHS2), 3.99 (s, 3H, –
OCH3), 3.95–3.97 (m, 1H, pro –CHF–), 3.42–3.70 (m, 2H, pro –
NCH2–), 2.68–2.90 (m, 4H, 2ꢂ –SCH2–), 2.43–2.66 (m, 3H, „CH,
pro –CH2–), 1.20–1.39 (m, 6H, –CH32); MS (ESI): m/z 479 (M+Na)+.
4.2.16. (2S,4R)-N-[4-{3-(17-Hydroxy-1,3,5-estratrien-3-yl)-oxy}-
propyloxy)-5-methoxy-2-nitrobenzoyl]-4-fluoropyrrolidine-2-
carboxaldehyde diethylthioacetal (13c)
This compound was prepared according to the method de-
scribed for compound 13a, employing compound (2S,4R)-N-[4-hy-
droxy-5-methoxy-2-nitrobenzoyl]-4-fluoropyrrolidine-2-carbox-
aldehyde diethylthioacetal 11b (418 mg, 1 mmol) and compound
6a (393 mg, 1 mmol). The crude product was purified by column
chromatography using ethyl acetate–hexane (65%) to afford the
compound 13c as a light yellow solid. Yield 576 mg, 79%; mp
100–102 °C; 1H NMR (CDCl3, 300 MHz): d 7.71 (s, 1H, ArH), 7.20
(d, 1H, J = 9.2 Hz, ArH), 6.79 (s, 1H, ArH), 6.70 (dd, 1H, J = 3.1,
9.2 Hz, ArH), 6.64 (s, 1H, ArH), 5.35 (br s, 1H, E2 –OH), 4.84 (d,
1H, J = 3.7 Hz, –CHS2), 4.67–4.77 (m, 1H, pro –NCH–), 4.27 (t, 2H,
J = 6.3 Hz, –OCH2–), 4.13 (t, 2H, J = 5.5 Hz, –OCH2–), 3.95 (s, 3H,
–OCH3), 3.69–3.77 (m, 1H, E2 –CY–OH), 3.17–3.29 (m, 3H, pro
–CYF–, pro –NCH2–), 2.67–2.91 (m, 6H, 2ꢂ –SCH2–, E2 –CH2–),
2.16–2.39 (m, 3H, E2 3ꢂ –CY–), 1.81–2.11 (m, 4H, pro –CY2–,
–CY2–), 1.19–1.70 (m, 16H, E2 5ꢂ –CY2–, –CH32), 0.78 (s, 3H, E2
–CH3); MS (ESI): m/z 731 (M+H)+.
4.2.13. (2S)-N-[5-Methoxy-2-nitrobenzoyl-4-(2-propynyloxy]-
4,4difluoropyrro-lidine-2-carboxaldehyde diethylthioacetal
(12c)
This compound was prepared according to the method
described for compound 12a, employing compound (2S)-N-[4-hy-
droxy-5-methoxy-2-nitrobenzoyl]-4,4-difluoropyrroli dine-2-car-
boxaldehyde diethylthioacetal 11c (435 mg, 1 mmol) and
propargyl bromide (305 mg, 2 mmol). The crude product was puri-
fied by column chromatography using ethyl acetate–hexane (40%)
to afford the compound 12c as a light yellow solid. Yield 388 mg,
85%; mp 97–99 °C; 1H NMR (CDCl3, 300 MHz): d 7.88 (s, 1H,
ArH), 6.82 (s, 1H, ArH), 4.87–4.95 (m, 1H, pro –NCH–), 4.84 (d,
2H, J = 2.3 Hz, –OCH2–), 4.78 (d, 1H, J = 3.0 Hz, –CHS2), 3.97 (s,
3H, –OCH3), 3.42–3.80 (m, 2H, pro –NCH2–), 2.70–2.92 (m, 4H,
2ꢂ –SCH2–), 2.40–2.69 (m, 3H, „CH, pro –CH2–), 1.31–1.42 (m,
6H, –CH32); MS (ESI): m/z 475 (M+H)+.

2574
A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2565–2581
4.2.17. (2S,4R)-N-[4-{3-(17-Hydroxy-1,3,5-estratrien-3-yl)-oxy)-
pentyloxy)-5-methoxy-2-nitrobenzoyl]-4-fluoropyrrolidine-2-
carboxaldehyde diethylthioacetal (13d)
potassium carbonate was removed by suction filtration and the
solvent was removed under vacuum. The crude product was puri-
fied by column chromatography using ethyl acetate–hexane (80%)
to afford the pure compound 14a as a light yellow solid. Yield
672 mg, 80%; mp 94–96 °C; 1H NMR (CDCl3, 300 MHz): d 7.70 (s,
1H, ArH), 7.20 (d, 1H, J = 8.8 Hz, ArH), 6.83 (s, 1H, ArH), 6.67 (dd,
1H, J = 2.2, 8.8 Hz, ArH), 6.61 (s, 1H, ArH), 5.34 (br s, 1H, E2 –OH),
4.88 (d, 1H, J = 3.7 Hz, –CHS2), 4.67–4.76 (m, 1H, pro – NCH–),
4.16 (t, 2H, J = 6.6, Hz, –OCH2–), 3.99 (t, 2H, J = 5.9, –OCH2–), 3.94
(s, 3H, –OCH3), 3.73 (t, 1H J = 8.8, E2 –CY–OH), 3.21–3.40 (m, 2H,
pro –NCH2–) 2.80–3.00 (m, 12H, –N(–CH2–CH2–)2N–, 2ꢂ –NCH2–
), 2.53–2.78 (m, 6H, 2ꢂ –SCH2–, E2 –CH2–), 1.75–2.41 (m, 11H, E2
3ꢂ –CY–, pro 2ꢂ –CY2–, 2ꢂ –CY2–), 1.15–1.69 (m, 16H, E2 5ꢂ –
CY2–, –CH32), 0.78 (s, 3H, E2 –CH3); MS (ESI): m/z 840 (M+H)+.
This compound was prepared according to the method de-
scribed for compound 13a, employing compound 11b (418 mg,
1 mmol) and compound 6b (423 mg, 1 mmol). The crude product
was purified by column chromatography using ethyl acetate–
hexane (66%) to afford the compound 13d as a light yellow solid.
Yield 622 mg, 82%; mp 99–101 °C; 1H NMR (CDCl3, 300 MHz): d
7.66 (s, 1H, ArH), 7.20 (d, 1H, J = 9.1 Hz, ArH), 6.89 (s, 1H, ArH),
6.70 (dd, 1H, J = 3.2, 9.1 Hz, ArH), 6.63 (s, 1H, ArH), 5.34 (br s, 1H,
E2 –OH), 4.76–4.84 (m, 1H, pro –NCH–), 4.61 (d, 1H, J = 6.8 Hz, –
CHS2), 4.08–4.46 (m, 2H, –OCH2–), 3.97 (s, 3H, –OCH3), 3.92–3.96
(m, 2H, –OCH2–), 3.73 (t, 1H, J = 8.3, E2 –CY–OH), 3.44–3.66 (m,
1H, pro –CHF–), 2.69–2.89 (m, 6H, 2ꢂ –SCH2–, pro –NCH2–),
2.47–2.66 (m, 2H, E2 –CH2–), 2.05–2.36 (m, 5H, E2 3ꢂ –CY–, pro
–CY2–), 1.79–2.01 (m, 6H, 3ꢂ –CY2–), 1.14–1.72 (m, 16H, E2 5ꢂ
–CY2–, –CH32), 0.77 (s, 3H, E2 –CH3); MS (ESI): m/z 759 (M+H)+.
4.2.21. (2S)-N-[4-{3-(17-Hydroxy-1,3,5-estratrien-3-yl)-oxy-
pentyl}-piperazino)-pentyloxy)-5-methoxy-2-nitrobenzoyl]-
pyrrolidine-2-carboxaldehyde diethylthioacetal (14b)
This compound was prepared according to the method
described for compound 14a, employing compound 11a (400 mg,
1 mmol) and compound 9b (576 mg, 1 mmol). The crude product
was purified by column chromatography using ethyl acetate–
hexane (85%) to afford the compound 14b as a light yellow solid.
Yield 736 mg, 85%; mp 97–99 °C; 1H NMR (CDCl3, 200 MHz): d
7.67 (s, 1H, ArH), 7.19 (d, 1H, J = 8.8 Hz, ArH), 6.82 (s, 1H, ArH),
6.69 (dd, 1H, J = 2.3, 8.8, Hz, ArH), 6.61 (s, 1H, ArH), 5.35 (br s,
1H, E2 –OH), 4.88 (d, 1H, J = 2.9 Hz, –CHS2), 4.69–4.73 (m, 1H, pro
–NCH–), 4.09–4.14 (m, 4H, 2ꢂ –OCH2–), 3.95 (s, 3H, –OCH3),
3.68–3.74 (m, 1H, E2 –CH–OH), 3.21–3.32 (m, 2H, pro –NCH2–),
2.68–2.89 (m, 12H, –N(–CH2–CH2–)2N–, –NCH2–), 2.38–2.60 (m,
2H, E2 –CH2), 2.06–2.36 (m, 7H, 2ꢂ –SCH2, E2 3ꢂ –CY–), 2.06–
2.23 (m, 4H, pro 2ꢂ –CH2), 1.88–2.02 (m, 6H, 3ꢂ –CH2), 1.75–
1.85 (m, 6H, 3ꢂ –CH2), 1.31–1.70 (m, 16H, E2 5ꢂ –CY2–, –CH32),
0.78 (s, 3H, E2 –CH3); MS (ESI): m/z 896 (M+H)+.
4.2.18. (2S)-N-[4-{3-(17-Hydroxy-1,3,5-estratrien-3-yl)-oxy}-
propyloxy)-5-methoxy-2-nitrobenzoyl]-4,4-difluoro-
pyrrolidine-2-carboxaldehyde diethylthioacetal (13e)
This compound was prepared according to the method
described for compound 13a, employing compound (2S)-N-[4-hy-
droxy-5-methoxy-2-nitrobenzoyl]-4,4-difluoropyrrolidine-2-car-
boxaldehyde diethylthioacetal 11c (436 mg, 1 mmol) and
compound 6a (393 mg, 1 mmol). The crude product was purified
by column chromatography using ethyl acetate–hexane (66%) to
afford the compound 13e as a light yellow solid. Yield 584 mg,
78%; mp 104–106 °C; 1H NMR (CDCl3, 300 MHz): d 7.73 (s, 1H,
ArH), 7.20 (d, 1H, J = 8.3 Hz, ArH), 6.77 (s, 1H, ArH), 6.71 (dd, 1H,
J = 2.6, 8.3, Hz, ArH), 6.65 (s, 1H, ArH), 5.32 (br s, 1H, E2 –OH),
4.91–4.95 (m, 1H, pro –NCH–), 4.83 (d, 1H, J = 3.8 Hz, –CHS2),
4.30 (t, 2H, J = 6.0 Hz, –OCH2–), 4.15 (t, 2H, J = 6.0 Hz, –OCH2–),
3.93 (s, 3H, –OCH3), 3.66–3.80 (m, 2H, pro –NCH2–), 3.64 (t, 1H,
J = 13.0 Hz, E2 –CY–OH), 2.62–2.92 (m, 6H, 2ꢂ –SCH2–, E2 –CH2–),
2.08–2.38 (m, 3H, E2 3ꢂ –CY–), 1.81–2.03 (m, 4H, pro –CY2–, –
4.2.22. (2S,4R)-N-[4-{3-(17-Hydroxy-1,3,5-estratrien-3-yl)-
oxypropyl}-piperazino)-propyloxy)-5-methoxy-2-nitrobenz-
oyl]-4-fluoropyrrolidine-2-carboxaldehyde diethyl thioacetal
(14c)
CH2–), 1.15–1.74 (m, 16H, E2 5ꢂ–CY2–, –CH32), 0.77 (s, 3H, E2
–
CH3); MS (ESI): m/z 749 (M+H)+.
This compound was prepared according to the method de-
scribed for compound 14a, employing compound 11b (418 mg,
1 mmol) and compound 9a (520 mg, 1 mmol). The crude product
was purified by column chromatography using ethyl acetate–
hexane (90%) to afford the compound 14c as a light yellow solid.
