Design, synthesis and biological evaluation of estradiol-chlorambucil hybrids as anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2010.01.053

A series of estradiol-chlorambucil hybrids was synthesized as anticancer drugs for site-directed chemotherapy of breast cancer. The novel compounds were synthesized in good yields through efficient modifications of estrone at position 16α of the steroid nucleus. The newly synthesized compounds were evaluated for their anticancer efficacy in different hormone-dependent and hormone-independent breast cancer cell lines. The novel hybrids showed significant in vitro anticancer activity when compared to chlorambucil. Structure-activity relationship (SAR) reveals the influence of the length of the spacer chain between carrier and drug molecule.

Full text of DOI:10.1016/j.bmcl.2010.01.053

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1614–1618
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of estradiol–chlorambucil hybrids
as anticancer agents
*
Atul Gupta, Pijus Saha, Caroline Descôteaux, Valérie Leblanc, Éric Asselin, Gervais Bérubé
Département de Chimie-Biologie, Groupe de Recherche en Oncologie et Endocrinologie Moléculaires, Université du Québec à Trois-Rivières, C.P. 500, Trois-Rivières,
Québec, Canada G9A 5H7
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of estradiol–chlorambucil hybrids was synthesized as anticancer drugs for site-directed chemo-
therapy of breast cancer. The novel compounds were synthesized in good yields through efficient mod-
Received 12 November 2009
Revised 12 January 2010
Accepted 13 January 2010
Available online 20 January 2010
ifications of estrone at position 16
a of the steroid nucleus. The newly synthesized compounds were
evaluated for their anticancer efficacy in different hormone-dependent and hormone-independent breast
cancer cell lines. The novel hybrids showed significant in vitro anticancer activity when compared to
chlorambucil. Structure–activity relationship (SAR) reveals the influence of the length of the spacer chain
between carrier and drug molecule.
Keywords:
Estradiol–chlorambucil hybrids
Anticancer agents
Estrogen receptor
Breast cancer
Ó 2010 Elsevier Ltd. All rights reserved.
Breast cancer, a leading pandemic, affects women of all ages.1 It
is the most frequent site of cancer in women. The successful treat-
ment of this disease is too often limited by the fact that essentially
all breast cancers become resistant to chemotherapy and endo-
crine therapy.2 Throughout a women lifetime, increased exposure
to the estradiol (1, Fig. 1) is an important factor for the develop-
ment of breast, uterine, and ovarian cancers. At menopausal and
post-menopausal conditions, despite low level of circulating estro-
gens, the tissular concentration of estrogens in tissues such as
breast, is significantly high due to its local production. The possible
therapies generally used for estrogen-dependent cancers such as
breast cancer are: (a) use of aromatase inhibitors3 or estrogen sul-
fatase inhibitors4 and (b) use of pure estrogen antagonists such as
fulvestrant5 or selective estrogen receptor modulators (SERMs)
such as tamoxifen.6 Furthermore, the use of estrogen–cytotoxic
conjugates linking platinum-based drugs7 and various alkylating
agents including chlorambucil (2) is the subject of intense
research.8
need to design new chemotherapeutic agents able to target not
only breast cancer, but also to display increased efficacy and over-
all decreased systemic toxicity.
In medicinal chemistry, prodrug approach provides a method to
reduce the gap between physiological and pharmacological doses
of a drug through its slow release in the body.11 Furthermore, if a
drug molecule is converted into a prodrug using a molecule having
affinity with the receptor for which the drug molecule have been
designed, can also improve the efficacy of the drug molecule.
Therefore, site-directed prodrug approach could be an alternative
of choice for the treatment of various diseases such as hormone-
dependent cancers which possibly could lead to increased activity
with reduced adverse side effects.