Yield 643 mg, 75%; mp 89–91 °C; mp 114–116 °C; 1H NMR (CDCl3,
200 MHz): d 7.66 (s, 1H, ArH), 7.22 (d, 1H, J = 8.4 Hz, ArH), 6.86 (s,
1H, ArH), 6.76 (dd, 1H, J = 2.2, 8.4 Hz, ArH), 6.62 (s, 1H, ArH), 5.33
(br s, 1H, E2 –OH), 4.71–4.77 (m, 1H, pro –NCH–), 4.54 (d, 1H,
J = 6.7 Hz, –CHS2), 4.26 (t, 2H, J = 6.0, Hz, –OCH2–), 4.00 (t, 2H,
J = 6.6, Hz, –OCH2–), 3.97 (s, 3H, –OCH3), 3.84–3.88 (m, 2H
J = 5.7 Hz, E2 –CH–OH, pro –CHF–), 3.40–3.64 (m, 2H, pro –NCH2–
), 2.70–3.21 (m, 12H, –N(–CH2–CH2–)2N–, 2ꢂ –NCH2–), 2.68–2.89
(m, 6H, 2ꢂ –SCH2–, E2 –CH2–), 2.42–2.66 (m, 9H, E2 3ꢂ –CY–, 2ꢂ
–CY2–, pro –CY2–), 1.14–1.78 (m, 16H, E2 5ꢂ –CY2–, –CH32), 0.78
(s, 3H, E2 –CH3); MS (ESI): m/z 857 (M+H)+.
4.2.19. (2S)-N-[4-{3-(17-Hydroxy-1,3,5-estratrien-3-yl)-oxy}-
pentyloxy)-5-methoxy-2-nitrobenzoyl]-4,4-difluoropyrro-
lidine-2-carboxaldehyde diethylthioacetal (13f)
This compound was prepared according to the method
described for compound 13a, employing compound 11c (436 mg,
1 mmol) and compound 6b (423 mg, 1 mmol). The crude product
was purified by column chromatography using ethyl acetate–hex-
ane (70%) to afford the compound 13f as a light yellow solid. Yield
687 mg, 86%; mp 91–93 °C; 1H NMR (CDCl3, 300 MHz): d 7.63 (s,
1H, ArH), 7.11 (d, 1H, J = 8.2 Hz, ArH), 6.72 (s, 1H, ArH), 6.61 (dd,
1H, J = 3.0, 8.2 Hz, ArH), 5.32 (br s, 1H, E2 –OH), 4.86–4.91 (m,
1H, pro –NCH–), 4.78 (d, 1H, J = 3.8 Hz, –CHS2), 4.11 (t, 2H,
J = 6.2 Hz, –OCH2–), 3.94–3.96 (m, 2H, –OCH2–), 3.93 (s, 3H, –
OCH3), 3.65–3.86 (m, 2H, pro –NCH2–), 3.61 (t, 1H, J = 13.4 Hz, E2
–CY–OH), 2.53–2.93 (m, 6H, 2ꢂ –SCH2–, E2 –CH2–), 2.13–2.25 (m,
3H, E2 3ꢂ –CY–), 1.75–2.10 (m, 8H, pro –CY2–, 3ꢂ –CY2–), 1.16–
1.71 (m, 16H, E2 5ꢂ –CY2–, –CH32), 0.77 (s, 3H, E2 –CH3); MS
(ESI): m/z 799 (M+ Na)+.
4.2.23. (2S,4R)-N-[4-{3-(17-Hydroxy-1,3,5-estratrien-3-yl)-
oxypentyl}-piperazino)-pentyloxy)-5-methoxy-2-nitrobenz-
oyl]-4-fluoropyrrolidine-2-carboxaldehyde diethyl thioacetal
(14d)
This compound was prepared according to the method de-
scribed for compound 14a, employing compound 11b (418 mg,
1 mmol) and compound 9b (576 mg, 1 mmol). The crude product
was purified by column chromatography using ethyl acetate–hexane
(90%) to afford the compound 14d as a yellow solid. Yield 618 mg,
70%; mp 100–102 °C; 1H NMR (CDCl3, 300 MHz): d 7.68 (s, 1H,
4.2.20. (2S)-N-[4-{3-(17-Hydroxy-1,3,5-estratrien-3-yl)oxypro-
pyl}-piperazino)-propyloxy)-5-methoxy-2-nitrobenzoyl]-
pyrrolidine-2-carboxaldehyde diethylthioacetal (14a)
To a solution of compound 11a (400 mg, 1 mmol) in acetonitrile
(20 mL) anhydrous K2CO3 (552 mg, 4 mmol) and the compound 9a
(520 mg, 1 mmol) were added. The reaction mixture was heated to
reflux for 30 h. After completion of the reaction as indicated by TLC,

A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2565–2581
2575
ArH), 7.20 (d, 1H, J = 8.2 Hz, ArH), 6.84 (s, 1H, ArH), 6.73 (dd, 1H,
J = 2.3, 8.2 Hz, ArH), 6.61 (s, 1H, ArH), 5.34 (br s, 1H, E2 –OH), 4.86
(d, 1H, J = 6.7 Hz, –CHS2), 4.70–4.72 (m, 1H, pro –NCH–), 4.08–4.15
(m, 4H, 2ꢂ –OCH2–), 3.96 (s, 3H, –OCH3), 3.68–3.73 (m, 2H, pro –
NCH2–), 3.20–3.31 (m, 2H, E2 –CH–OH, pro –CHF–), 2.70–2.92 (m,
12H, –N(–CH2–CH2–)2N–, 2ꢂ –NCH2–), 2.38–2.60 (m, 2H, E2 –CH2–
), 2.06–2.36 (m, 7H, 2ꢂ –SCH2, E2 3ꢂ –CY–), 1.88–2.23 (m, 10H,
4ꢂ –CH2, pro –CH2), 1.76–1.84 (m, 4H, 2ꢂ –CH2), 1.16–1.71 (m,
16H, E2 5ꢂ –CY2–, –CH32), 0.77 (s, 3H, E2 –CH3); MS (ESI): m/z 913
(M+H)+.
through a Celite bed. The combined organic layer were washed
with brine (2 ꢂ 30 mL), dried over anhydrous Na2SO4, and solvent
was evaporated under reduced pressure to get the crude product.
This was further purified by column chromatography using ethyl
acetate–hexane (65%) to afford the compound 15a as a yellow so-
lid. Yield 718 mg, 88%; mp 103–105 °C; 1H NMR (CDCl3, 300 MHz):
d 7.90 (s, 1H, triazole –H), 7.69 (s, 1H, ArH), 7.21 (d, 1H, J = 9.1 Hz,
ArH), 6.84 (s, 1H, ArH), 6.62 (dd, 1H, J = 3.0, 9.1 Hz, ArH), 6.61 (s, 1H,
ArH), 5.34 (br s, 1H, E2 –OH), 5.31 (s, 2H, –OCH2–), 4.88 (d, 1H,
J = 3.77 Hz, –CHS2), 4.68–4.74 (m, 1H, pro –NCH–), 4.49 (t, 2H,
J = 7.5 Hz, –OCH2–), 3.93 (s, 3H, –OCH3), 3.74 (t, 1H, J = 8.3 Hz, E2
–CY–OH), 3.40 (t, 2H, J = 5.7 Hz, –CH2N–), 3.18–3.33 (m, 2H, pro
–CH2N–), 2.66–2.90 (m, 2H, E2 –CH2–), 2.24–2.48 (m, 4H, 2ꢂ –
SCH2–), 2.02–2.22 (m, 3H, E2 3ꢂ –CY–), 1.70–2.00 (m, 6H, pro 2ꢂ
–CY2–, –CY2–), 1.12–1.68 (m, 16H, E2 5ꢂ –CY2–, –CH32), 0.77 (s,
3H, E2 –CH3); MS (ESI): m/z 817 (M+Na)+.
4.2.24. (2S)-N-[4-{3-(17-Hydroxy-1,3,5-estratrien-3-yl)-oxy-
propyl}-piperazino)-propyloxy)-5-methoxy-2-nitrobenzoyl]-
4,4-difluoropyrrolidine-2-carboxaldehyde diethyl thioacetal
(14e)
This compound was prepared according to the method
described for compound 14a, employing compound 11c (436 mg,
1 mmol) and compound 9a (520 mg, 1 mmol). The crude product
was purified by column chromatography using ethyl acetate–
hexane (92%) to afford the compound 14e as a light yellow solid.
Yield 683 mg, 78%; mp 90–92 °C; 1H NMR (CDCl3, 200 MHz): d
7.72 (s, 1H, ArH), 7.19 (d, 1H, J = 8.8 Hz, ArH), 6.83 (s, 1H, ArH),
6.69 (dd, 1H, J = 2.2, 8.8 Hz, ArH), 6.61 (s, 1H, ArH), 5.32 (br s, 1H,
E2 –OH), 4.86 (d, 1H, J = 4.0 Hz, –CHS2), 4.66–4.77 (m, 1H, pro –
NCH–), 4.17 (t, 2H, J = 6.6, Hz, –OCH2–), 3.98 (t, 2H, J = 5.9, Hz, –
OCH2–), 3.93 (s, 3H, –OCH3), 3.72 (t, 1H J = 8.8, E2 –CY–OH),
3.21–3.40 (m, 2H, pro –NCH2–) 2.82–3.10 (m, 12H, –N(–CH2–
CH2–)2N–, 2ꢂ –NCH2–), 2.54–2.79 (m, 6H, 2ꢂ –SCH2–, E2 –CH2–),
1.60–2.51 (m, 9H, E2 3ꢂ –CY–, pro –CY2–, 2ꢂ –CY2–), 1.12–1.55
(m, 16H, E2 5ꢂ –CY2–, –CH32), 0.78 (s, 3H, E2 –CH3); MS (ESI):
m/z 876 (M+H)+.
4.2.27. (2S)-N-[4-{3-(17-Hydroxy-1,3,5-estratrien-3-yl}-oxy-
pentyl)-1H-1,2,3-triazol-4-yl]-methoxy-5-methoxy-2-nitro-
benzoyl]-pyrrolidine-2-carboxaldehyde diethylthio acetal (15b)
This compound was prepared according to the method
described for compound 15a, employing compound 12a (439 mg,
1 mmol) and compound 10b (384 mg, 1 mmol). The crude product
was purified by column chromatography using ethyl acetate–
hexane (70%) to afford the compound 15b as a yellow solid. Yield
739 mg, 90%; mp 110–112 °C; 1H NMR (CDCl3, 300 MHz): d 7.91
(s, 1H, 1H, triazole –H), 7.68 (s, 1H, ArH), 7.19 (d, 1H, J = 8.5 Hz,
ArH), 6.85 (s, 1H, ArH), 6.68 (dd, 1H, J = 2.3, 8.5 Hz, ArH), 6.61 (s,
1H, ArH), 5.34 (br s, 1H, E2 –OH), 5.30 (s, 2H, –OCH2–), 4.87 (d,
1H, J = 3.8 Hz, –CHS2), 4.67–4.74 (m, 1H, pro –NCH–), 4.40 (t, 2H,
J = 7.2 Hz, –OCH2–), 3.94 (s, 3H, –OCH3), 3.73 (t, 1H, J = 8.7 Hz, E2
–CY–OH), 3.39 (t, 2H, J = 6.9 Hz, –CH2N–), 3.20–3.30 (m, 2H, pro
–CH2N–), 2.68–2.90 (m, 2H, E2 –CH2–), 2.23–2.43 (m, 4H, 2ꢂ –
SCH2–), 2.05–2.21 (m, 3H, E2 3ꢂ –CY–), 1.72–2.20 (m, 10H, pro
2ꢂ –CY2–, 3ꢂ –CY2–), 1.13–1.68 (m, 16H, E2 5ꢂ –CY2–, –CH32),
0.77 (s, 3H, E2 –CH3); MS (ESI): m/z 822 (M+ꢀ).