In our relentless effort to design new anticancer drugs for site-
directed chemotherapy, we have reported a unique class of estra-
diol–Pt(II) hybrid derivatives which showed good in vitro12–17
and in vivo18 potential for the treatment of hormone-dependent
cancers, in particular breast cancer, without any apparent side ef-
fects. Their interactions with DNA and RNA was also studied using
various spectroscopic methods.19–21
Chlorambucil (2) is an alkylating agent of the nitrogen mustard
group and is used as cytostatic drug in cancer therapy.8 In general,
alkylating agents are both mutagenic and genotoxic.9 The alkylat-
ing agents form adducts with DNA. However, they also form ad-
ducts with RNA and protein which are likely to contribute to the
overall cytotoxicity.9
In our quest to develop alternative anticancer agents for site-di-
rected chemotherapy of hormone-dependent cancers, we have de-
signed a series of estradiol–chlorambucil hybrids of type 4. In the
present approach, chlorambucil (2), a known nitrogen mustard
drug was linked to the estradiol framework of type (3) at position
The main side effects of chlorambucil (2) are bone marrow sup-
pression, anemia, and weak immune system.10 Therefore, there is a
16a (Fig. 1). This position albeit close to the estrogenic binding site
apparently does not affect its binding mode. From our previous
studies, we observed that the use of compound 3 as a carrier moi-
ety seemed, in many cases, to be even superior over estradiol 1 in
* Corresponding author. Tel.: +1 819 376 5011x3353.
E-mail address: Gervais.Berube@uqtr.ca (G. Bérubé).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.053

A. Gupta et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1614–1618
OH
1615
OH
Cl
OH
H
N
O
Cl
HO
HO
HO
2
3
1
OH
Cl
OH
N
O
Cl
n
NH
HO
4, n = 2, 4, 6
Figure 1. Structures of estradiol (1), chlorambucil (2), and estradiol derivatives 3 and 4.
O
O
a
HO
THPO
5
6
b
O
O
O
O
c
OCH3
OCH3
n
Br
THPO
8
7
d
O
O
O
O
e
OCH3
OCH3
n
n
NH2
N3
RO
RO
10 i) R = THP
9
i) R = THP
ii)R = OH; n = 4
ii)R = OH; n = 4
O
O
Cl
Cl
OCH3
N
O
n
NH
RO
i) R = THP
ii)R = OH; n = 4
11
g
h
OH
O
4, n = 2, 4, 6
(see figure 1)
Cl
OCH3
N
O
Cl
4
NH
12
HO
Scheme 1. Reagent and conditions: (a) dihydropyran, pyridinium-p-toluenesulfonate, dichloromethane (DCM), 22 °C; (b) dimethyl carbonate, KH, dry tetrahydrofuran (THF),
reflux; (c) dibromoalkane, 10% aq NaOH solution, benzyltriethylammonium chloride (Bn(Et)3N+ClÀ), DCM, reflux; (d) sodium azide (NaN3), methanol, reflux, 20 h; (e) 10% Pd/
C, H2 gas, 22 °C; (f) chlorambucil, HOBt, DCC, Et3N, dry dimethylformamide (DMF), 22 °C; (g) (i) LiBH4 excess, dry diethyl ether (Et2O), 22 °C; (ii) PPTs, EtOH, reflux, 3 h; (h) see
condition g with 1 equiv LiBH4.

1616
A. Gupta et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1614–1618
term of receptor binding affinity. This study presents the design,
synthesis and biological activities of these new estradiol–chloram-
bucil hybrid molecules (4).