4.2.25. (2S)-N-[4-{3-(17-Hydroxy-1,3,5-estratrien-3-yl)-oxy-
pentyl}-piperazino)-pentyloxy)-5-methoxy-2-nitrobenzoyl]-
4,4-difluoropyrrolidine-2-carboxaldehyde diethyl thioacetal
(14f)
This compound was prepared according to the method
described for compound 14a, employing compound 11c (436 mg,
1 mmol) and compound 9b (576 mg, 1 mmol). The crude product
was purified by column chromatography using ethyl acetate–
hexane (95%) to afford the compound 14f as a yellow solid. Yield
659 mg, 73%; mp 102–103 °C; 1H NMR (CDCl3, 200 MHz): d 7.69
(s, 1H, ArH), 7.19 (d, 1H, J = 8.7 Hz, ArH), 6.81 (s, 1H, ArH), 6.70
4.2.28. (2S,4R)-N-[4-{3-(17-Hydroxy-1,3,5-estratrien-3-yl}-oxy-
propyl)-1H-1,2,3-triazol-4-yl]-methoxy-5-methoxy-2-nitro-
benzoyl]-4-fluoropyrrolidine-2-carboxaldehyde diethylthio-
acetal (15c)
This compound was prepared according to the method
described for compound 15a, employing compound 12b (457 mg,
1 mmol) and compound 10a (356 mg, 1 mmol). The crude product
was purified by column chromatography using ethyl acetate–
hexane (75%) to afford the compound 15c as a yellow solid. Yield
663 mg, 82%; mp 98–100 °C; 1H NMR (CDCl3, 300 MHz): d 7.90
(s, 1H, triazole –H), 7.68 (s, 1H, ArH), 7.20 (d, 1H, J = 8.6 Hz), 6.89
(s, 1H, ArH), 6.67 (dd, 1H, J = 3.1, 8.6 Hz, ArH), 6.61 (s, 1H, ArH),
5.36 (br s, 1H, E2 –OH), 5.32 (s, 2H, –OCH2–), 4.87 (d, 1H,
J = 3.8 Hz, –CHS2), 4.55–4.66 (m, 1H, pro –NCH–), 4.58 (t, 2H,
J = 7.0 Hz, –OCH2–), 3.97 (s, 3H, –OCH3), 3.89–3.95 (m, 2H, –
CH2N–), 3.45–3.79 (m, 2H, E2 –CY–OH, pro –CYF–), 3.20–3.30 (m,
2H, pro –CH2N–), 2.75–2.99 (m, 6H, E2 –CH2–, 2ꢂ –SCH2–), 2.38–
2.52 (m, 3H, E2 –CY–), 1.82–2.30 (m, 4H, pro –CY2–, –CY2–),
1.14–1.73 (m, 16H, E2 5ꢂ –CY2–, –CH32), 0.77 (s, 3H, E2 –CH3);
MS (ESI): m/z 835 (M+Na)+.
(dd, 1H, J = 3.1, 8.7 Hz, ArH), 6.62 (s, 1H, ArH), 5.34 (br s, 1H, E2
–
OH), 4.87 (d, 1H, J = 3.9 Hz, –CHS2), 4.70–4.74 (m, 1H, pro –NCH–
), 4.08–4.15 (m, 4H, 2ꢂ –OCH2–), 3.97 (s, 3H, –OCH3), 3.70–3.77
(m, 3H, pro –NCH2–, E2 –CY–OH), 2.77–3.50 (m, 12H, –N(–CH2–
CH2–)2N–, 2ꢂ –NCH2–), 2.40–2.72 (m, 6H, 2ꢂ –SCH2–, E2 –CH2–),
2.37–2.61 (m, 3H, E2 3ꢂ –CY–), 1.89–2.00 (m, 10H, 4ꢂ –CH2– pro
–CY2–), 1.76–1.86 (m, 4H, 2ꢂ –CH2), 1.32–1.72 (m, 16H, E2 5ꢂ –
CY2–, –CH32), 0.78 (s, 3H, E2 –CH3); MS (ESI): m/z 932 (M+H)+.
4.2.26. (2S)-N-[4-{3-(17-Hydroxy-1,3,5-estratrien-3-yl}oxy-
propyl)-1H-1,2,3-triazol-4-yl]-methoxy-5-methoxy-2-nitro-
benzoyl]-pyrrolidine-2-carboxaldehyde diethylthio acetal (15a)
To a solution of nitro diethylthioacetal 12a, (439 mg 1 mmol)
and compound 10a (356 mg, 1 mmol) were dissolved in t-BuOH,
H2O 1:1 ratio (20 mL). Freshly prepared sodium ascorbate solution
(19.01 mg, 5 mol %) was added followed by CuSO4ꢀ5H2O (1 mol %)
the heterogeneous mixture was stirred at room temperature for
12 h, after completion of reaction, 2 mL of 3% ammonia solution
was added to the reaction mixture for quenching of excess
CuSO4ꢀ5H2O and stirred for further 10 min, t-BuOH was removed
under reduced pressure, the aqueous layer was diluted with ethyl
acetate (30 mL) stirred for another 10 min and then filtered
4.2.29. (2S,4R)-N-[4-{3-(17-Hydroxy-1,3,5-estratrien-3-yl}-
oxypentyl)-1H-1,2,3-triazol-4-yl]-methoxy-5-methoxy-2-
nitrobenzoyl]-4-fluoropyrrolidine-2-carboxaldehyde
diethylthioacetal (15d)
This compound was prepared according to the method de-
scribed for compound 15a, employing compound 12b (457 mg,
1 mmol) and compound 10b (384 mg, 1 mmol). The crude product

2576
A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2565–2581
was purified by column chromatography using ethyl acetate–
hexane (83%) to afford the compound 15d as a yellow solid. Yield
733 mg, 85%; mp 104–106 °C; 1H NMR (CDCl3, 300 MHz): d 7.90
(s, 1H, triazole –H), 7.69 (s, 1H, ArH), 7.19 (d, 1H, J = 8.3 Hz, ArH),
6.88 (s, 1H, ArH), 6.68 (dd, 1H, J = 3.1, 8.3 Hz, ArH), 6.63 (s, 1H,
ArH), 5.35 (s, 1H, E2 –OH), 5.30 (s, 2H, –OCH2–), 4.88 (d, 1H,
J = 3.0 Hz, –CHS2), 4.59–4.65 (m, 1H, pro –NCH–), 4.57 (t, 2H,
J = 7.0 Hz, –OCH2–), 3.98 (s, 3H, –OCH3), 3.87–3.96 (m, 2H,
–CH2N–), 3.44–3.80 (m, 2H, E2 –CY–OH, pro –CYF–), 3.19–3.29
(m, 2H, pro –CH2N–), 2.74–2.98 (m, 6H, E2 –CH2–, 2ꢂ –SCH2–),
2.38–2.52 (m, 3H, E2 3ꢂ –CY–), 1.78–2.35 (m, 8H, pro –CY2–, 3ꢂ
–CY2–), 1.13–1.71 (m, 16H, E2 5ꢂ –CY2–, –CH32), 0.78 (s, 3H, E2
–CH3); MS (ESI): m/z 862 (M+Na)+.
phase was dried over anhydrous Na2SO4 and evaporated under
vacuum to afford the amino diethylthio acetal, which due to poten-
tial stability problems preceded for the next step. A solution of
amino diethylthioacetal (0.82 mmol), HgCl2 (2.10 mmol) and
CaCO3 (2.10 mmol) in CH3CN–H2O (4:1) was stirred slowly at room
temperature until TLC indicated complete loss of starting material
(12 h). The reaction mixture was diluted with ethyl acetate (25 mL)
and filtered through a Celite bed. The clear light yellow organic
supernatant was washed with saturated 5% NaHCO3 (20 mL) and
brine (20 mL), and the combined organic phase was dried over
anhydrous Na2SO4. The organic layer was evaporated in vacuum
and purified by column chromatography using MeOH/CHCl3
(1.2%) to give the final product 3a as a white solid. Yield 318 mg,
68%; mp 88–90 °C; 1H NMR (CDCl3, 200 MHz): d 7.66 (d, 1H,
J = 4.40 Hz, imine –H), 7.51 (s, 1H, ArH), 7.19 (d, 1H, J = 8.1 Hz,
ArH), 6.83 (s, 1H, ArH), 6.70 (dd, 1H, J = 2.2, 8.1 Hz, ArH), 6.63 (s,
1H, ArH), 5.32 (br s, 1H, E2 –OH), 4.26 (t, 2H, J = 4.1 Hz, –OCH2–),
4.14 (t, 2H, J = 5.1 Hz, –OCH2–), 3.93 (s, 3H, –OCH3), 3.53–3.86
(m, 3H, pro –NCH2–, –NCH–), 2.96–3.00 (m, 1H, E2 –CY–OH),
2.80–2.86 (m, 2H, E2 –CH2–), 2.28–2.33 (m, 3H, E2 3ꢂ –CY–),
2.10–2.20 (m, 4H, pro 2ꢂ –CH2–), 1.81–1.98 (m, 2H, –CH2–),
1.16–1.70 (m, 10H, E2 5ꢂ –CH2–), 0.77 (s, 3H, E2 –CH3); 13C NMR
(CDCl3, 75 MHz): d 164.5, 162.3, 156.4, 150.6, 147.6, 140.4, 137.8,
132.6, 126.2, 120.0, 114.3, 111.9, 111.4, 110.4, 81.6, 65.4, 64.0,
56.0, 53.6, 49.8, 46.6, 43.8, 43.1, 38.7, 36.6, 30.6, 29.6, 28.9, 27.1,
4.2.30. (2S)-N-[4-{3-(17-Hydroxy-1,3,5-estratrien-3-yl}-oxy-
propyl)-1H-1,2,3-triazol-4-yl]-methoxy-5-methoxy-2-nitro-
benzoyl]-4,4-difluoropyrrolidine-2-carboxaldehyde diethyl-
thioacetal (15e)
This compound was prepared according to the method
described for compound 15a, employing compound 12c (475 mg,
1 mmol) and compound 10a (356 mg, 1 mmol). The crude product
was purified by column chromatography using ethyl acetate–
hexane (85%) to afford the compound 15e as a yellow solid. Yield
682 mg, 80%; mp 109–111 °C; 1H NMR (CDCl3, 200 MHz): d 7.87
(s, 1H, triazole –H), 7.67 (s, 1H, ArH), 7.19 (d, 1H, J = 8.6 Hz, ArH),
6.88 (s, 1H, ArH), 6.67 (dd, 1H, J = 3.1, 8.6, Hz, ArH), 6.61 (s, 1H,
ArH), 5.36 (br s, 1H, E2 –OH), 5.32 (s, 2H, –OCH2–), 4.86 (d, 1H,
J = 3.8 Hz, –CHS2), 4.54–4.65 (m, 1H, pro –NCH–), 4.49 (t, 2H,
J = 7.0 Hz, –OCH2–), 3.98 (s, 3H, –OCH3), 3.88–3.95 (m, 2H, –
CH2N–), 3.67–3.79 (m, 1H, E2 –CY–OH), 3.19–3.29 (m, 2H, pro –
CH2N–), 2.74–2.99 (m, 6H, E2 –CH2–, 2ꢂ –SCH2–), 2.36–2.51 (m,
3H, E2 3ꢂ –CY–), 1.77–2.10 (m, 4H, pro –CY2–, –CY2–), 1.15–1.72
(m, 16H, E2 5ꢂ –CY2–, –CH32), 0.79 (s, 3H, E2 –CH3); MS (ESI):
m/z 853 (M+Na)+.
26.2, 24.0, 23.0, 22.5, 10.9; IR (KBr) (m
max/cm–1): 3356 (br), 2932,
2872, 1604, 1501, 1433, 1254, 1056, 755; MS (ESI): m/z 559
(M+H)+; Anal. Calcd for C34H42N2O5: C, 73.69; H, 7.78; N, 5.13.
Found: C, 73.62; H, 7.72; N, 5.08.
4.2.33. 7-Methoxy-8-{3-(17-Hydroxy-1,3,5-estratrien-3-yl)-
oxy}-pentyloxy)-(11aS)-1,2,3,11a-tetrahydro-5H-
pyrrolo[2,1-c][1,4]benzodiazepin-5-one (3b)
The compound 3b was prepared according to the method
described for the compound 3a, employing the compound 13b
(650 mg, 0.88 mmol). The crude product was purified by column
chromatography using MeOH/CHCl3 (1.4%) to afford the compound
3b as a white solid. Yield 356 mg, 70%; mp 92–94 °C; 1H NMR
(CDCl3, 300 MHz): d 7.69 (d, 1H, J = 3.90 Hz, imine –H), 7.51 (s,
1H, ArH), 7.19 (d, 1H, J = 7.8 Hz, ArH), 6.85 (s, 1H, ArH), 6.69 (dd,
1H, J = 1.95,7.81 Hz, ArH), 6.62 (s, 1H, ArH), 5.30 (br s, 1H, E2
–OH), 4.13 (t, 2H, J = 6.8 Hz, –OCH2–), 4.06 (t, 2H, J = 6.9 Hz, –
OCH2–), 3.94 (s, 3H, –OCH3), 3.73–3.84 (m, 2H, E2 –CY–OH, pro
4.2.31. (2S)-N-[4-{3-(17-Hydroxy-1,3,5-estratrien-3-yl}oxy-
pentyl)-1H-1,2,3-triazol-4-yl]-methoxy-5-methoxy-2-nitro-
benzoyl]-4,4-difluoropyrrolidine-2-carboxaldehyde diethyl-
thioacetal (15f)
This compound was prepared according to the method
described for compound 15a, employing compound 12c (475 mg,
1 mmol) and compound 10b (384 mg, 1 mmol). The crude product
was purified by column chromatography using ethyl acetate–
hexane (85%) to afford the compound 15f as a yellow solid. Yield
712 mg, 83%; mp 119–121 °C; 1H NMR (CDCl3, 300 MHz): d 7.88
(s, 1H, triazole –H), 7.61 (s, 1H, ArH), 7.11 (d, 1H, J = 8.7 Hz, ArH),
6.74 (s, 1H, ArH), 6.59 (dd, 1H, J = 8.7, 2.4 Hz, ArH), 6.51 (s, 1H,
ArH), 5.33 (br s, 1H, E2 –OH), 5.30 (s, 2H, –OCH2–), 4.84–4.89 (m,
1H, pro –NCH–), 4.77 (d, 1H, J = 2.4 Hz, –CHS2), 4.40 (t, 2H,
J = 7.2 Hz, –OCH2–), 3.93 (s, 3H, –OCH3), 3.89 (t, 2H, J = 6.0 Hz, –
CH2N–), 3.69–3.74 (m, 1H, E2 –CY–OH), 3.21–3.32 (m, 2H, pro –
CH2N–), 2.47–2.94 (m, 6H, E2 –CH2–, 2ꢂ –SCH2–), 2.02 –2.34 (m,
5H, E2 3ꢂ –CY–, pro –CY2–), 1.76–1.95 (m, 6H, 3ꢂ –CY2–), 1.12–
1.70 (m, 16H, E2 5ꢂ –CY2–, –CH32), 0.77 (s, 3H, E2 –CH3); MS
(ESI): m/z 859 (M+H)+.
–NCH–), 3.55–3.61 (m, 2H, pro –NCH2–), 2.80–2.86 (m, 2H, E2
–
CH2–), 2.29–2.34 (m, 3H, E2 3ꢂ –CY–), 2.01–2.22 (m, 4H, pro
–CY2–), 1.64–1.73 (m, 6H, 3ꢂ –CY2–), 1.16–1.62 (m, 10H, E2 5ꢂ –
CY2–), 0.77 (s, 3H, E2 –CH3); 13C NMR (CDCl3, 75 MHz): d 164.3,
162.3, 156.3, 150.4, 147.7, 140.3, 137.4, 132.2, 125.7, 120.0,
113.9, 112.0, 111.5, 110.3, 80.9, 67.1, 66.8, 55.8, 53.1, 49.6, 46.6,
43.5, 42.8, 38.4, 36.3, 33.3, 31.4, 30.6, 29.9, 29.3, 28.8, 26.8, 25.9,
22.6, 22.1, 10.7; IR (KBr) (m
max/cmꢁ1): 3348 (br), 2927, 2869,
1604, 1502, 1433, 1254, 1058, 755; MS (ESI): m/z 587 (M+H)+;
Anal. Calcd for C36H46N2O5: C, 73.67; H, 7.91; N, 4.72. Found: C,
73.61; H, 7.86; N, 4.66.
4.2.34. 7-Methoxy-8-{3-(17-Hydroxy-1,3,5-estratrien-3yl)-oxy}-
propyloxy)-(2R,11aS)-2-fluoro-1,2,3,11a-tetrahydro-5H-
pyrrolo[2,1-c][1,4]benzodiazepin-5-one (3c)
The compound 3c was prepared according to the method de-
scribed for the compound 3a, employing the compound 13c
(550 mg, 0.76 mmol). The crude product was purified by column
chromatography using MeOH/CHCl3 (1.6%) to afford the compound
3c as a white solid. Yield 285 mg, 66%; mp 113–115 °C; 1H NMR
(CDCl3, 200 MHz): d 7.77 (d, 1H, J = 3.69 Hz, imine –H), 7.49 (s,
4.2.32. Synthesis of 7-methoxy-8-{3-(17-hydroxy-1,3,5-
estratrien-3-yl)-oxy}-propyloxy)-(11aS)-1,2,3,11a-tetrahydro-
5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (3a)
To a solution of compound 13a (600 mg, 0.84 mmol) in metha-
nol (20 mL), SnCl2ꢀ2H2O (5 mmol) was added and refluxed for 2 h.
The methanol was evaporated in vacuum and the aqueous layer
was carefully adjusted to pH 7 with 10% NaHCO3 solution and then
extracted with ethyl acetate (3 ꢂ 30 mL). The combined organic

A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2565–2581
2577
1H, ArH), 7.19 (d, 1H, J = 7.7 Hz, ArH), 6.84 (s, 1H, ArH), 6.70 (dd,
4.2.37. 7-Methoxy-8-{3-(17-hydroxy-1,3,5-estratrien-3-yl)-
1H, J = 2.2, 7.7 Hz, ArH), 6.63 (s, 1H, ArH), 5.34 (br s, 1H, E2 –OH),
4.24 (t, 2H, J = 6.3 Hz, –OCH2–), 4.11 (t, 2H, J = 6.3 Hz, –OCH2–),
3.94 (s, 3H, –OCH3), 3.82–3.87 (m, 2H, pro –CHF–, E2 –CY–OH),
3.71–3.76 (m, 3H, – pro NCH–, –NCH2–), 3.37–3.43 (m, 2H, E2
–CH2–), 2.80–2.86 (m, 3H, E2 3ꢂ –CY–), 2.25–2.39 (m, 2H,
pro –CH2–), 1.84–2.20 (m, 2H, –CH2–), 1.15–1.69 (m, 10H, E2 5ꢂ
–CH2–), 0.77 (s, 3H, E2 –CH3); 13C NMR (CDCl3, 75 MHz): d 164.4,
162.4, 156.4, 150.9, 147.4, 140.2, 137.8, 132.6, 126.1, 120.1,
114.4, 111.8, 111.4, 110.5, 90.2, 81.6, 65.5, 64.0, 56.0, 53.2, 49.8,
46.6, 43.7, 43.1, 38.6, 36.5, 30.3, 29.6, 28.9, 27.1, 26.1, 24.0, 23.0,
oxy}-pentyloxy)-(11aS)-2,2-difluoro-1,2,3,11a-tetrahydro-5H-
pyrrolo[2,1-c][1,4]benzodiazepin-5-one (3f)
The compound 3f was prepared according to the method de-
scribed for the compound 3a, employing the compound 13f
(650 mg, 0.84 mmol). The crude product was purified by column
chromatography using MeOH/CHCl3 (2.2%) to afford the compound
3f as a white solid. Yield 323 mg, 62%; mp 121–123 °C; 1H NMR
(CDCl3, 300 MHz): d 7.79 (d, 1H, J = 3.6 Hz, imine –H), 7.48 (s, 1H,
ArH), 7.19 (d, 1H, J = 9.0 Hz, ArH), 6.82 (s, 1H, ArH), 6.69 (dd, 1H,
J = 2.7, 9.0 Hz, ArH), 6.62 (s, 1H, ArH), 5.34 (br s, 1H, E2 –OH),
4.13 (t, 2H, J = 6.3 Hz, –OCH2–), 4.02 (t, 2H, J = 6.4 Hz, –OCH2–),
3.94 (s, 3H, –OCH3), 3.83–3.86 (m, 1H, pro –NCH–), 3.71–3.74 (m,
1H, E2 –CY–OH), 3.35–3.40 (m, 2H, pro –NCH2–), 2.80–2.86 (m,
2H, E2–CH2–), 2.28–2.33 (m, 3H, E2 3ꢂ –CY–), 2.10–2.20 (m, 6H,
pro –CH2–, 2ꢂ –CH2–), 1.81–1.98 (m, 2H, –CH2–), 1.16–1.71 (m,
22.9, 11.0; IR (KBr) (m
max/cmꢁ1): 3354 (br), 2928, 2870, 1606,
1501, 1433, 1253, 1058, 755; MS (ESI): m/z 577 (M+H)+; Anal. Calcd
for C34H41FN2O5: C, 70.78; H, 7.27; N, 4.81. Found: C, 70.71; H,
7.22; N, 4.74.
4.2.35. 7-Methoxy-8-{3-(17-hydroxy-1,3,5-estratrien-3-yl)-
oxy}-pentyloxy)-(2R,11aS)-2-fluoro-1,2,3,11a-tetrahydro-5H-
pyrrolo[2,1-c][1,4]benzodiazepin-5-one (3d)
10H, E2 5ꢂ –CH2–), 0.77 (s, 3H, E2 –CH3); IR (KBr) (
m
max/cmꢁ1):
3360 (br), 2938, 2869, 1606, 1501, 1432, 1252, 1133, 1058, 756;
MS (ESI): m/z 623 (M+H)+; Anal. Calcd for C36H44F2N2O5: C,
70.23; H, 7.12; N, 4.50. Found: C, 70.16; H, 7.06; N, 4.43.
The compound 3d was prepared according to the method de-
scribed for the compound 3a, employing the compound 13d
(600 mg, 0.80 mmol). The crude product was purified by column
chromatography using MeOH/CHCl3 (1.7%) to afford the compound
3d as a white solid. Yield 329 mg, 69%; mp 128–130 °C; 1H NMR
(CDCl3, 300 MHz): d 7.85 (d, 1H, J = 3.69 Hz, imine –H), 7.51 (s,
1H, ArH), 7.19 (d, 1H, J = 6.2 Hz, ArH), 6.83 (s, 1H, ArH), 6.70 (dd,
1H, J = 2.2, 6.2 Hz, ArH), 6.63 (s, 1H, ArH), 5.31 (br s, 1H, E2 –OH),
4.13 (t, 2H, J = 6.2 Hz, –OCH2–), 4.06 (t, 2H, J = 6.4 Hz, –OCH2–),
3.94 (s, 3H, –OCH3), 3.84–3.87 (m, 2H, pro –CHF–, E2 –CY–OH),
4.2.38. 7-Methoxy-8-{3-(17-hydroxy-1,3,5-estratrien-3-yl)-
oxypropyl}-piperazino)-propyloxy)-(11aS)-1,2,3,11a-tetra-
hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (4a)
The compound 4a was prepared according to the method de-
scribed for the compound 3a, employing the compound 14a
(650 mg, 0.78 mmol). The crude product was purified by column
chromatography using MeOH/CHCl3 (3.1%) to afford the compound
4a as a white solid. Yield 266 mg, 50%; mp 82–84 °C; 1H NMR
(CDCl3, 300 MHz): d 7.67 (d, 1H, J = 4.5 Hz, imine –H), 7.52 (s, 1H,
ArH), 7.19 (d, 1H, J = 8.3 Hz, ArH), 6.83 (s, 1H, ArH), 6.67 (dd, 1H,
J = 2.2, 8.3 Hz, ArH), 6.60 (s, 1H, ArH), 5.29 (br s, 1H, E2 –OH),
4.15 (t, 2H, J = 6.0 Hz, –OCH2–), 4.00 (t, 2H, J = 5.3 Hz, –OCH2–),
3.94 (s, 3H, –OCH3), 3.78–3.87(m, 2H, pro –NCH–, E2 –CY–OH),
3.69–3.77 (m, 2H, pro –NCH2–), 3.54–3.63 (m, 4H, 2ꢂ –NCH2–),
3.66–3.75 (m, 3H, pro –NCH–, –NCH2–), 3.35–3.40 (m, 2H, E2
–
CH2–), 2.81–2.87 (m, 3H, E2 3ꢂ –CY–), 2.29–2.35 (m, 2H, pro
–CH2–), 1.85–2.20 (m, 6H, 3ꢂ –CH2–), 1.15–1.65 (m, 10H, E2 5ꢂ
–CH2–), 0.78 (s, 3H, E2 –CH3); 13C NMR (CDCl3, 75 MHz): d 164.6,
162.1, 156.7, 151.0, 147.7, 140.5, 137.7, 132.4, 126.1, 121.7,
114.3, 111.8, 111.3, 110.4, 90.5, 81.7, 68.7, 67.7, 56.0, 52.7, 49.8,
46.6, 43.8, 43.1, 38.7, 36.5, 32.2, 30.4, 29.6, 28.9, 28.5. 27.1, 26.2,
2.70–2.98 (m, 8H, –N(–CH2–CH2–)2N–), 2.28–2.36 (m, 5H, E2
–
23.0, 22.9, 22.4, 11.0; IR (KBr) (
m
max/cmꢁ1): 3387 (br), 2928,
CH2–, E2 3ꢂ –CY–), 1.82–2.26 (m, 8H, pro 2ꢂ –CH2–, 2ꢂ –CH2–),
1.16–1.73 (m, 10H, E2 5ꢂ –CH2–), 0.77 (s, 3H, E2 –CH3); 13C NMR
(CDCl3, 75 MHz): d 164.4, 162.7, 156.6, 149.9, 146.86, 140.6,
138.0, 133.1, 125.1, 120.0, 114.4, 111.8, 111.2, 110.2, 81.5, 65.6,
64.9, 56.1, 53.2, 50.1, 49.1, 48.9, 48.1, 47.9, 46.5, 43.6, 43.1, 38.6,
36.6, 31.2, 30.6, 29.6, 28.9, 27.1, 26.2, 24.0, 23.0, 22.5, 10.9; IR
2866, 1607, 1502, 1430, 1254, 1057, 785; MS (ESI): m/z 605
(M+H)+; Anal. Calcd for C36H45FN2O5: C, 71.49; H, 7.51; N, 4.64.