(IC50 = 40 lM) as compared with chlorambucil (2) (IC50 >160 lM)
on MDA-MB-436 cell line (Fig. 2). In general, compounds inhibited
effectively cellular proliferation of estrogen receptor negative can-
cer cell lines as compared with estrogen receptor positive cancer
cell lines (Figs. 2–4). However for the ERÀ MDA-MB-231 breast
cancer cells, the various derivatives were completely inactive while
The synthesis of the target compounds of type 4 was initiated
from estrone (5) (Scheme 1). As previously reported, estrone (5)
was protected using dihydropyran in dichloromethane at room
temperature (22 °C) yielding 3-tetrahydropyranyloxy estrone (6)
in 100% yield.12 Compound 6 was activated at position 16 through
the formation of the b-ketoester (7). Compound 7 was obtained
from the reaction of 6 with dimethyl carbonate in presence of
potassium hydride (KH) in dry THF at reflux in 90% yield. Alkyl-
ation of compound 7 with an appropriate dibromoalkane, in bipha-
sic medium using 10% aqueous solution of sodium hydroxide and
dichloromethane, in presence of benzyltriethylammonium chlo-
ride (Bn(Et)3N+ClÀ, a phase transfer catalyst) at reflux gave com-
pound 8 in 50–85% yield.12 Nucleophilic substitution reaction of
compound 8 with sodium azide (NaN3) in methanol at reflux gave
compound 9 in 75–85% yield. Catalytic reduction of compound 9
by hydrogen gas in presence of 10% Pd/C gave compound 10 quan-
titatively. Condensation of compound 10 with chlorambucil in
presence of 1-hydroxybenzotriazole (HOBt), dicyclohexylcarbodi-
imide (DCC) and triethylamine at room temperature (22 °C)
yielded compound 11 in 55–70% yield. Borohydride reduction of
compound 11 with excess of lithium borohydride (LiBH4) in dry
diethyl ether (Et2O) at room temperature (22 °C) and subsequent
deprotection of the tetrahydropyranyl ether group by refluxing
with ethanol in the presence of pyridinium-p-toluenesulfonate
(PPTs) gave the targeted compounds 4 (a–c) in good yields. It is
noteworthy that reduction with a limited amount of lithium boro-
hydride under similar reaction conditions yielded, after deprotec-
tion, the estradiol–chlorambucil conjugate, compound 12, on
which the carboxymethyl group at position 16b of estradiol skele-
ton is retained. The compound 12 was also obtained as side prod-
uct during the synthesis of 4b. The estradiol–chlorambucil hybrids
4 (a–c) (Table 1) were fully characterized by the use of infrared
(IR), nuclear magnetic resonance spectroscopy (NMR) and their
respective high resolution mass analysis.22
chlorambucil itself showed an IC50 of about 130
shown).
lM (data not
The observed biological activity of the hybrids in hormone-
dependent cancer cell line could be due to the potential estrogenic
effect of the estradiol (1) carrier molecule which may trigger coun-
teracting effect over the cytotoxicity of the hybrids leading to over-
all moderate activities in ER+ cancer cell line (MCF-7).
A complete structure–activity relationship (SAR) was attempted
to establish in terms of length of spacer between the carrier
150
2
4a
4b
4c
12
125
100
75
50
25
0
0
40
80 120 160 200 240 280 320
Doses (uM)
In addition, for complete characterization, deprotected com-
pounds 9ii, 10ii and 11ii were synthesized. It was observed that
the coupling reaction after deprotection (between 10ii and chlor-
ambucil) gave an hybrid that was more distinct on thin layer chro-
matography and easier to purify than the coupling of 10i with
chlorambucil but the yield in both cases was almost the same.
However, the reduction of the hybrids with LiBH4 was easier before
deprotection of the hydroxy function at position 3 of the steroid
nucleus.
The newly synthesized compounds were evaluated for their
in vitro cytotoxic activity using estrogen receptor positive (MCF-
7) and negative (MDA-MB-436, MDA-MB-468, MDA-MB-231)
breast cancer cell lines. Chlorambucil (2) was used as positive
control. MTT assays showed that the newly synthesized estra-
diol–chlorambucil hybrids of type 4 (Table 1) possess moderate
to significant cytotoxic activity.23,24
Figure 2. Percent of cell survival in MDA-MB-436 cell line at different concentra-
tions of synthesized compounds after 72 h.
2
4a
4b
4c
150
125
100
75
12
Biological activity results of the compounds showed cytotoxic
activity at higher doses than that of chlorambucil except for com-
pound 4a, which showed anticancer activity at lower dose
50
Table 1
25
Compound numbers and descriptors
Compound No.
Compound descriptors
0
2
Chlorambucil
n = 2
n = 4
n = 6
n = 4
0
40
80 120 160 200 240 280 320
Doses (uM)
4a
4b
4c
12
Figure 3. Percent of cell survival in MDA-MB-468 cell line at different concentra-
tions of synthesized compounds after 72 h.

A. Gupta et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1614–1618
1617
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Relat. Cancer 2008, 15, 27; (b) Lombardi, P. Biochim. Biophys. Acta 2002, 1587,
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R.; Possinger, K.; Rutquist, L. E. Eur. J. Cancer 2005, 41, 346.