Found: C, 71.45; H, 7.46; N, 4.59.
4.2.36. 7-Methoxy-8-{3-(17-hydroxy-1,3,5-estratrien-3-yl)-
oxy}-propyloxy)-(11aS)-2,2-difluoro-1,2,3,11a-tetrahydro-5H-
pyrrolo[2,1-c][1,4]benzodiazepin-5-one (3e)
(KBr) (
1024, 755; MS (ESI): m/z 685 (M+H)+; Anal. Calcd for
41H56N4O5: C, 71.81; H, 8.32; N, 8.29. Found: C, 71.76; H, 8.25;
m
max/cmꢁ1): 3381 (br), 2945, 2870, 1603, 1500, 1434, 1253,
The compound 3e was prepared according to the method de-
scribed for the compound 3a, employing the compound 13e
(550 mg, 0.74 mmol). The crude product was purified by column
chromatography using MeOH/CHCl3 (2%) to afford the compound
3e as a white solid. Yield 263 mg, 60%; mp 115–117 °C; 1H NMR
(CDCl3, 300 MHz): d 7.79 (d, 1H, J = 3.66 Hz, imine –H), 7.47 (s,
1H, ArH), 7.19 (d, 1H, J = 8.8 Hz, ArH), 6.86 (s, 1H, ArH), 6.71 (dd,
1H, J = 2.2, 8.8 Hz, ArH), 6.64 (s, 1H, ArH), 5.36 (s, 1H, –E2 OH),
4.25 (t, 2H, J = 7.3 Hz, –OCH2–), 4.14 (t, 2H, J = 7.3 Hz, –OCH2–),
3.93 (s, 3H, –OCH3), 3.82–3.87 (m, 1H, pro –NCH–), 3.71–3.74
(m, 1H, E2 –CY–OH), 3.36–3.41 (m, 2H, pro –NCH2–), 2.79–2.87
(m, 2H, E2 –CH2–), 2.26–2.40 (m, 3H, E2 3ꢂ –CY–), 1.83–2.19
(m, 4H, pro –CH2–, –CH2–), 1.16–1.70 (m, 10H, E2 5ꢂ –CH2–),
0.76 (s, 3H, E2 –CH3); 13C NMR (CDCl3, 75 MHz): d 163.8, 160.4,
156.5, 151.3, 146.4, 140.3, 138.0, 132.8, 126.3, 120.6, 114.4,
111.9, 111.5, 110.6, 96.7, 81.8, 65.6, 64.0, 56.1, 53.1, 49.9, 43.9,
43.2, 38.8, 37.2, 36.6, 30.5, 29.7, 29.0, 27.2, 26.2, 23.0, 22.5, 11.0;
C
N, 8.22.
4.2.39. 7-Methoxy-8-{3-(17-hydroxy-1,3,5-estratrien-3-yl)-
oxypentyl}-piperazino)-pentyloxy)-(11aS)-1,2,3,11a-tetra-
hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (4b)
The compound 4b was prepared according to the method de-
scribed for the compound 3a, employing the compound 14b
(700 mg, 0.78 mmol). The crude product was purified by column
chromatography using MeOH/CHCl3 (3.2%) to afford the compound
4b as a white solid. Yield 317 mg, 55%; mp 88–90 °C; 1H NMR
(CDCl3, 200 MHz): d 7.70 (d, 1H, J = 3.9 Hz, imine –H), 7.50 (s, 1H,
ArH), 7.20 (d, 1H, J = 7.2 Hz, ArH), 6.84 (s, 1H, ArH), 6.66 (d, 1H,
J = 7.7 Hz, ArH), 6.61 (s, 1H, ArH), 5.28 (br s, 1H, E2 –OH), 3.99–
4.13 (m, 4H, 2ꢂ –OCH2–), 3.93 (s, 3H, –OCH3), 3.78–3.88(m, 2H,
pro –NCH–, E2 –CY–OH), 3.63–3.75 (m, 2H, pro –NCH2–), 3.54–
3.63 (m, 4H, 2ꢂ –NCH2–), 2.70–2.98 (m, 8H, –N(–CH2–CH2–)2N–),
2.26–2.38 (m, 5H, E2 –CH2–, E2 3ꢂ –CY–), 1.82–2.26 (m, 12H, pro
2ꢂ –CH2–, 4ꢂ–CH2–), 1.71–1.80 (m, 4H, 2ꢂ –CH2–), 1.16–1.69
IR (KBr) (
1256, 1055, 757; MS (ESI): m/z 595 (M+H)+; Anal. Calcd for
34H40F2N2O5: C, 68.95; H, 7.21; N, 4.91. Found: C, 68.88; H,
7.15; N, 4.82.
m
max/cmꢁ1): 3584 (br), 2930, 2870, 1609, 1501, 1435,
C
(m, 10H, E2 5ꢂ –CH2–), 0.77 (s, 3H, E2 –CH3); IR (KBr) (mmax
/
cmꢁ1): 3378 (br), 2951, 2861, 1605, 1530, 1428, 1264, 1031, 754;

2578
A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2565–2581
MS (ESI): m/z 742 (M+H)+; Anal. Calcd for C45H64N4O5: C, 72.93; H,
8.64; N, 7.57. Found: C, 72.88; H, 8.58; N, 8.51.
CH2–)2N–), 2.25–2.40 (m, 5H, E2 –CH2–, E2 3ꢂ –CY–), 1.83–2.27
(m, 6H, pro –CH2–, 2ꢂ –CH2–), 1.14–1.70 (m, 10H, E2 5ꢂ –CH2–),
0.77 (s, 3H, E2 –CH3); IR (KBr) (m
max/cmꢁ1): 3412 (br), 3020, 2870,
4.2.40. 7-Methoxy-8-{3-(17-hydroxy-1,3,5-estratrien-3-yl)-
oxypropyl}-piperazino)-propyloxy)-(2R,11aS)-2-fluoro-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodia zepin-
5-one (4c)
1612, 1517, 1425, 1215, 1049, 758; MS (ESI): m/z 721 (M+H)+;
Anal. Calcd for C41H54F2N4O5: C, 68.42; H, 7.55; N, 7.63. Found: C,
68.38; H, 7.48; N, 7.57.
The compound 4c was prepared according to the method de-
scribed for the compound 3a, employing the compound 14c
(600 mg, 0.70 mmol). The crude product was purified by column
chromatography using MeOH/CHCl3 (3.4%) to afford the compound
4c as a white solid. Yield 235 mg, 48%;mp 84–86 °C; 1H NMR
(CDCl3, 300 MHz): d 7.84 (d, 1H, J = 4.3 Hz, imine –H), 7.51 (s, 1H,
ArH), 7.19 (d, 1H, J = 8.7 Hz, ArH), 6.86 (s, 1H, ArH), 6.69 (dd, 1H,
J = 2.4, 8.7 Hz, ArH), 6.62 (s, 1H, ArH), 5.31 (br s, 1H, E2 –OH),
4.04–4.27 (m, 4H, 2ꢂ –OCH2–), 3.95 (s, 3H, –OCH3), 3.85–3.92
(m, 3H, E2–CY–OH, pro –CYF–, pro –NCH–), 3.62–3.79 (m, 4H,
2ꢂ –NCH2–), 3.36–3.41 (m, 2H, pro –NCH2–), 2.80–2.88 (m, 8H, –
N(–CH2–CH2–)2N–), 2.55–2.67 (m, 5H, E2 –CH2–, E2 3ꢂ –CY–),
1.83–2.27 (m, 6H, pro –CH2–, 2ꢂ –CH2–), 1.16–1.72 (m, 10H, E2
5ꢂ –CH2–), 0.77 (s, 3H, E2 –CH3); 13C NMR (CDCl3, 75 MHz): d
164.5, 162.5, 156.6, 149.8, 146.6, 140.1, 138.3, 133.2, 125.1,
120.0, 114.6, 111.6, 111.1, 110.6, 92.3, 81.6, 65.7, 65.2, 56.0, 53.3,
50.2, 49.2, 48.7, 48.0, 46.9, 46.4, 43.6, 43.1, 38.6, 36.6, 30.6, 29.4,
4.2.43. 7-Methoxy-8-{3-(17-hydroxy-1,3,5-estratrien-3-yl)-
oxypentyl}-piperazino)-pentyloxy)-(11aS)-2,2-difluoro-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodia zepin-5-
one (4f)
The compound 4f was prepared according to the method de-
scribed for the compound 3a, employing the compound 14f
(600 mg, 0.64 mmol). The crude product was purified by column
chromatography using MeOH/CHCl3 (3.8%) to afford the compound
4f as a white solid. Yield 248 mg, 50%; mp 111–113 °C; 1H NMR
(CDCl3, 300 MHz): d 7.78 (d, 1H, J = 3.9 Hz, imine –H), 7.48 (s, 1H,
ArH), 7.20 (d, 1H, J = 7.7 Hz, ArH), 6.84 (s, 1H, ArH), 6.70 (dd, 1H,
J = 3.1, 7.7 Hz, ArH), 6.62 (s, 1H, ArH), 5.30 (br s, 1H, E2 –OH),
3.99–4.32 (m, 4H, 2ꢂ –OCH2–) 3.94 (s, 3H, –OCH3), 3.80–3.90 (m,
2H, E2 –CY–OH, pro –NCH–), 3.68–3.73 (m, 4H, 2ꢂ –NCH2–),
3.47–3.49 (m, 2H, pro –NCH2–), 2.79–2.86 (m, 8H, –N(–CH2–CH2–
)2N–), 2.34–2.48 (m, 5H, E2 –CH2–, E2 3ꢂ –CY–), 1.82–2.26 (m,
14H, pro –CH2–, 6ꢂ –CH2–), 1.15–1.75 (m, 10H, E2 5ꢂ –CH2–),
28.9, 27.0, 26.2, 24.1, 23.1, 22.5, 11.0; IR (KBr) (
m
max/cmꢁ1): 3382
0.79 (s, 3H, E2 –CH3); IR (KBr) (m
max/cmꢁ1): 3360 (br), 2932, 2863,
(br), 2929, 2863, 1605, 1501, 1432, 1215, 1056, 755; MS (ESI):
m/z 703 (M+H)+; Anal. Calcd for C41H55FN4O5: C, 70.46; H, 7.89;
N, 7.91. Found: C, 70.39; H, 7.83; N, 7.87.