150
125
100
75
2
4a
4b
4c
12
6. Jordan, V. C. J. Med. Chem. 2003, 46, 1081.
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9. Drabløs, F.; Feyzi, E.; Aas, P. A.; Vaagbø, C. B.; Kavli, B.; Bratlie, M. S.; Peña-Diaz,
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10. (a) Dorr, R. T.; Fritz, W. L. Cancer Chemotherapy Handbook; Elsevier Science:
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11. Ettmayer, P.; Amidon, G. L.; Clement, B.; Testa, B. J. Med. Chem. 2004, 47, 2393.
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Asselin, É.; Bérubé, G. Bioorg. Med. Chem. Lett. 2003, 13, 3927.
13. Gagnon, V.; St-Germain, M.-È.; Descôteaux, C.; Provencher-Mandeville, J.;
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5919.
0
0
40
80 120 160 200 240 280 320
Doses (uM)
Figure 4. Percent of cell survival in MCF-7 cell line at different concentrations of
synthesized compounds after 72 h.
14. Perron, V.; Rabouin, D.; Asselin, É.; Parent, S.; Gaudreault, R.-C.; Bérubé, G.
Bioorg. Chem. 2005, 33, 1.
15. Bérubé, G. U.S. patent 7153,844, 2006.
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Steroids 2008, 73, 1077.
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É.; Bérubé, G. Bioorg. Med. Chem. Lett. 2008, 18, 2282.
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Bérubé, G.; Asselin, É. Endocr. Relat. Cancer 2009, 16, 1185.
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G. DNA Cell Biol. 2008, 27, 101.
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H.-A.; Bérubé, G. DNA Cell Biol. 2008, 27, 337.
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Bérubé, G.; Tajmir-Riahi, H.-A. DNA Cell Biol. 2009, 28, 31.
22. Typical procedure for the synthesis of estradiol–chlorambucil conjugate 4: The
synthesis of derivatives 3-tetrahydropyranyloxy-1,3,5(10)-estratrien-17-one
estradiol framework of type (3) and cytotoxic molecule chloram-
bucil (2). Cytotoxic activity of these compounds in hormone-inde-
pendent cancer cell lines (MDA-MB-436 and MDA-MB-486)
showed the effect of chain length. In fact, there is a gradual de-
crease in activity of the hybrids with an increase in spacer length.
Compound 4a, with a four carbon atoms spacer, was found to be
most active in hormone-independent cell lines. However, such
structure–activity relationship could not be established for these
compounds in hormone-dependent cell line (MCF-7). In MCF-7 cell
line, the hybrid with six carbon atoms spacer was found to be most
active followed by compound with four carbon atoms spacer
among this series of hybrids. It is noteworthy that derivative 12,
bearing a carboxymethyl function at position 16b, is essentially
inactive in all the breast cancer cells tested. This further indicates
that the hydroxymethyl function at position 16b is beneficial for
biological activity of the hybrids 4.
(6) and 3-tetrahydropyranyloxy-16
a,b-methoxycarbonyl-1,3,5(10)-estratrien-
17-one (7) was already described in an earlier report.16
Synthesis of 3-tetrahydropyranyloxy-16b-(methoxycarbonyl)-16a-(6-bromoh-
exyl)-1,3,5(10)-estratrien-17-one (8): Compound
7 (2.01 g, 4.87 mmol) was
dissolved in 58 mL of dichloromethane, to it 1,6-dibromohexane (29.22 mmol),
triethylbenzyl-ammonium chloride (525 mg) and 30 mL of 10% w/v NaOH
solution were added. The reaction mixture was refluxed for 24 h with vigorous
stirring, diluted with ether, washed with saturated NH4Cl solution and then
with water (4Â). The organic phase was dried over MgSO4, filtered, evaporated
and the residue was purified by column chromatography using acetone-
hexane (1:9) as the eluent to give 8 in 50% yield. Spectral data for 8 (n = 4): IR
In conclusions, the new estradiol–chlorambucil hybrids
4
showed moderate to significant cytotoxic activity in hormone-
dependent (MCF-7) and hormone-independent (MDA-MB-436
and MDA-MB-486) breast cancer cell lines. The novel hybrids were
synthesized in good yields through modification of estrone (5) at
position 16 using simple and efficient chemistry. The MTT cytotox-
icity studies of the compounds indicated that these novel hybrid
molecules of type 4 have chemotherapeutic potential which could
be used as cytotoxic agent in treatment of cancer. The true capacity
of the molecules to treat hormone-dependent cancer will be more
clearly expressed in vivo. Further evaluation of these compounds
would be useful for the development of new anticancer drugs.
(m
max, cmÀ1): 1751 (C@O, ester), 1724 (C@O, ketone), 1611 (C@C); 1H NMR
(200 MHz, CDCl3, d ppm): 7.18 (d, J = 8.60 Hz, 1H, ArH), 6.88–6.79 (m, 2H, ArH),
5.39 (m, 1H, OCHO), 3.91–3.77 and 3.69–3.55 (two m, 2H, OCH2) 3.72 (s, 3H,
OCH3), 3.38 (t, J = 6.64 Hz, 2H, CH2Br), 2.89 (m, 2H, CH2), 2.44–1.22 (several m,
27H, 3 Â CH and 12 Â CH2), 0.92 (s, 3H, CH3); 13C NMR (50 MHz, CDCl3, d ppm):
214.3, 172.1, 155.3, 137.8, 132.9, 126.4, 116.7, 114.3, 96.6, 62.2, 60.4, 52.9,
49.8, 46.2, 44.3, 38.1, 35.6, 34.1, 32.9, 32.3, 30.8, 30.6, 29.8, 29.1, 28.1, 26.7,
25.9, 25.5, 18.9, 14.3; ESI+ HRMS: (M+H)+ and (M+Na)+ calculated for
C31H43BrO5: 575.2367 and 597.2186; found = 575.2369 and 597.2184,
respectively.
Synthesis of 3-tetrahydropyranyloxy-16b-(methoxycarbonyl)-16a-(6-azidohexyl)-
1,3,5(10)-estratrien-17-one (9i): Compound 8 (1.5 g, 2.61 mmol) was dissolved
in methanol (20 mL) and sodium azide (NaN3, 5 equiv) was added to this
solution. The solution was heated to reflux for 20 h with stirring. The solvent
was evaporated and diethyl ether was added to the residue. The ethereal
solution was washed with water, dried over magnesium sulfate, filtered and
concentrated to the oily product. The crude oily product was purified by flash
chromatography using acetone–hexane (1:9) as the eluent which yielded
Acknowledgments
The authors wish to thank the Fonds de Recherche sur la Nature
et les Technologies du Québec (FQRNT) for valuable financial sup-
port. We also wish to thank the following B.Sc. students who par-
ticipated to the synthesis of the starting material: David Marcoux,
Nicolas Bruneau-Latour, Martine Loranger, Yancee Bourassa.
compound 9i in 85% yield. Spectral data for 9i (n = 4): IR (m
max, cmÀ1): 2094
(N3), 1752 (C@O, ester), 1725 (C@O, ketone), 1609 (C@C); 1H NMR (200 MHz,
CDCl3, d ppm): 7.18 (d, J = 8.60 Hz, 1H, ArH), 6.88–6.79 (m, 2H, ArH), 5.38 (m,
1H, OCHO), 3.97–3.85 and 3.69–3.55 (two m, 2H, OCH2), 3.72 (s, 3H, OCH3),
3.25 (t, J = 6.64 Hz, 2H, CH2N3), 2.88 (m, 2H, CH2), 2.44–1.22 (several m, 27H,
3 Â CH and 12 Â CH2), 0.92 (s, 3H, CH3); ESI+ HRMS: (M+Na)+ calculated for
C31H43N3O5: 560.31010; found = 560.30988. A portion of this product was
deprotected (reflux with ethanol in presence of pyridinium-p-toluenesulfonate
References and notes
(PPTs) for 3 h) for full characterization: 3-hydroxy-16b-(methoxycarbonyl)-16
(6-azidohexyl)-1,3,5(10)-estratrien-17-one (9ii, 3-OH): IR (
max, cmÀ1): 3435
(OH), 2093 (N3), 1750 (C@O, ester), 1722 (C@O, ketone), 1611 (C@C); 1H NMR
a-
m
1. (a) Polyak, K. Biochim. Biophys. Acta 2001, 1552, 1; (b) Esteva, F. J.; Hortobagyi,