1607, 1509, 1432, 1258, 1054, 771; MS (ESI): m/z 777 (M+H)+;
Anal. Calcd for C45H62F2N4O5: C, 69.62; H, 8.34; N, 7.23. Found: C,
69.58; H, 8.31; N, 7.18.
4.2.41. 7-Methoxy-8-{3-(17-hydroxy-1,3,5-estratrien-3-yl)-
oxypentyl}-piperazino)-pentyloxy)-(2R,11aS)-2-fluoro-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodia zepin-5-
one (4d)
4.2.44. 7-Methoxy-8-{3-(17-hydroxy-1,3,5-estratrien-3-yl}-
oxypropyl)-1H-1,2,3-triazole-4-yl-methoxy}-(11aS)-1,2,3,11a-
tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (5a)
The compound 5a was prepared according to the method de-
scribed for the compound 3a, employing the compound 15a
(650 mg, 0.88 mmol). The crude product was purified by column
chromatography using MeOH/CHCl3 (2.6%) to afford the compound
5a as a white solid. Yield 318 mg, 61%; mp 98–100 °C; 1H NMR
(CDCl3, 300 MHz): d 7.79 (d, 1H, J = 3.4 Hz, imine –H), 7.65 (s, 1H,
triazole –H), 7.52 (s, 1H, ArH), 7.19 (d, 1H, J = 9.1 Hz, ArH), 6.98
(s, 1H, ArH), 6.69 (dd, 1H, J = 3.0, 9.1 Hz, ArH), 6.63 (s, 1H, ArH),
5.32 (br s, 1H, E2 –OH), 5.30 (s, 2H, –OCH2–), 4.58 (t, 2H,
J = 6.8 Hz, –OCH2–), 3.95 (t, 2H, J = 5.3 Hz, –CH2N–), 3.92 (s, 3H, –
OCH3), 3.71–3.86 (m, 2H, E2 –CY–OH, pro –NCH–), 3.53–3.62(m,
2H, pro –NCH2–), 2.80–2.87 (m, 2H, E2 –CH2–), 2.27–2.40 (m, 3H,
E2 3ꢂ –CY–), 1.70–2.12 (m, 6H, pro 2ꢂ –CH2–, –CH2–), 1.16–1.66
(m, 10H, E2 5ꢂ –CH2–), 0.77 (s, 3H, E2 –CH3); 13C NMR (CDCl3,
75 MHz): d 164.5, 162.7, 156.7, 156.0, 147.7, 144.4, 138.0, 137.6,
133.0, 126.3, 123.7, 119.7, 114.2, 111.7, 111.2, 110.5, 81.6, 67.8,
63.7, 56.0, 49.8, 49.3, 47.1, 43.7, 43.1, 38.6, 36.5, 30.6, 30.3, 29.7,
The compound 4d was prepared according to the method de-
scribed for the compound 3a, employing the compound 14d
(550 mg, 0.60 mmol). The crude product was purified by column
chromatography using MeOH/CHCl3 (3.5%) to afford the compound
4d as a white solid. Yield 208 mg, 46%; mp 99–101 °C; 1H NMR
(CDCl3, 200 MHz): d 7.79 (d, 1H, J = 4.1 Hz, imine –H), 7.50 (s, 1H,
ArH), 7.20 (d, 1H, J = 7.8 Hz, ArH), 6.84 (s, 1H, ArH), 6.67 (d, 1H,
J = 7.8 Hz, ArH), 6.61 (s, 1H, ArH), 5.30 (br s, 1H, E2 –OH), 3.99–
4.13 (m, 4H, 2ꢂ –OCH2–) 3.94 (s, 3H, –OCH3), 3.80–3.90 (m, 3H,
E2 –CY–OH, pro –CYF–, pro –NCH–), 3.63 –3.75 (m, 4H, 2ꢂ
–NCH2–), 3.46–3.59 (m, 2H, pro –NCH2–), 2.78–2.86 (m, 8H,
–N(–CH2–CH2–)2N–), 2.44–2.58 (m, 5H, E2 –CH2–, E2 3ꢂ –CY–),
1.82–2.26 (m, 14H, pro –CH2–, 6ꢂ –CH2–), 1.16–1.71 (m, 10H, E2
5ꢂ –CH2–), 0.77 (s, 3H, E2 –CH3); IR (KBr) (
m
max/cmꢁ1): 3356 (br),
2929, 2864, 1604, 1507, 1433, 1264, 1065, 788; MS (ESI): m/z
760 (M+H)+; Anal. Calcd for C45H63FN4O5: C, 71.52; H, 8.49; N,
7.61. Found: C, 71.46; H, 8.41; N, 7.54.
28.9, 27.0, 26.1, 24.0, 23.0, 22.9, 10.9; IR (KBr) (m
max/cmꢁ1): 3332
(br), 2931, 2872, 1605, 1503, 1434, 1254, 1054, 754; MS (ESI):
m/z 640 (M+H)+; Anal. Calcd for C37H45N5O5: C, 69.46; H, 7.09; N,
10.95. Found: C, 69.41; H, 7.02; N, 10.89.
4.2.42. 7-Methoxy-8-{3-(17-hydroxy-1,3,5-estratrien-3-yl)-
oxypropyl}-piperazino)-propyloxy)-(11aS)-2,2-difluoro-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodia zepin-5-
one (4e)
4.2.45. 7-Methoxy-8-{3-(17-hydroxy-1,3,5-estratrien-3-yl}-
oxypentyl)-1H-1,2,3-triazole-4-ylmethoxy}-(11aS)-1,2,3,11a-
tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (5b)
The compound 5b was prepared according to the method de-
scribed for the compound 3a, employing the compound 15b
(700 mg, 0.85 mmol). The crude product was purified by column
chromatography using MeOH/CHCl3 (2.8%) to afford the compound
5b as a white solid. Yield 367 mg, 65%; mp 116–118 °C; 1H NMR
(CDCl3, 300 MHz): d 7.80 (d, 1H, J = 3.5 Hz, imine –H), 7.66 (s, 1H,
triazole –H), 7.53 (s, 1H, ArH), 7.20 (d, 1H, J = 8.7 Hz, ArH), 6.99
(s, 1H, ArH), 6.66 (dd, 1H, J = 2.5, 8.7 Hz, ArH), 6.61 (s, 1H, ArH),
5.32 (br s, 1H, E2 –OH), 5.29 (s, 2H, –OCH2–), 4.56 (t, 2H,
The compound 4e was prepared according to the method de-
scribed for the compound 3a, employing the compound 14e
(650 mg, 0.74 mmol). The crude product was purified by column
chromatography using MeOH/CHCl3 (3.6%) to afford the compound
4e as a white solid. Yield 266 mg, 50%; mp 105–107 °C; 1H NMR
(CDCl3, 300 MHz): d 7.79 (d, 1H, J = 3.7 Hz, imine –H), 7.47 (s, 1H,
ArH), 7.19 (d, 1H, J = 8.8 Hz, ArH), 6.86 (s, 1H, ArH), 6.71 (dd, 1H,
J = 2.0, 8.8 Hz, ArH), 6.64 (s, 1H, ArH), 5.31 (br s, 1H, E2 –OH),
4.00–4.35 (m, 4H, 2ꢂ –OCH2–), 3.94 (s, 3H, –OCH3), 3.83–3.86
(m, 2H, E2 –CY–OH, pro –NCH–), 3.70–3.75 (m, 4H, 2ꢂ –NCH2–),
3.37–3.40 (m, 2H, pro –NCH2–), 2.80–2.87 (m, 8H, –N(–CH2–

A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2565–2581
2579
J = 6.2 Hz, –OCH2–), 3.94 (s, 3H, –OCH3), 3.84–3.92 (m, 2H, –CH2N–),
3.71–3.79 (m, 2H, E2 –CY–OH, pro –NCH–), 3.55–3.61 (m, 2H,
–NCH2–), 2.80–2.86 (m, 2H, E2 –CH2–), 2.27–2.40 (m, 3H, E2 3ꢂ
–CY–), 2.00–2.21 (m, 4H, pro 2ꢂ –CH2–), 1.72–1.90 (m, 6H, 3ꢂ
–CH2–), 1.16–1.68 (m, 10H, E2 5ꢂ –CH2–), 0.77 (s, 3H, E2 –CH3);
13C NMR (CDCl3, 75 MHz): d 164.5, 162.7, 155.8, 151.8, 145.0,
144.2, 137.9, 136.8, 133.1, 126.2, 122.5, 119.3, 114.4, 111.9,
111.0, 110.2, 81.5, 68.1, 61.7, 55.9, 49.8, 49.3, 47.1, 43.7, 43.0,
38.5, 36.7, 31.3, 30.2, 29.6, 28.2, 27.0, 26.1, 24.1, 22.9, 22.4, 10.9;
4.2.48. 7-Methoxy-8-{3-(17-hydroxy-1,3,5-estratrien-3-yl}-
oxypropyl)-1H-1,2,3-triazole-4-ylmethoxy}-(11aS)-2,2-difluoro-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]be nzodiazepin-5-
one (5e)
The compound 5e was prepared according to the method de-
scribed for the compound 3a, employing the compound 15e
(650 mg, 0.78 mmol). The crude product was purified by column
chromatography using MeOH/CHCl3 (3.4%) to afford the compound
5e as a white solid. Yield 315 mg, 60%; mp 113–115 °C; 1H NMR
(CDCl3, 300 MHz): d 7.80 (d, 1H, J = 3.9 Hz, imine –H), 7.66, (s,
1H, triazole –H), 7.51 (s, 1H, ArH), 7.20 (d, 1H, J = 7.7 Hz, ArH),
7.00 (s, 1H, ArH), 6.67 (dd, 1H, J = 2.8, 7.7 Hz, ArH), 6.61 (s, 1H,
ArH), 5.32 (br s, 1H, E2 –OH), 5.30 (s, 2H, –OCH2–), 4.37 (t, 2H,
J = 7.5 Hz, –OCH2–), 3.94 (s, 3H, –OCH3), 3.80–3.91 (m, 2H, –
CH2N–), 3.70–3.77 (m, 2H, E2 –CY–OH, pro –NCH–), 3.52–3.62
(m, 2H, pro –NCH2–), 2.22–2.87 (m, 2H, E2 –CH2 –), 2.29–2.36 (m,
3H, E2 3ꢂ –CY–), 1.91–2.17 (m, 4H, pro –CH2–, –CH2–), 1.16–1.74
(m, 10H, E2 5ꢂ –CH2–), 0.77 (s, 3H, E2 –CH3); 13C NMR (CDCl3,
75 MHz): d 164.4, 162.5, 156.7, 156.1, 147.6, 144.1, 138.1, 136.5,
133.3, 126.4, 123.5, 119.6, 114.3, 111.9, 111.2, 110.4, 92.5, 81.8,
63.8, 62.3, 56.1, 49.9, 49.2, 47.2, 43.8, 43.2, 38.7, 36.6, 30.5, 29.4,
IR (KBr) (
1256, 1055, 755; MS (ESI): m/z 668 (M+H)+; Anal. Calcd for
39H49N5O5: C, 70.34; H, 7.40; N, 10.49. Found: C, 70.28; H, 7.35;
m
max/cmꢁ1): 3353 (br), 2925, 2868, 1604, 1503, 1433,
C
N, 10.42.
4.2.46. 7-Methoxy-8-{3-(17-hydroxy-1,3,5-estratrien-3-yl}-
oxypropyl)-1H-1,2,3-triazole-4-ylmethoxy}-(2R,11aS)-2-fluoro-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]be nzodiazepin-5-
one (5c)
The compound 5c was prepared according to the method de-
scribed for the compound 3a, employing the compound 15c
(600 mg, 0.74 mmol). The crude product was purified by column
chromatography using MeOH/CHCl3 (3%) to afford the compound
5c as a white solid. Yield 267 mg, 55%; mp 84–86 °C; 1H NMR (CDCl3,
300 MHz): d 7.83 (d, 1H, J = 4.53 Hz, imine –H), 7.68 (s, 1H, triazole –
H), 7.54 (s, 1H, ArH), 7.20 (d, 1H, J = 9.06 Hz, ArH), 7.01 (s, 1H, ArH),
28.7, 27.1, 26.2, 24.1, 23.0, 22.4, 11.1; IR (KBr) (m
max/cmꢁ1): 3381
(br), 3020, 2936, 1607, 1500, 1431, 1216, 1050, 756; MS (ESI):
m/z 676 (M+H)+; Anal. Calcd for C37H43F2N5O5: C, 65.72; H, 6.49;
N, 10.38. Found: C, 65.65; H, 6.42; N, 10.31.