G. N. Sci. Am. 2008, 58; (c) Baselga, J.; Norton, L. Cancer Cell 2002, 1, 319.

1618
A. Gupta et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1614–1618
(11ii, 3-OH): IR (
max, cmÀ1): 3296 (OH), 1752 (C@O, ester), 1723 (C@O, ketone);
(200 MHz, CDCl3, d ppm): 7.15 (d, J = 8.60 Hz, 1H, ArH), 6.70–6.58 (m, 2H, ArH),
m
5.00 (br s, 1H, OH), 3.72 (s, 3H, OCH3), 3.24 (t, J = 6.64 Hz, 2H, CH2N3), 2.88 (m,
1H NMR (200 MHz, CDCl3, d ppm): 1H NMR (200 MHz, CDCl3, d ppm): 7.14–7.04
(m, 3H, ArH), 6.68–6.61 (m, 4H, ArH), 5.46 (br s, 1H, NH), 3.71(s, 3H, –OCH3),
3.70–3.61 (m, 9H, N(CH2CH2Cl)2, OH hidden) 3.22 (q, J = 6.2 Hz, 2H, CH2N), 2.82
(m, 2H, CH2), 2.55 (t, J = 7.40 Hz, 2H, CH2CONH), 2.43–1.09 (several m, 25H,
3 Â CH, 11 Â CH2), 0.92 (s, 3H, CH3). 13C NMR (50 MHz, CDCl3, d ppm): 214.2,
172.9, 171.9, 154.0, 144.2, 137.8, 131.5, 130.8, 129.7, 126.4, 115.4, 113.0, 112.3,
60.1, 53.6, 52.6, 49.5, 49.3, 46.0, 44.0, 40.5, 39.5, 37.9, 36.0, 35.4, 34.1, 33.8, 32.1,
30.7, 29.6, 29.4, 27.4, 26.6, 25.8, 25.6, 25.3, 24.9, 14.1; ESI+ HRMS: (M+H)+ and
(M+Na)+ calculated for C40H54Cl2N2O5: 713.3483 and 7735.3302;
found = 713.3484 and 735.3293, respectively.
2H, CH2), 2.44–1.10 (several m, 21H, 3 Â CH and 9 Â CH2), 0.92 (s, 3H, CH3); 13
C
NMR (50 MHz, CDCl3, d ppm): 214.9, 172.1, 153.9, 138.2, 132.0, 126.7, 115.5,
113.1, 60.4, 52.9, 51.6, 49.8, 46.2, 44.2, 38.1, 35.7, 32.3, 30.8, 29.6, 29.5, 29.0,
26.7, 26.0, 25.5, 14.3; ESI+ HRMS: (M+Na)+ calculated for C26H35N3O4:
476.2520; found = 476.2519.
Synthesis of 3-tetrahydropyranyloxy-16b-(methoxycarbonyl)-16a-(6-aminohexyl)-
1,3,5(10)-estratrien-17-one (10i): To the solution of compound 9i (0.856 g,
1.67 mmol) in tetrahydrofuran (THF, 15 mL), 10% Pd/C (0.150 g) was added.
Hydrogen gas was bubbled to this solution for 10 min and the solution stirred for
24 h. The reaction mixture was filtered through a pad of Celite™ that washed with
excess diethyl ether to recover the product. The solvent was evaporated and the
crude product was used as such in the next reaction (99% yield). Spectral data for
Synthesis of 3-hydroxy-16b-hydroxymethyl-16a-(4,4-([bis-(2-chloroethyl)-amino]-
phenyl)-butanoylaminohexyl)-1,3,5(10)-estratrien-17-ol 4b (4, n = 4): In a round
bottomed flask, compound 11i (0.45 g, 0.56 mmol) was dissolved in dry diethyl
ether (10 mL), to this solution an excess of lithium borohydride (6 equiv LiBH4)
was added in portions at 0 °C. The reaction mixture was stirred at room
temperature (22 °C) for 24 h. After completion of the reaction, the reaction
mixture was neutralized with saturated ammonium chloride solution and
extracted with ether. The organic layer was separated and dried over
magnesium sulfate. The ether layer was concentrated on rotatory evaporator
and the crude residue was then refluxed with pyridinium-p-toluenesulfonate in
ethanol for 3 h; neutralized with saturated ammonium chloride solution, diluted
with diethyl ether, then washed with saturated ammonium chloride solution 2Â
and water 4Â, respectively. Thereafter organic phase was dried over MgSO4,
evaporated and purified by column chromatography on silica gel using acetone–
hexane (4:6) as the eluent which gave pure compound with 44% yield. Spectral
10i (n = 4): IR (m
max, cmÀ1): 3374 (NH), 1754 (C@O, ester), 1725 (C@O, ketone),
1612 (C@C);1H NMR (200 MHz, CDCl3, d ppm): 7.18(d,J = 8.60 Hz, 1H, ArH), 6.88–
6.81(m,2H,ArH),5.39(m,1H,OCHO),3.96–3.85and3.62–3.56(twom,2H,OCH2),
3.73 (s, 3H, OCH3), 3.00 (m, 2H, CH2NH2), 2.89 (m, 2H, CH2), 2.65 (m, 2H, CH2NH2),
2.37–1.25 (several m, 27H, 3 Â CH, 12 Â CH2), 0.93 (s, 3H, CH3).
For full characterization the deprotected derivative (10ii) was prepared from 9i
using PPTs as described above: 3-hydroxy-16b-(methoxycarbonyl)-16a-(6-
aminohexyl)-1,3,5(10)-estratrien-17-one (10ii, 3-OH): 1H NMR (200 MHz, CDCl3,
d ppm): 7.08 (d, J = 8.60 Hz, 1H, ArH), 6.62–6.56 (m, 2H, ArH), 3.71 (s, 3H, OCH3),
3.60 (m, 2H, CH2NH2), 2.85 (m, 2H, CH2), 2.71 (m, 3H, OH and CH2NH2), 2.43–1.17
(several m, 21H, 3 Â CH, 9 Â CH2), 0.91 (s, 3H, CH3); 13C NMR (50 MHz, CDCl3, d
ppm): 214.2, 171.9, 154.5, 137.7, 131.0, 126.3, 115.5, 113.1, 60.1, 52.6, 49.5, 46.0,
44.0, 41.8, 38.0, 35.5, 33.0, 32.1, 30.6, 29.5, 29.4, 26.5, 25.7, 25.6, 25.3, 14.1; ESI+
HRMS: (M+H)+ and (M+Na)+ calculated for C26H37NO4: 428.2795 and 450.2615;
found = 428.2795 and 450.2611, respectively.
data for 4b: IR (m
max, cmÀ1): 3306 (OH); 1H NMR (200 MHz, CDCl3, d ppm): 7.14–
7.04 (m, 3H, ArH), 6.65–6.57 (m, 4H, ArH), 6.10(brs, 1H, OH), 5.58 (t, J = 5.6 Hz, 1H,
NH), 3.82–3.44 (m, 13H, N(CH2CH2Cl)2, CH, CH2OH, OH), 3.22 (q, J = 6.2 Hz, 2H,
CH2N), 2.79 (m, 2H, CH2), 2.55 (t, J = 7.40 Hz, 2H, CH2CONH), 2.30–1.03 (several m,
25H, 3 Â CH, 11 Â CH2), 0.86 (s, 3H, CH3). 13C NMR (50 MHz, CDCl3, d ppm): 173.4,
154.2, 144.6, 138.2, 132.3, 130.9, 129.9, 126.5, 115.6, 113.1, 112.4, 90.8, 67.2, 53.8,
47.9, 47.0, 45.1, 44.1, 40.8, 39.7, 39.5, 38.2, 36.3, 34.3, 33.4, 30.1, 29.8, 27.7, 27.0,
26.5, 24.8, 12.2. ESI+ HRMS: (M+H)+ and (M+Na)+ calculated for C39H56Cl2N2O4:
687.36899 and 709.35093; found = 687.36964 and 709.35232, respectively.
Synthesis of 3-tetrahydropyranyloxy-16b-methoxycarbonyl-16a-(4,4-([bis-(2-chloro-
ethyl)-amino]-phenyl)-butanoylaminobutyl)-1,3,5(10)-estratrien-17-one (11i):
Chlorambucil (0.192 g, 0.63 mmol), 1-hydroxybenzotriazole (HOBt, 0.085 g,
0.63 mmol) and dicyclohexylcarbodiimide (DCC, 0.130 g, 0.63 mmol) were
dissolved in dry dimethylformamide (DMF, 1.5 mL) and stirred at room
temperature (22 °C) for 15–20 min until a white precipitate was separated out.