6.68 (d, 1H, J = 9.06 Hz, ArH), 6.61 (s, 1H, ArH), 5.32 (br s, 1H, E2
–
OH), 5.29 (s, 2H, –OCH2–), 4.39 (t, 2H, J = 6.38 Hz, –OCH2–), 3.94 (s,
3H, –OCH3), 3.81–3.86 (m, 2H, –CH2N–), 3.71–3.80 (m, 3H, E2 –CY–
OH, pro –CHF–, –NCH–), 3.38–3.44 (m, 2H, pro –NCH2–), 2.81–2.87
(m, 2H, E2 –CH2–), 2.24–2.38 (m, 3H, E2 3ꢂ –CY–), 1.86–2.20 (m,
4H, pro –CH2–, –CH2–), 1.16–1.69 (m, 10H, E2 5ꢂ –CH2–), 0.78 (s,
3H, E2 –CH3); 13C NMR (CDCl3, 75 MHz): d 164.3, 162.5, 158.5,
156.0, 147.2, 143.4, 138.0, 136.7, 133.1, 126.3, 123.5, 119.5, 114.2,
111.8, 111.1, 110.1, 85.3, 81.6, 63.7, 62.3, 56.0, 49.8, 49.2, 47.2,
43.7, 43.1, 38.6, 36.5, 30.3, 29.5, 28.3, 27.1, 25.4, 24.1, 23.0, 22.9,
4.2.49. 7-Methoxy-8-{3-(17-hydroxy-1,3,5-estratrien-3-yl}-
oxypentyl)-1H-1,2,3-triazole-4-ylmethoxy}-(11aS)-2,2-difluoro-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-
one (5f)
The compound 5f was prepared according to the method de-
scribed for the compound 3a, employing the compound 15f
(650 mg, 0.75 mmol). The crude product was purified by column
chromatography using MeOH/CHCl3 (3.5%) to afford the compound
5f as a white solid. Yield 310 mg, 59%; mp 116–118 °C; 1H NMR
(CDCl3, 300 MHz): d 7.81 (d, 1H, J = 3.9 Hz, imine –H), 7.69 (s, 1H,
triazole –H), 7.53 (s, 1H, ArH), 7.19 (d, 1H, J = 8.3 Hz, ArH), 6.99
(s, 1H, ArH), 6.67 (dd, 1H, J = 3.3, 8.3 Hz, ArH), 6.60 (s, 1H, ArH),
5.31 (br s, 1H, E2 –OH), 5.29 (s, 2H, –OCH2–), 4.38 (t, 2H,
J = 6.8 Hz, –OCH2–), 3.94 (s, 3H, –OCH3), 3.79–3.91 (m, 2H, –
CH2N–), 3.71–3.76 (m, 2H, E2 –CY–OH, pro –NCH–), 3.53–3.62
(m, 2H, pro –NCH2–), 2.79–2.86 (m, 2H, E2 –CH2–), 2.28–2.35 (m,
3H, E2 3ꢂ –CY–), 2.08–2.25 (m, 4H, 2ꢂ –CH2–), 1.75–2.01 (m, 4H,
pro –CH2–, –CH2–), 1.17–1.60 (m, 10H, E2 5ꢂ –CH2–), 0.77 (s, 3H,
E2 –CH3); 13C NMR (CDCl3, 75 MHz): d 164.5, 162.8, 158.6, 156.5,
147.0, 143.2, 137.9, 136.7, 132.6, 126.2, 123.3, 119.2, 114.3,
111.8, 111.0, 110.2, 90.5, 81.7, 67.1, 62.3, 56.1, 50.3, 49.1, 47.1,
43.8, 43.1, 38.7, 36.6, 30.4, 29.7, 28.5, 27.1, 26.2, 25.6, 25.2, 24.2,
10.9; IR (KBr) (
1429, 1253, 1053, 754; MS (ESI): m/z 658 (M+H)+; Anal. Calcd for
37H44FN5O5: C, 67.64; H, 6.64; N, 10.44. Found: C, 67.59; H, 6.58;
m
max/cmꢁ1): 3386 (br), 2930, 2870, 1607, 1501,
C
N, 10.37.
4.2.47. 7-Methoxy-8-{3-(17-hydroxy-1,3,5-estratrien-3-yl}-
oxypentyl)-1H-1,2,3-triazole-4-ylmethoxy}-(2R,11aS)-2-fluoro-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]be nzodiazepin-5-
one (5d)
The compound 5d was prepared according to the method de-
scribed for the compound 3a, employing the compound 15d
(700 mg, 0.83 mmol). The crude product was purified by column
chromatography using MeOH/CHCl3 (3.1%) to afford the compound
5d as a white solid. Yield 352 mg, 62%; mp 118–120 °C; 1H NMR
(CDCl3, 300 MHz): 7.85 (d, 1H, J = 3.9 Hz, imine –H), 7.65 (s, 1H, tri-
azole –H), 7.52 (s, 1H, ArH), 7.19 (d, 1H, J = 8.8 Hz, ArH), 7.01 (s, 1H,
ArH), 6.67 (dd, 1H, J = 8.7 Hz, ArH), 6.60 (s, 1H, ArH), 5.35 (br s, 1H,
E2 –OH), 5.32 (s, 2H, –OCH2–), 4.37 (t, 2H, J = 6.4 Hz, –OCH2–), 3.94
(s, 3H, –OCH3), 3.86–3.92 (m, 2H, –CH2N–), 3.71–3.75 (m, 1H, pro –
CHF–), 3.62–3.68 (m, 2H, E2 –CY–OH, pro –NCH–), 3.36–3.42 (m,
2H, pro –NCH2–), 2.81–2.86 (m, 2H, E2 –CH2–), 2.29–2.56 (m, 3H,
E2 3ꢂ –CY–), 2.09–2.22 (m, 4H, 2ꢂ –CH2–), 1.76–2.03 (m, 4H, pro
–CH2–, –CH2–), 1.15–1.72 (m, 10H, E2 5ꢂ –CH2–), 0.78 (s, 3H, E2
–CH3); 13C NMR (CDCl3, 75 MHz): d 164.3, 162.7, 158.6, 156.4,
147.8, 143.6, 137.8, 136.6, 132.6, 126.2, 123.3, 119.6, 114.3,
111.8, 110.2, 108.2, 84.7, 81.6, 67.1, 62.2, 56.0, 50.2, 49.8, 47.1,
43.8, 43.1, 38.7, 36.4, 31.4, 30.5, 29.8, 28.5, 27.1, 26.2, 25.4, 23.2,
23.0, 22.9, 11.0; IR (KBr) (m
max/cmꢁ1): 3387 (br), 2981, 2876,
1609, 1531, 1464, 1252, 1051, 777; MS (ESI): m/z 704 (M+H)+;
Anal. Calcd for C39H47F2N5O5: C, 66.71; H, 6.63; N, 9.76. Found: C,
66.64; H, 6.58; N, 9.71.
4.3. Cell culture
MCF-7 (breast carcinoma cells) were incubated by using DMEM
media, supplemented with 10% fetal calf serum, 100
lg/mL
pencillin-G and 100 g/mL streptomycin sulfate. The cell line was
l
maintained at 37 °C in a humidified atmosphere containing 5%
CO2 in the incubator.
4.4. MTT cell viability assay
23.0, 22.5, 11.0; IR (KBr) (m
max/cmꢁ1): 3408 (br), 2927, 2866,
1609, 1503, 1430, 1253, 1054, 787; MS (ESI): m/z 686 (M+H)+;
Anal. Calcd for C39H48FN5O5: C, 68.70; H, 7.45; N, 10.31. Found:
C, 68.62; H, 7.37; N, 10.24.
Cell viability was assessed by the MTT based assay, a mitochon-
drial function assay. It is based on the ability of viable cells to re-
duce the MTT to insoluble formazan crystals by mitochondrial

2580
A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2565–2581
dehydrogenase. In this assay MCF-7 cells were seeded in a 96-well
plate at a density of 10,000 cells/well. After overnight incubation
cells were treated with E2 (1), DC-81 (2), 4a, 4c, 5c, 5d and 5f at
HDAC-1 and HDAC-3 were purchased from Millipore company
Ltd. Mouse monoclonal antibodies such as NF-kB (p65), procas-
pase-7, Rabbit polyclonal antibodies such as b-actin were pur-
chased from Imgenex company. Mouse monoclonal p53 and
Rabbit polyclonal Bax antibodies were purchased from santacruz
Biotech Company. Membranes were washed with TBST three times
for 15 min and the blots were visualized with chemiluminescence
reagent (Thermo Fischer Scientifics Ltd). The X-ray films were
developed with developer and fixed with fixer solution purchased
from Kodak Company.
4
l
M concentration and incubated for 24 h. Then the medium
was discarded and replaced with 10 L MTT dye. Plates were incu-
bated at 37 °C for 2 h. The resulting formazan crystals were solubi-
lized in 100 L extraction buffer. The optical density (O.D.) was
l
l
read at 570 nm with micro plate reader.
4.5. Cell cycle analysis
5 ꢂ 105 MCF-7 cells were seeded in 60 mm dish and were al-
4.8. Transfection studies
lowed to grow for 24 h, 4 lM concentration of E2 (1), DC-81 (2),
4a, 4c, 5c, 5d and 5f compounds were added to the culture media,
and the cells were incubated for an additional 24 h. Cells were har-
vested with Trypsin-EDTA, fixed with ice-cold 70% ethanol at 4 °C
for 30 min, washed with PBS and incubated with 1 mg/mL RNAase
solution (Sigma) at 37 °C for 30 min. Cells were collected by centri-
MCF-7 cells were transfected with 1.5
l
g of p21 (ꢁ208/+8)
promoter fused with luciferase reporter gene in one experiment
pertaining to observe p21 promoter activity in 6 well plate and
transfected into MCF-7 cells using lipofectamine 2000 (invitro-
gen) according to manufacturer’s recommendations. After trans-
fections cells were allowed to grow for 24 h. Then they were
subjected with compound treatment E2 (1), DC-81 (2), 5c and
5d for 24 h at 4 lM concentration. Then the cells were harvested
and lysates were subjected to luciferase assay, which is a lumi-
nescence based assay.
fugation at 2000 rpm for 5 min and further stained with 250 lL of
DNA staining solution [10 mg of propidium iodide (PI), 0.1 mg of
trisodium citrate, and 0.03 mL of Triton X-100 were dissolved in
100 mL of sterile MilliQ water at room temperature for 30 min in
the dark]. The DNA contents of 20,000 events were measured by
flow cytometer (DAKO CYTOMATION, Beckman Coulter, Brea,
CA). Histograms were analyzed using Summit Software.
4.9. Tunel assay
4.6. Immunofluorescence microscopy studies for tubulin
polymerization
Tunel assay (Terminal Transferase dUTP Nick End Labeling) was
conducted by using the Apoalert DNA fragmentation Assay kit
(Clone tech). Apoptosis induced nuclear DNA fragmentation was
determined using this assay. This assay was conducted according
to the manufacturer’s recommendations and is based on the prin-
ciple of terminal deoxy nucleotidyl transferase (TdT)-mediated
dUTP nick-end-labeling. TdT catalyzes incorporation of fluores-
cein-dUTP at the free 30-hydroxyl ends of fragmented DNA.
Flouroscein-labeled DNA can be detected via confocal microscope.