To this mixture, compound 10i (0.322 g, 0.63 mmol), dissolved in dry DMF
(1.5 mL), and 2–4 drops of triethylamine were added under constant stirring for
20 h. The excess solvent was evaporated under rotary evaporator. The residue
wasdissolved in ether andextracted with water. Theorganic layerwas driedover
magnesium sulfate and concentrated to oily residue. The crude product was
purified by flash chromatography using acetone-hexane (3:7) as the eluent
which gave pure compound 11i with 55% yield. Spectral data for 11i (n = 4): IR
Synthesis of 3-hydroxy-16b-methoxycarbonyl-16a-(4,4-([bis-(2-chloroethyl)-amino]-
phenyl)-butanoylaminohexyl)-1,3,5(10)-estratrien-17-ol (12): This derivative was
synthesized using the same procedure as above for derivative 4b, in this case, only
1 equiv of LiBH4. Spectral data: IR (m
max, cmÀ1): 3325 (OH), 1730 (C@O, ester), 1704
(C@O, ketone); NMR (200 MHz, CDCl3, d ppm): 7.11–7.04 (m, 3H, ArH), 6.64–6.59
(m,4H,ArH),5.80(brs,1H,OH),5.48(m,1H,NH),4.65(d,J = 7.4 Hz, 1H, CHOH), 3.71
(s, 3H, OCH3), 3.69–3.40 (m, 9H, N(CH2CH2Cl)2, OH), 3.22 (q, J = 6.3 Hz, 2H, CH2N),
2.80 (m, 2H, CH2), 2.54 (t, J = 7.40 Hz, 2H, CH2CONH), 1.94–1.03 (several m, 25H,
3 Â CH, 11 Â CH2), 0.72 (s, 3H, CH3). 13C NMR (50 MHz, CDCl3, d ppm): 178.9, 173.2,
154.1, 144.6, 138.1, 132.3, 130.9, 129.9, 126.7, 115.5, 113.1, 112.4, 90.0, 66.9, 53.8,
52.3, 52.0, 46.6, 45.3, 44.2, 42.4, 40.8, 39.7,38.4, 37.2, 36.6, 36.3,34.3, 29.8, 27.7, 27.5,
26.9, 26.4, 25.6, 11.9. ESI+ HRMS: (M+H)+ and (M+Na)+ calculated for
C40H56Cl2N2O5: 715.36390 and 737.34585; found = 715.36324 and 737.34571,
respectively.
(m
max, cmÀ1): 3315 (NH), 1752 (C@O, ester), 1725 (C@O, ketone); 1H NMR
(200 MHz, CDCl3, d ppm): 7.14–7.04 (m, 3H, ArH), 6.65–6.57 (m, 4H, ArH), 5.45
(br s, 1H, NH), 5.38 (m, 1H, OCHO), 3.95–3.85 and 3.69–3.55 hidden (two m, 2H,
OCH2), 3.71 (s, 3H, OCH3), 3.66–3.49 (m, 8H, N(CH2CH2Cl)2, 3.20 (m, 2H, CH2N),
2.80 (m, 2H, CH2), 2.54 (t, J = 7.20 Hz, 2H, CH2CONH), 2.41–1.10 (several m, 31H,
3 Â CH, 14 Â CH2), 0.92 (s, 3H, CH3); ESI+ HRMS: (M+H)+ calculated for
C45H62Cl2N2O6: 797.40630; found = 797.40651.
For full characterization deprotected derivative (11ii) was synthesized from the
coupling reaction between 10ii and chlorambucil using the same procedure as
23. Carmichael, J.; DeGrapp, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell, J. B. Cancer
Res. 1987, 47, 936.
for the preparation of 11i: 3-hydroxy-16b-methoxycarbonyl-16
chloroethyl)-amino]-phenyl)-butanoylaminobutyl)-1,3,5(10)-estratrien-17-one
a
-(4,4-([bis-(2-
24. Ford, C. H. J.; Richardson, V. J.; Tsaltas, G. Cancer Chemother. Pharmacol. 1989,
24, 295.