MCF-7 cells were seeded on glass cover slips, incubated for 24 h
in the presence or absence of test compounds E2 (1), DC-81 (2), 5c
and 5d at 4 lM concentration. Following the termination of incu-
bation, cells were fixed with 4% paraformaldehyde, 0.02% glutaral-
dehyde in PBS and permeabilized by dipping the cells in 100%
methanol (ꢁ20 °C). Later, cover slips were blocked with 1% BSA
in phosphate buffered saline for 1 h followed by incubation with
a primary antitubulin (mouse monoclonal) antibody followed by
Cy3 conjugated secondary mouse anti-IgG antibody. At the end
of experiments, cells were washed and fixed Photographs were ta-
ken using the confocal microscope (Olympus), equipped with Cy3
settings and the pictures were analyzed for the integrity of micro-
Acknowledgements
The authors M.K.R., Rajender, J.S.N.R., Y.V.V., D.D.G. and E.V.B. are
grateful to CSIR, and UGC New Delhi, for the award of research fel-
lowships. We are also grateful to the Department of Biotechnology,
New Delhi; for financial assistance.
tubule network. Here Nocodazole (Noc) (4 lM) was used as posi-
tive control for analyzing microtubule integrity. b-Tubulin
antibody was purchased from Abcam Company.
References and notes
4.7. Protein extraction and Western blot analysis
1. Thurston, D. E. Molecular Aspects of Anticancer Drug–DNA Interactions; The
Macmillan Press Ltd: London, UK, 1993. p 54.
2. Denny, W. A. Curr. Med. Chem. 2001, 8, 533.
3. Hadjivassileva, T.; Thurston, D. E.; Taylor, P. W. J. Antimicrob. Chemother. 2005,
56, 513.
4. Inaba, M. Clin. Calcium 2004, 14, 27.
5. Kamal, A.; Reddy, P. S. M. M.; Reddy, D. R. Bioorg. Med. Chem. Lett. 2004, 14,
2669.
6. Kamal, A.; Reddy, J. S.; Ramaiah, M. J.; Bharathi, E. V.; Dastagiri, D.; Reddy, M.
K.; Pushpavalli, S. N. C. L. V.; Pal-Bhadra, M. Bioorg. Med. Chem. Lett. 2010, 20,
5232.
7. Kamal, A.; Kumar, P. P.; Seshadri, B. N.; Srinivas, O.; Kumar, M. S.; Sen, S.;
Kurian, N.; Juvekar, A.; Zingde, S. Bioorg. Med. Chem. 2008, 16, 3895.
8. Kamal, A.; Bharathi, E. V.; Ramaiah, M. J.; Reddy, J. S.; Dastagiri, D.; Viswanath,
A.; Sultana, F.; Pushpavalli, S. N. C. L. V.; Pal-Bhadra, M.; Juvekar, A.; Sen, S.;
Zingde, S. Bioorg. Med. Chem. Lett. 2010, 20, 3310.
9. Kamal, A.; Ramu, R.; Venkatesh, T.; Khanna, G. B. R.; Barkume, M. S.; Juvekar, A.;
Zingde, S. Bioorg. Med. Chem. 2008, 16, 7218.
10. (a) Hou, X.; Ge, Z.; Wang, T.; Guo, W.; Cui, J.; Cheng, T.; Lai, C.; Li, R. Bioorg. Med.
Chem. Lett. 2006, 16, 4214; (b) Shchekotikhin, A. E.; Shtil, A. A.; Luzikov, Y. N.;
Bobrysheva, T. V.; Buyanov, V. N.; Preobrazhenskaya, M. N. Bioorg. Med. Chem.
2005, 13, 2285.
11. Wang, S.; Wang, Q.; Wang, Y.; Liu, L.; Weng, X.; Zhang, G. L. X.; Zhou, X. Bioorg.
Med. Chem. Lett. 2008, 18, 6505.
12. Michejda, C.; Cholody, W. M.; Hernandez, L.; Hassner, L.; Scudiero, D. A.;
Djurickovic, D. B. J. Med. Chem. 1995, 38, 3043.
5 ꢂ 105 MCF-7 cells were seeded in 60 mm dish and were al-
lowed to grow for 24 h, 4 lM concentration of E2 (1), DC-81 (2),
5c and 5d compounds were added to the culture media, and the
cells were incubated for an additional 24 h. Total cell lysates from
cultured MCF-7 cells were obtained by lysing the cells in ice-cold
RIPA buffer (1ꢂ PBS, 1% NP-40, 0.5% sodium deoxycholate and
0.1% SDS) and containing 100
l
g/mL PMSF, 5
l
l
g/mL Aprotinin,
g/mL NaF. After
5
lg/mL leupeptin, 5
lg/mL pepstatin and 100
centrifugation at 12,000 rpm for 10 min, the protein in supernatant
was quantified by Bradford method (BIO-RAD) using Multimode
varioskan instrument (Thermo-Fischer Scientifics). Fifty micro-
grams of protein per lane was applied in 12% SDS–polyacrylamide
gel. After electrophoresis, the protein was transferred to polyvi-
nylidine difluoride (PVDF) membrane (Amersham Biosciences).
The membrane was blocked at room temperature for 2 h in 1ꢂ
TBS + 0.1% Tween20 (TBST) containing 5% blocking powder (Santa-
cruz). The membrane was washed with TBST for 5 min, and pri-
mary antibody was added and incubated at 4 °C overnight.
Mouse monoclonal antibodies p21, Cdk1 and Rabbit polyclonal

A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2565–2581
2581
13. (a) Kamal, A.; Babu, A. H.; Ramana, K. V.; Bharathi, E. V.; Kumar, M. S. Bioorg.
Med. Chem. Lett. 2005, 15, 2621; (b) Kamal, A.; Reddy, P. S. M. M.; Reddy, D. R.;
Laxman, E. Bioorg. Med. Chem. Lett. 2006, 14, 385.
14. Aufort, M.; Herscovici, J.; Bouhours, P.; Moreau, N.; Girard, C. Bioorg. Med. Chem.
Lett. 2008, 18, 1195.
15. Charansingh, G.; Jadhav, G.; Shaikh, M.; Rajesh, R.; Anant, G.; Nagargoje, D.;
Mahendra, S. Bioorg. Med. Chem. Lett. 2008, 18, 6244.
16. Kamal, A.; Shankaraiah, N.; Devaiah, V.; Reddy, K. L.; Juvekar, A.; Kurian, N.;
Zingde, S. Bioorg. Med. Chem. Lett. 2008, 18, 1468.
17. Cargill, C.; Bachmann, E.; Zbinden, G. J. Natl. Cancer Inst. 1974, 53, 481.
18. In Metal Complexes in Cancer Chemotherapy; Keppler, S., Ed.; Weinheim, VCH;
Angerer, V. E., 1993; p 75.
19. Kounelis, S.; Kapranos, N.; Kouri, E.; Coppola, D.; Papadaki, H.; Jones, M. W.
Mod. Pathol. 2000, 13, 379.
30. (a) Kamal, A.; Shankaraiah, N.; Devaiah, V.; Reddy, K. L. Tetrahedron Lett. 2006,
47, 6553; (b) Kamal, A.; Khan, M. N. A.; Srikanth, Y. V. V.; Reddy, K. S.; Juvekar,
A.; Sen, S.; Kurian, N.; Zingde, S. Bioorg. Med. Chem. 2008, 16, 7804.
31. (a) Thurston, D. E.; Murty, V. S.; Langley, D. R.; Jones, G. B. Synthesis 1990, 81;
(b) O’ Neil, I. A.; Thompson, S.; Kalindjian, S. B.; Jenkins, T. C. Tetrahedron Lett.
2003, 44, 7809.
32. Kamal, A.; Rajender; Reddy, R.; Reddy, M. K.; Balakishan, G.; Shaik, T. B.;
Chourasia, M.; Sastry, G. N. Bioorg. Med. Chem. 2009, 17, 1557.
33. Jordan, M. A. Curr. Med. Chem. Anti-cancer Agents 2002, 2, 1.
34. (a) Kanthou, C.; Greco, O.; Stratford, A.; Cook, I.; Knight, R.; Benzakour, O.;
Tozer, G. Am. J. Pathol. 2004, 165, 1401; (b) Wang, L. G.; Liu, X. M.; Kries, W.;
Budman, D. R. Cancer Chemother. Pharmacol. 1999, 44, 355.
35. O’Connor, P. M.; Ferris, D. K.; Pagano, M.; Draetta, G.; Pines, J.; Hunter, T.;
Longo, D. L.; Kohn, K. W. J. Biol. Chem. 1993, 268, 8298.
20. Rao, B. R.; Slotman, B. J. Endocr. Rev. 1991, 12, 14.
21. Canadian Cancer Society, Statistics for 2004, at www.cancer.ca.
22. Burke, P. J.; Koch, T. H. J. Med. Chem. 2004, 47, 1193.
36. Marko, D.; Schatzle, S.; Friedel, A.; Genzlinger, A.; Zanki, H.; Meijer, L.;
Eisenbrand, G. Br. J. Cancer 2001, 84, 283.
37. Basu, A.; Halder, S. Mol. Hum. Reprod. 1998, 4, 1099.
23. Liu, C.; Strobl, J. S.; Bane, S.; Schilling, J. K.; Mc Cracken, M.; Chatterjee, S. K.;
Rabim-Bata, R.; Kingston, D. G. I. J. Nat. Prod. 2004, 67, 152.
24. Do Jun, Y.; Park, H. S.; Kim, J. S.; Park, W.; Ho Song, B.; Kim, H. S.; Taub, D.; Ho
Kim, Y. Toxicol. Appl. Pharmacol. 2008, 231, 401.
25. Liu, H.; Lee, E. S.; Gajdos, C.; Pearce, S. T.; Chen, B.; Sipco, C.; Loweth, J.; McKian,
K.; Alexander, D. L. R.; Wing, L.; Jordan, V. C. J. Natl. Cancer Inst. 2010, 95,
1586.
26. (a) Bérubé, G.; Gupta, A.; Saha, P.; Descoteaux, C.; Leblanc, V.; Asselin, E. Bioorg.
Med. Chem. 2010, 20, 1614; (b) Bérubé, G.; Rabouina, D.; Perrona, V.; Nzembaa,
B.; Gaudreaultb, R.; Parenta, S.; Asselina, E. Steroids 2006, 71, 911.
27. Umezawa, K.; Morioka, M.; Kamizono, A.; Takikawa, H.; Mori, A.; Ueno, H.;
Kadowaki, S.; Nakao, Y.; Kato, K. Bioorg. Med. Chem. 2010, 18, 1143.
28. Barnes, R. K.; Kutikov, A.; Lippard, S. J. Chem. Biol. 2004, 11, 557.
29. (a) Kamal, A.; Laxman, E.; Reddy, P. S. M. M. Tetrahedron Lett. 2000, 41,
7743; (b) Kamal, A.; Reddy, G. S. K.; Raghavan, S. Bioorg. Med. Chem. Lett. 2001,
13, 387.
38. Thornberry, N. A.; Lazebnik, Y. Science 1998, 281, 1312.
39. (a) Kagawa, S.; Fujiwara, S. T.; Hizuta, A.; Yasuda, T.; Zhang, W. W.; Roth, J. A.;
Tanaka, N. Oncogene 1997, 15, 1903; (b) Taguchi, T.; Kato, Y.; Baba, Y.;
Nishimura, Y. G.; Tanigaki, Y.; Horiuchi, C.; Mochimatsu, I.; Tsukuda, M. Oncol.
Rep. 2004, 11, 421.
40. (a) Macleod, K. F.; Sherry, N.; Hannon, G.; Beach, D.; Tokino, T.; Kinzler, K.;
Vogelstein, B.; Jacks, T. Genes Dev. 1995, 9, 935; (b) El-Deiry, W. S.; Tokino, T.;
Velculescu, V.; Levy, D.; Parsons, R.; Trent, J. Cell 1993, 75, 817.
41. Margueron, R.; Duong, V.; Castet, A.; Cavailles, V. Biochem. Pharmacol. 2004, 68,
1239.
42. Yin, L.; Laevsky, G.; Giardina, C. J. Biol. Chem. 2001, 276, 44641.
43. Shishodia, S.; Amin, H.; Lai, R.; Aggarwal, B. B. Biochem. Pharmacol. 2005, 70, 700.
44. Viatour, P.; Merville, M. P.; Bours, V.; Chariot, A. Trends Biochem. Sci. 2005, 30,
43.
45. Takada, Y.; Gillenwater, A.; Ichikawa, H.; Aggarwal, B. B. J. Biol. Chem. 2006, 281,
5612.